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Diese Zusammenstellung basiert auf Befunden einer laufenden Plagiatsanalyse (Stand: 2016-02-17) – es handelt sich insofern nicht um einen abschließenden Bericht. Zur weiteren Meinungsbildung wird daher empfohlen, den jeweiligen Stand der Analyse auf der Seite http://de.vroniplag.wikia.com/wiki/Ww zum Vergleich heranzuziehen.

Eine kritische Auseinandersetzung mit der Dissertation von Wen Wang: Validation of shRNA clones for gene silencing in 293FT cells

Inaugural-Dissertation zur Erlangung der Doktorwürde der Medizinischen Fakultät der Bayerischen Julius-Maximilians-Universität Würzburg. Tag der mündlichen Prüfung: 21.01.2008. Referent: PD Dr. Norbert Schütze, Koreferent: Prof. Dr. med Franz Jakob.
→ Nachweis: Deutsche Nationalbibliothek "Diese Publikation ist aus rechtlichen Gründen gesperrt. Ursprünglich als Dissertation veröffentlicht, Doktorgrad wurde laut Universität Würzburg am 21.01.2019 entzogen."
→ Nachweis: UB Würzburg "Aus rechtlichen Gründen wurde der Zugriff auf den Volltext zu diesem Dokument gesperrt."

Der Barcode drückt den Anteil der Seiten aus, die Fremdtextübernahmen enthalten, nicht den Fremdtextanteil am Fließtext. Je nach Menge des übernommenen Textes werden drei Farben verwendet:

  • schwarz: bis zu 50 % Fremdtextanteil auf der Seite
  • dunkelrot: zwischen 50 % und 75 % Fremdtextanteil auf der Seite
  • hellrot: über 75 % Fremdtextanteil auf der Seite

Weiße Seiten wurden entweder noch nicht untersucht oder es wurde nichts gefunden. Blaue Seiten umfassen Titelblatt, Inhaltsverzeichnis, Literaturverzeichnis, Vakatseiten und evtl. Anhänge, die in die Berechnung nicht einbezogen werden.

Der Barcode stellt den momentanen Bearbeitungsstand dar. Er gibt nicht das endgültige Ergebnis der Untersuchung wieder, da Untersuchungen im VroniPlag Wiki stets für jeden zur Bearbeitung offen bleiben, und somit kein Endergebnis existiert.

24 Seiten mit Plagiatstext

Seiten mit weniger als 50% Plagiatstext

9 Seiten: 001 024 025 027 062 063 016 041 064

Seiten mit 50%-75% Plagiatstext

2 Seiten: 014 015

Seiten mit mehr als 75% Plagiatstext

13 Seiten: 013 026 002 003 004 005 008 010 011 012 007 029 065

Befunde

  • Die Dissertation enthält zahlreiche wörtliche und sinngemäße Textübernahmen, die nicht als solche kenntlich gemacht sind. Als betroffen festgestellt wurden bisher (Stand: 17. Februar 2016) folgende Kapitel, die sich teilweise als vollständig übernommen erwiesen haben – siehe Klammervermerke:
  • 1. Introduction
  • 1.1 dsRNA, siRNA, miRNA and shRNA
  • 1.1.2 siRNA (S. 1-2): Seiten 1, 2 – [vollständig]
  • 1.1.3 miRNA [Anf.] (S. 2): Seite 2
  • 1.1.3.1 Formation and processing of miRNA (S. 2-4): Seiten 2, 3, 4 – [vollständig]
  • 1.1.3.2 Cellular functions of miRNA (S. 4-5): Seiten 4, 5 – [vollständig]
  • 1.1.4 shRNA (S. 5-7): Seiten 5, 7
  • 1.2 RNA interference [Anf.] (S. 7-8): Seiten 7 – [vollständig]
  • 1.2.1 Mechanism of RNAi [Anf.] (S. 8): Seite 8 – [vollständig (wörtlich)]
  • 1.2.1.1 Initiation phase: dsRNA processing into siRNAs (S. 8-11): Seiten 8, 10, 11 – [vollständig (Text)]
  • 1.2.1.2 Execution phase: assembly of siRNA containing Silencing complexes (S. 11-12): Seiten 11, 12 – [vollständig]
  • 1.2.2 Therapeutic applications of RNAi [Anf.] (S. 12): Seite 12 – [vollständig (wörtlich)]
  • 1.2.2.1 Cancer (S. 13): Seite 13 – [vollständig]
  • 1.2.2.2 Infectious diseases (S. 14): Seite 14 – [vollständig (wörtlich)]
  • 1.2.2.4 Drawbacks of RNAi therapeutics (S. 15-16): Seiten 15, 16 – [vollständig]
  • 1.4 293FT cell lines [Anf.] (S. 24): Seite 24 – [vollständig]
  • 1.4.1 Origins of 293 cell lines (S. 24-26): Seiten 24, 25, 26
  • 1.4.2 Applications of 293 cell lines (S. 26-27): Seiten 26, 27 – [vollständig]
  • 2. Materials
  • 2.1 shRNA bacterial glycerol stocks (S. 29-30): Seite 29
  • 3. Methods
  • 3.2 Purification and sequencing of plasmid DNA
  • 3.2.7 Midi-purification of plasmid DNA (S. 41): Seite 41
  • 5. Discussion
  • 5.2 PLCβ4 is a multifunction protein [Anf.] (S. 62-63): Seiten 62
  • 5.2.2 Activation of PLCβ4 by Gɑq subunits (S. 63-64): Seite 63
  • 5.3 Possible reasons for low efficiency of RNAi
  • 5.3.1 Drawbacks of shRNA (S. 64-65): Seiten 64, 65.

Herausragende Quellen

Herausragende Fundstellen

  • Fragment 014 01: Ein wörtlich übernommener Abschnitt, bei dem die Quelle ungenannt bleibt. Auch zahlreiche Literaturverweise sind übernommen.
  • Fragment 026 01: Eine ganzseitige Übernahme aus der Wikipedia, die ungenannt bleibt.

Andere Beobachtungen

  • Die Seitenangaben im Inhaltsverzeichnis stimmen zum Teil nicht.
  • Die zum Zeitpunkt der Abgabe der Dissertation gültige Promotionsordnung für die Medizinische Fakultät der Julius-Maximilians-Universität Würzburg vom 29. März 1983 (→ PDF der geänderten Fassung vom 1.10.2001) enthält u.a. folgende Aussagen und Bestimmungen:
  • § 4 Zulassung
    "(3) Der Antrag auf Zulassung zum Promotionsverfahren ist unter Angabe des angestrebten Doktorgrades schriftlich beim Dekanat der Medizinischen Fakultät einzureichen. Ihm sind beizufügen: [...]
    3. die ehrenwörtliche Erklärung, dass der Bewerber die Dissertation selbständig angefertigt hat und keine anderen als die von ihm angegebenen Quellen und Hilfsmittel benutzt hat [...]."
  • § 6 Dissertation
    "(1) Die Dissertation ist eine wissenschaftliche Abhandlung, durch welche der Bewerber seine Fähigkeit nachweist, wissenschaftliche Probleme selbständig und methodisch einwandfrei zu bearbeiten. Sie sollte in der Regel nicht mehr als 40 Seiten umfassen. [...]"
  • § 11 Ungültigkeit von Promotionsleistungen
    "(2) Wird die Täuschung erst nach Aushändigung der Urkunde bekannt, so kann nachträglich die Doktorprüfung für Nichtbestanden erklärt werden. Die Entziehung des Doktorgrades erfolgt nach dem Gesetz über die Führung akademischer Grade vom 7. Juni 1939 (BayBSErgB S.115).
    (5) Im Übrigen richtet sich der Entzug des Doktorgrades nach den gesetzlichen Bestimmungen (Art. 48 und 49 BayVwVfG)."
  • Auf der Seite iii der Dissertation findet sich eine "Declaration". Dort heißt es u.a.:
    "I declare that the submitted dissertation was completed by myself and none other, and I have not used any sources or materials other than those enclosed."

Statistik

  • Es sind bislang 33 gesichtete Fragmente dokumentiert, die als Plagiat eingestuft wurden. Bei 31 von diesen handelt es sich um Übernahmen ohne Verweis auf die Quelle („Verschleierungen“ oder „Komplettplagiate“). Bei 2 Fragmenten ist die Quelle zwar angegeben, die Übernahme jedoch nicht ausreichend gekennzeichnet („Bauernopfer“).
  • Die untersuchte Arbeit hat 70 Seiten im Hauptteil. Auf 24 dieser Seiten wurden bislang Plagiate dokumentiert, was einem Anteil von 34.3 % entspricht.
    Die 70 Seiten lassen sich bezüglich des Textanteils, der als Plagiat eingestuft ist, wie folgt einordnen:
Plagiatsanteil Anzahl Seiten
keine Plagiate dokumentiert 46
0 % - 50 % Plagiatsanteil 9
50 % - 75 % Plagiatsanteil 2
75 % - 100 % Plagiatsanteil 13
Ausgehend von dieser Aufstellung lässt sich abschätzen, wieviel Text der untersuchten Arbeit gegenwärtig als plagiiert dokumentiert ist: Es sind, konservativ geschätzt, rund 18 % des Textes im Hauptteil der Arbeit.


Illustration

Folgende Grafik illustriert das Ausmaß und die Verteilung der dokumentierten Fundstellen. Die Farben bezeichnen den diagnostizierten Plagiatstyp:
(grau=Komplettplagiat, rot=Verschleierung, gelb=Bauernopfer)

Ww col

Die Nichtlesbarkeit des Textes ist aus urheberrechtlichen Gründen beabsichtigt.

Zum Vergrößern auf die Grafik klicken.


Anmerkung: Die Grafik repräsentiert den Analysestand vom 17. Februar 2016.

Definition von Plagiatkategorien

Die hier verwendeten Plagiatkategorien basieren auf den Ausarbeitungen von Wohnsdorf / Weber-Wulff: Strategien der Plagiatsbekämpfung, 2006. Eine vollständige Beschreibung der Kategorien findet sich im VroniPlag-Wiki. Die Plagiatkategorien sind im Einzelnen:

Übersetzungsplagiat

Ein Übersetzungsplagiat entsteht durch wörtliche Übersetzung aus einem fremdsprachlichen Text. Natürlich lässt hier die Qualität der Übersetzung einen mehr oder weniger großen Interpretationsspielraum. Fremdsprachen lassen sich zudem höchst selten mit mathematischer Präzision übersetzen, so dass jede Übersetzung eine eigene Interpretation darstellt. Zur Abgrenzung zwischen Paraphrase und Kopie bei Übersetzungen gibt es ein Diskussionsforum.

Komplettplagiat

Text, der wörtlich aus einer Quelle ohne Quellenangabe übernommen wurde.

Verschleierung

Text, der erkennbar aus fremder Quelle stammt, jedoch umformuliert und weder als Paraphrase noch als Zitat gekennzeichnet wurde.

Bauernopfer

Text, dessen Quelle ausgewiesen ist, der jedoch ohne Kenntlichmachung einer wörtlichen oder sinngemäßen Übernahme kopiert wurde.

Quellen nach Fragmentart

Die folgende Tabelle schlüsselt alle gesichteten Fragmente zeilenweise nach Quellen und spaltenweise nach Plagiatskategorien auf.

Tabelle: Ww: Quellen / Fragmente (dynamische Auszählung)
Quelle
Jahr ÜP
KP
VS
BO
KW
KeinP

ZuSichten
Unfertig
Geley and Mueller 2004 0 2 1 0 0 0 3 0 0
Hammond 2005 0 1 4 0 0 0 5 0 0
Kilic and Yuan 2007 0 0 1 0 0 0 1 0 0
Krueger 2007 0 0 0 0 0 0 0 1 0
McIntyre and Fanning 2006 0 0 0 1 0 0 1 0 0
Milhavet et al 2003 0 2 0 0 0 0 2 0 0
Paddison et al 2002 0 0 0 1 0 0 1 0 0
Rebecchi Pentyala 2000 0 1 0 0 0 0 1 0 0
Root et al 2006 0 0 1 0 0 0 1 0 0
Stevenson 2004 0 2 2 0 0 0 4 0 0
Wikipedia HEK 293 cells 2007 0 0 4 0 0 0 4 0 0
Wikipedia Phosphoinositide phospholipase C 2007 0 1 0 0 0 0 1 0 0
Wikipedia Small hairpin RNA 2007 0 1 1 0 0 0 2 0 0
Wikipedia Small interfering RNA 2007 0 0 2 0 0 0 2 0 0
Wikipedia microRNA 2007 0 0 4 0 0 0 4 0 0
Zou and Yoder 2005 0 0 1 0 0 0 1 0 0
- 0 10 21 2 0 0 33 1 0

Fragmentübersicht

33 gesichtete, geschützte Fragmente

FragmentSeiteArbeitZeileArbeitQuelleSeiteQuelleZeileQuelleTypus
Ww/Fragment 001 13113-21Wikipedia Small interfering RNA 20071 (Internetquelle)-Verschleierung
Ww/Fragment 002 0121-9Wikipedia Small interfering RNA 20071 (Internetquelle)-Verschleierung
Ww/Fragment 002 17217-29Wikipedia microRNA 20071 (Internetquelle)-Verschleierung
Ww/Fragment 003 0131ff (komplett)Wikipedia microRNA 20071 (Internetquelle)-Verschleierung
Ww/Fragment 004 0141 ff. (komplett)Wikipedia microRNA 20071 (Internetquelle)-Verschleierung
Ww/Fragment 005 0151-18Wikipedia microRNA 20071 (Internetquelle)-Verschleierung
Ww/Fragment 005 20520-24Wikipedia Small hairpin RNA 20071 (Internetquelle)-Verschleierung
Ww/Fragment 007 0171-5Wikipedia Small hairpin RNA 20071 (Internetquelle)-KomplettPlagiat
Ww/Fragment 007 0777-21Zou and Yoder 2005211l.col: 2ffVerschleierung
Ww/Fragment 007 22722-28Geley and Mueller 2004985r.col: 12ffKomplettPlagiat
Ww/Fragment 008 0181-20Geley and Mueller 2004985, 986985: r.col: letzte Zeilen; 986: l.col: 1ffKomplettPlagiat
Ww/Fragment 008 22822-28Hammond 20055822, 58235822: r.col. 36 ff.; 5823: l.col.: 26 ff.Verschleierung
Ww/Fragment 010 01101 ff. (komplett)Hammond 20055823 f5823: l.col. 30 ff.; r.col. 4 ff.; 5824 l.col. last linesVerschleierung
Ww/Fragment 011 01111-10Hammond 20055824r.col: 20ffVerschleierung
Ww/Fragment 011 111111-24Geley and Mueller 2004987l.col: 1ffVerschleierung
Ww/Fragment 011 251125-30Hammond 20055825r.col. 14 ff.KomplettPlagiat
Ww/Fragment 012 01121-20Hammond 20055825 f.5825: r.col. last lines; 5826: l.co. 1 ff., 26 ff.Verschleierung
Ww/Fragment 012 211221 ff.Stevenson 20041773r.col: 4ffKomplettPlagiat
Ww/Fragment 013 01131-11Milhavet et al 2003643643:li.Sp. 21ffKomplettPlagiat
Ww/Fragment 013 121312-29Stevenson 200417731773: r.col: 19ffVerschleierung
Ww/Fragment 014 01141-18Milhavet et al 2003643-644643:re.Sp. 51ff - 644:li.Sp. 1ffKomplettPlagiat
Ww/Fragment 015 111511-31Stevenson 20041776l.col: 20ffKomplettPlagiat
Ww/Fragment 016 01161-10Stevenson 20041776r.col: 12ffVerschleierung
Ww/Fragment 024 112411-21Wikipedia HEK 293 cells 20071 (Internetquelle)-Verschleierung
Ww/Fragment 025 242524-29Wikipedia HEK 293 cells 20071 (Internetquelle)-Verschleierung
Ww/Fragment 026 01261 ff. (komplett)Wikipedia HEK 293 cells 20071 (Internetquelle)-Verschleierung
Ww/Fragment 027 01271-14Wikipedia HEK 293 cells 20071 (Internetquelle)-Verschleierung
Ww/Fragment 029 03293-11Root et al 2006716figure 1Verschleierung
Ww/Fragment 041 114111-17Kilic and Yuan 20071 (online source)0028Verschleierung
Ww/Fragment 062 146214-24Wikipedia Phosphoinositide phospholipase C 20071 (Internetquelle)-KomplettPlagiat
Ww/Fragment 063 156315-28Rebecchi Pentyala 20001298 , 12991298: l.col. last lines; r.col. last lines; 1299: l.col: 1ffKomplettPlagiat
Ww/Fragment 064 156415-22Paddison et al 2002951l..col: 38ffBauernOpfer
Ww/Fragment 065 08658-31McIntyre and Fanning 20061, 2, 3, 41: abstract: 2ff, r.col: 5ff; 2: l.col: 1ff; 3: l.col: letzte Zeile; 4: r.col: 21ffBauernOpfer

Textfragmente

Anmerkung zur Farbhinterlegung

Die Farbhinterlegung dient ausschließlich der leichteren Orientierung des Lesers im Text. Das Vorliegen einer wörtlichen, abgewandelten oder sinngemäßen Übernahme erschließt sich durch den Text.

Hinweis zur Zeilenzählung

Bei der Angabe einer Fundstelle wird alles, was Text enthält (außer Kopfzeile mit Seitenzahl), als Zeile gezählt, auch Überschriften. In der Regel werden aber Abbildungen, Tabellen, etc. inklusive deren Titel nicht mitgezählt. Die Zeilen der Fußnoten werden allerdings beginnend mit 101 durchnummeriert, z. B. 101 für die erste Fußnote der Seite.

33 gesichtete, geschützte Fragmente

[1.] Ww/Fragment 001 13

Verschleierung
Untersuchte Arbeit:
Seite: 1, Zeilen: 13-21
Quelle: Wikipedia Small interfering RNA 2007
Seite(n): 1 (Internetquelle), Zeilen: -
1.1.2 siRNA

Small interfering RNAs (siRNAs), sometimes known as short interfering RNA or silencing RNAs, represent a class of 20-25 nucleotide-long double-stranded RNA molecules that play a variety of roles in biology.

siRNAs have a well defined structure: a short (usually 21-nt) double-strand of RNA (dsRNA) with 2-nt 3' overhangs on either end. Each strand has a 5' phosphate group and a 3' hydroxyl (-OH) group. This structure is the result of processing by Dicer, an enzyme that converts either long dsRNA or hairpin RNAs into siRNAs (Baulcombe et al, 1999).

Most notably, siRNAs are involved in the RNA interference (RNAi) pathway [where the siRNA interferes with the expression of a specific gene (Tuschl et al, 2001).]

Small interfering RNA (siRNA), sometimes known as short interfering RNA or silencing RNA, are a class of 20-25 nucleotide-long double-stranded RNA molecules that play a variety of roles in biology. Most notably, siRNA is involved in the RNA interference (RNAi) pathway where the siRNA interferes with the expression of a specific gene. [...]

[...]

SiRNAs have a well defined structure: a short (usually 21-nt) double-strand of RNA (dsRNA) with 2-nt 3' overhangs on either end:

[...]

Each strand has a 5' phosphate group and a 3' hydroxyl (-OH) group. This structure is the result of processing by dicer, an enzyme that converts either long dsRNAs or small hairpin RNAs into siRNAs.[3]


3. Bernstein E, Caudy A, Hammond S, Hannon G (2001). "Role for a bidentate ribonuclease in the initiation step of RNA interference". Nature 409 (6818): 363–6. PMID 11201747.

Anmerkungen

Kein Hinweis auf die Quelle.


[2.] Ww/Fragment 002 01

Verschleierung
Untersuchte Arbeit:
Seite: 2, Zeilen: 1-9
Quelle: Wikipedia Small interfering RNA 2007
Seite(n): 1 (Internetquelle), Zeilen: -
[Most notably, siRNAs are involved in the RNA interference (RNAi) pathway] where the siRNA interferes with the expression of a specific gene (Tuschl et al, 2001). In addition to their role in the RNAi pathway, siRNAs also act in RNAi-related pathways, as an antiviral mechanism or in shaping the chromatin structure of a genome. siRNAs can also be exogenously (artificially) introduced into cells by various transfection methods to bring about the specific knockdown of a gene of interest. Essentially any gene of which the sequence is known can thus be targeted based on sequence complementarity with tailored siRNA. This has made siRNA an important tool for gene function and drug target validation studies in the post-genomic era. Most notably, siRNA is involved in the RNA interference (RNAi) pathway where the siRNA interferes with the expression of a specific gene. In addition to their role in the RNAi pathway, siRNAs also act in RNAi-related pathways, e.g. as an antiviral mechanism or in shaping the chromatin structure of a genome; the complexity of these pathways is only now being elucidated.

[...]

[...] SiRNAs can also be exogenously (artificially) introduced into cells by various transfection methods to bring about the specific knockdown of a gene of interest. Essentially any gene of which the sequence is known can thus be targeted based on sequence complementarity with an appropriately tailored siRNA. This has made siRNAs an important tool for gene function and drug target validation studies in the post-genomic era.

Anmerkungen

Kein Hinweis auf die Quelle.


[3.] Ww/Fragment 002 17

Verschleierung
Untersuchte Arbeit:
Seite: 2, Zeilen: 17-29
Quelle: Wikipedia microRNA 2007
Seite(n): 1 (Internetquelle), Zeilen: -
In genetics, miRNAs are single-stranded RNA molecules of about 21-23 nucleotides in length, which regulate gene expression. miRNAs are encoded by genes that are transcribed from DNA but not translated into protein (non-coding RNA); instead they are processed from primary transcripts known as pri-miRNAs to short stem-loop structures called pre-miRNAs and finally to function miRNAs. Mature miRNA molecules are partially complementary to one or more messenger RNA (mRNA) molecules, and their main function is to down-regulate gene expression.

1.1.3.1 Formation and processing of miRNA

The genes encoding miRNAs are much longer than the processed mature miRNA molecule; miRNAs are first transcribed as primary transcripts or pri-miRNAs with a cap and poly-A tail and processed to short, 70-nucleotide stem-loop structures known as pre-miRNAs in the cell nucleus. This [processing is performed in animals by a protein complex known as the Microprocessor complex, consisting of the nuclease Drosha and the and the double-stranded RNA binding protein Pasha (Denli et al, 2004).]

In genetics, microRNAs (miRNA) are single-stranded RNA molecules of about 21-23 nucleotides in length regulating gene expression. miRNAs are encoded by genes that are transcribed from DNA but not translated into protein (non-coding RNA); instead they are processed from primary transcripts known as pri-miRNA to short stem-loop structures called pre-miRNA and finally to functional miRNA. Mature miRNA molecules are partially complementary to one or more messenger RNA (mRNA) molecules, and their main function is to downregulate gene expression. [...]

[...]

The genes encoding miRNAs are much longer than the processed mature miRNA molecule; miRNAs are first transcribed as primary transcripts or pri-miRNA with a cap and poly-A tail and processed to short, 70-nucleotide stem-loop structures known as pre-miRNA in the cell nucleus. This processing is performed in animals by a protein complex known as the Microprocessor complex, consisting of the nuclease Drosha and the double-stranded RNA binding protein Pasha.[3]


3. Denli AM, Tops BB, Plasterk RH, Ketting RF, Hannon GJ. (2004). Nature 432(7014):231-5.

Anmerkungen

Kein Hinweis auf die Quelle.


[4.] Ww/Fragment 003 01

Verschleierung
Untersuchte Arbeit:
Seite: 3, Zeilen: 1ff (komplett)
Quelle: Wikipedia microRNA 2007
Seite(n): 1 (Internetquelle), Zeilen: -
[This] processing is performed in animals by a protein complex known as the Microprocessor complex, consisting of the nuclease Drosha and the double-stranded RNA binding protein Pasha (Denli et al, 2004). These pre-miRNAs are then processed to mature miRNAs in the cytoplasm by interaction with the endonuclease Dicer, which also initiates the formation of the RNA-induced silencing complex (RISC) (Bernstein et al, 2001). This complex is responsible for the gene silencing observed due to miRNA expression and RNA interference. The pathway in plants varies slightly due to the lack of Drosha homologs; instead, Dicer homologs alone affect several processing steps (Kurihara and Watanabe, 2004).

It has been shown that the efficient processing of pre-miRNAs by Drosha requires the presence of extended single-stranded RNAs on both 3’- and 5’-ends of hairpin molecules (Zeng et al, 2005). This study showed that these motifs could be of different composition while their defined length is of high importance for processing to take place. Findings were confirmed in another work by Han et al (2004). Using bioinformatics tools the folding of 321 human and 68 fly pri-miRNAs was analysed. 280 human and 55 fly pri-miRNAs were selected for further study excluding those molecules where folding showed the presence of multiple loops. All human and fly pri-miRNAs contained very similar structural regions, which the authors called ‘’basal segments’’, ‘’lower stem’’, ’’upper stem’’ and ‘’terminal loop’’. Based on the encoding position of miRNAs, in the 5’-strand (5’-donors) or 3’-strand (3’-donors), thermodynamic profiles of pri-miRNAs were determined (Zeng et al, 2005). Subsequent experiments showed that Drosha complex cleaves RNA molecules ~2 helical turns away from the terminal loop and ~1 turn away from basal segments. In most analyzed molecules this region contains unpaired nucleotides and the free energy of the duplex is relatively high compared to lower and upper stem regions (Zeng and Cullen, 2005).

Most pre-miRNAs don’t have a perfect double-stranded RNA (dsRNA) structure topped by a terminal loop. There are few possible explanations for [such selectivity.]

This processing is performed in animals by a protein complex known as the Microprocessor complex, consisting of the nuclease Drosha and the double-stranded RNA binding protein Pasha.[3] These pre-miRNAs are then processed to mature miRNAs in the cytoplasm by interaction with the endonuclease Dicer, which also initiates the formation of the RNA-induced silencing complex (RISC).[4] This complex is responsible for the gene silencing observed due to miRNA expression and RNA interference. The pathway in plants varies slightly due to their lack of Drosha homologs; instead, Dicer homologs alone effect several processing steps.[5]

Zeng et al. have shown that efficient processing of pre-miRNA by Drosha requires presence of extended single-stranded RNA on both 3'- and 5'-ends of hairpin molecule. They demonstrated that these motifs could be of different composition while their length is of high importance if processing is to take place at all. Their findings were confirmed in another work by Han et al. Using bioinformatical tools Han et al. analysed folding of 321 human and 68 fly pri-miRNAs. 280 human and 55 fly pri-miRNAs were selected for further study, excluding those molecules which folding showed presence of multiple loops. All human and fly pri-miRNA contained very similar structural regions, which authors called 'basal segments', 'lower stem', 'upper stem' and 'terminal loop'. Based on the encoding position of miRNA, i.e. in the 5'-strand (5'-donors) or 3'-strand (3'-donors), thermodynamical profiles of pri-miRNA were determined. Following experiments have shown that Drosha complex cleaves RNA molecule ~2 helical turns away from the terminal loop and ~1 turn away from basal segments. In most analysed molecules this region contains unpaired nucleotides and the free energy of the duplex is relatively high compared to lower and upper stem regions.

Most pre-miRNAs don't have a perfect double-stranded RNA (dsRNA) structure topped by a terminal loop. There are few possible explanations for such selectivity. One could be that dsRNAs longer than 21 base pairs activate interferon response and anti-viral machinery in the cell.


3. Denli AM, Tops BB, Plasterk RH, Ketting RF, Hannon GJ. (2004). Nature 432(7014):231-5.

4. Bernstein E, Caudy AA, Hammond SM, Hannon GJ. (2001). Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 409(6818):363-6.

5. Kurihara Y, Watanabe Y. (2004). Arabidopsis micro-RNA biogenesis through Dicer-like 1 protein functions. Proc Natl Acad Sci USA 101(34):12753-8.

Anmerkungen

Kein Hinweis auf die Quelle.


[5.] Ww/Fragment 004 01

Verschleierung
Untersuchte Arbeit:
Seite: 4, Zeilen: 1 ff. (komplett)
Quelle: Wikipedia microRNA 2007
Seite(n): 1 (Internetquelle), Zeilen: -
[There are few possible explanations for] such selectivity. One could be that dsRNAs longer than 21 base pairs activate an interferon response and the anti-viral machinery in the cell. Another plausible explanation could be that the thermodynamical profile of

pre-miRNAs determines which strand will be incorporated into the Dicer complex. Indeed, the aforementioned study by Han et al. demonstrated very clear similarities between pri-miRNAs encoded in respective (5’- or 3’-) strands.

When Dicer cleaves the pre-miRNA stem-loop, two complementary short RNA molecules are formed, but only one becomes integrated into the RISC complex. This strand is known as the guide strand and is selected by the argonaute protein, the catalytically active RNase in the RISC complex, on the basis of the stability of the 5’ end (Preall et al, 2006). The remaining strand, known as the anti-guide or passenger strand is degraded as a RISC complex substrate (Gregory et al, 2005). After integration into the active RISC complex, miRNAs base pair with their complementary mRNA molecules and induce mRNA degradation by argonaute proteins. It is as yet unclear how the activated RISC complex locates the mRNA targets in the cell, though it has been shown that the process is not coupled to ongoing protein translation from the mRNA (Sen et al, 2005).

1.1.3.2 Cellular functions of miRNA

The miRNAs appear to be important for gene regulation. An individual miRNA is complementary to a part of one or more messenger RNAs (mRNAs). Animal miRNAs are usually complementary to a site in the 3’ UTR whereas plant miRNAs are usually complementary to coding regions of mRNAs. The annealing of the miRNA to the mRNA then inhibits protein translation, but sometimes facilitates cleavage of the mRNA. This is thought to be the primary mode of action of plant miRNAs. In such cases, the formation of the double-stranded RNA through the binding of the miRNA triggers the degradation of the mRNA transcript through a process similar to RNAi, though [in other cases it is believed that the miRNA complex blocks the protein translation machinery or otherwise prevents protein translation without causing the mRNA to be degraded.]

There are few possible explanations for such selectivity. One could be that dsRNAs longer than 21 base pairs activate interferon response and anti-viral machinery in the cell. Another plausible explanation could be that thermodynamical profile of pre-miRNA determines which strand will be incorporated into Dicer complex. Indeed, aforementioned study by Han et al. demonstrated very clear similarities between pri-miRNAs encoded in respective (5'- or 3'-) strands.

When Dicer cleaves the pre-miRNA stem-loop, two complementary short RNA molecules are formed, but only one is integrated into the RISC complex. This strand is known as the guide strand and is selected by the argonaute protein, the catalytically active RNase in the RISC complex, on the basis of the stability of the 5' end.[6] The remaining strand, known as the anti-guide or passenger strand, is degraded as a RISC complex substrate.[7] After integration into the active RISC complex, miRNAs base pair with their complementary mRNA molecules and induce mRNA degradation by argonaute proteins, the catalytically active members of the RISC complex. It is as yet unclear how the activated RISC complex locates the mRNA targets in the cell, though it has been shown that the process is not coupled to ongoing protein translation from the mRNA.[8]

Cellular functions

The function of miRNAs appears to be in gene regulation. For that purpose, a miRNA is complementary to a part of one or more messenger RNAs (mRNAs). Animal miRNAs are usually complementary to a site in the 3' UTR whereas plant miRNAs are usually complementary to coding regions of mRNAs. The annealing of the miRNA to the mRNA then inhibits protein translation, but sometimes facilitates cleavage of the mRNA. This is thought to be the primary mode of action of plant miRNAs. In such cases, the formation of the double-stranded RNA through the binding of the miRNA triggers the degradation of the mRNA transcript through a process similar to RNA interference (RNAi), though in other cases it is believed that the miRNA complex blocks the protein translation machinery or otherwise prevents protein translation without causing the mRNA to be degraded.


6. Preall JB, He Z, Gorra JM, Sontheimer EJ. (2006). Short interfering RNA strand selection is independent of dsRNA processing polarity during RNAi in Drosophila. Curr Biol 16(5):530-5.

7. Gregory RI, Chendrimada TP, Cooch N, Shiekhattar R. (2005). Human RISC couples microRNA biogenesis and posttranscriptional gene silencing. Cell 123(4):631-40.

8. Sen GL, Wehrman TS, Blau HM. (2005). mRNA translation is not a prerequisite for small interfering RNA-mediated mRNAs cleavage. Differentiation 73(6):287-93.

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[In such cases, the formation of the double-stranded RNA through the binding of the miRNA triggers the degradation of the mRNA transcript through a process similar to RNAi, though] in other cases it is believed that the miRNA complex blocks the protein translation machinery or otherwise prevents protein translation without causing the mRNA to be degraded. miRNAs may also target methylation of genomic sites which correspond to targeted mRNAs. miRNAs function in association with a complement of proteins collectively termed the miRNP (micro-ribonucleic protein).

This effect was first described for the worm C. elegans in 1993 (Lee et al., 1993). As of 2002, miRNAs have been confirmed in various plants and animals, including C. elegans, human and the plant Arabidopsis thaliana. Genes have been found in bacteria that are similar in the sense that they control mRNA abundance or translation by binding a mRNA by base pairing, however they are not generally considered to be miRNAs because the Dicer enzyme is not involved.

In plants, similar RNA species termed short-interfering RNAs are used to prevent the transcription of viral RNA. While this siRNA is double-stranded, the mechanism seems to be closely related to that of miRNAs, especially taking the hairpin structures into account. siRNAs are also used to regulate cellular genes, as miRNAs do.

In such cases, the formation of the double-stranded RNA through the binding of the miRNA triggers the degradation of the mRNA transcript through a process similar to RNA interference (RNAi), though in other cases it is believed that the miRNA complex blocks the protein translation machinery or otherwise prevents protein translation without causing the mRNA to be degraded. miRNAs may also target methylation of genomic sites which correspond to targeted mRNAs. miRNAs function in association with a complement of proteins collectively termed the miRNP.

This effect was first described for the worm C. elegans in 1993 by Victor Ambros and coworkers (Lee et al., 1993). As of 2002, miRNAs have been confirmed in various plants and animals, including C. elegans, human and the plant Arabidopsis thaliana. Genes have been found in bacteria that are similar in the sense that they control mRNA abundance or translation by binding an mRNA by base pairing, however they are not generally considered to be miRNAs because the Dicer enzyme is not involved.

In plants, similar RNA species termed short-interfering RNAs siRNAs are used to prevent the transcription of viral RNA. While this siRNA is double-stranded, the mechanism seems to be closely related to that of miRNA, especially taking the hairpin structures into account. siRNAs are also used to regulate cellular genes, as miRNAs do.

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Short hairpin RNAs (shRNAs) are a sequence of RNAs that makes a tight hairpin turn and are transcribed by RNA polymerase Ⅲ. It uses a vector introduced into cells and utilizes the U6 promoter to ensure that the shRNA is always expressed and can be cleaved by the cellular machinery into siRNA, which is then bound to the RISC silencing gene expression via RNAi (Brummelkamp et al., 2002; Lee et al., 2002; Miyagishi and Tiara, 2002; [Paddison, et al. 2002; Paul et al., 2002; Sui et al., 2002; Cao et al, 2005; Harper et al., 2005; McIntyre and Fanning, 2006).] A short hairpin RNA (shRNA) is a sequence of RNA that makes a tight hairpin turn that can be used to silence gene expression via RNA interference. shRNA uses a vector introduced into cells and utilizes the U6 promoter to ensure that the shRNA is always expressed. This vector is usually passed on to daughter cells, allowing the gene silencing to be inherited. The shRNA hairpin structure is cleaved by the cellular machinery into siRNA, which is then bound to the RNA-induced silencing complex (RISC).

[...]

References

McIntyre G, Fanning G (2006). "Design and cloning strategies for constructing shRNA expression vectors". BMC Biotechnol. 6: 1. PMID 16396676.

Harper S, Staber P, He X, Eliason S, Martins I, Mao Q, Yang L, Kotin R, Paulson H, Davidson B (2005). "RNA interference improves motor and neuropathological abnormalities in a Huntington's disease mouse model". Proc. Natl. Acad. Sci. U.S.A. 102 (16): 5820–5. PMID 15811941.

Nielsen M, Pedersen F, Kjems J (2005). "Molecular strategies to inhibit HIV-1 replication". Retrovirology 2: 10. PMID 15715913.

Paddison P, Caudy A, Bernstein E, Hannon G, Conklin D (2002). "Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells". Genes Dev. 16 (8): 948–58. PMID 11959843.

Cao W, Hunter R, Strnatka D, McQueen C, Erickson R (2005). "DNA constructs designed to produce short hairpin, interfering RNAs in transgenic mice sometimes show early lethality and an interferon response". J. Appl. Genet. 46 (2): 217–25. PMID 15876690.

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shRNAs can also be made for use in plants and other systems, and are not necessarily driven by a U6 promoter. In plants the traditional promoter for strong constitutive [sic] expression (in most plant species) is the cauliflower mosaic virus 35S promoter (CaMV35S), in which case RNA polymerase II is used to express the transcript destined to initiate RNAi. shRNAs can also be made for use in plants and other systems, and are not necessarily driven by a U6 promoter. In plants the traditional promoter for strong consitutive expression (in most plant species) is the cauliflower mosaic virus 35S promoter (CaMV35S), in which case RNA Polymerase II is used to express the transcript destined to initiate RNAi
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RNA interference (RNAi) was a term coined by Fire and coworkers to describe the inhibition of gene expression by double-stranded RNAs (dsRNAs) when introduced into nematode worms (Caenorhabditis elegans). Following on from the studies of Guo and Kemphues (1995), who had found that sense RNA was as effective as antisense RNA for suppressing gene expression in worms, Fire et al. (1998) applied single-stranded antisense RNA and double stranded RNA in their experiments. To their surprise, they found that dsRNA was more effective in producing interference than was either strand individually. After injection into adult C. elegans, single-stranded antisense RNA had a modest effect in diminishing specific gene expression, whereas double-stranded mixtures caused potent and specific interference.

Today we know that RNAi is a multistep process involving the generation of small interfering RNAs (siRNAs) in vivo through the action of the RNase III endonuclease “Dicer”. The resulting 21- to 23-nt siRNAs mediate the degradation of their complementary RNA (Shi, 2003).

RNA interference (RNAi) was a term coined by Fire and coworkers (1998) to describe the inhibition of gene expression by double-stranded RNAs (dsRNAs) when introduced into nematode worms (Caenorhabditis elegans). Following on from the studies of Guo and Kemphues (1995), who had found that sense RNA was as effective as antisense RNA for suppressing gene expression in worms, Fire et al. (1998) applied single-stranded antisense RNA and double stranded RNA in their experiments. To their surprise, they found that dsRNA was more effective at producing interference than was either strand individually. After injection into adult C. elegans, single-stranded antisense RNA had a modest effect in diminishing specific gene expression, whereas double-stranded mixtures caused potent and specific interference (Fire et al., 1998). RNAi is a multistep process involving the generation of small interfering RNAs (siRNAs) in vivo through the action of the RNase III endonuclease ‘Dicer’. The resulting 21- to 23-nt siRNAs mediate degradation of their complementary RNA (Shi, 2003).
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A major breakthrough in the elucidation of the underlying mechanism was the biochemical analysis of RNAi using Drosophila embryo or cell extracts (Tuschl et al., 1999; Hammond et al., 2000; Zamore et al., 2000), which led to the identification of the dsRNA processing enzyme Dicer (Bernstein et al., 2001a) as well as the RNA induced silencing complex, RISC (Hammond et al., 2000), which executes RNAi by using the small dsRNA species generated by Dicer as guidance molecules to target the homologous, endogenous mRNA for [degradation (Elbashir et al., 2001b,c; Zamore et al., 2000).] A major breakthrough in the elucidation of the underlying mechanism was the biochemical analysis of RNAi using Drosophila embryo or cell extracts (Hammond et al., 2000; Tuschl et al., 1999; Zamore et al., 2000), which led to the identification of the dsRNA processing enzyme Dicer (Bernstein et al., 2001a) as well as the RNA induced silencing complex, RISC (Hammond et al., 2000), which executes RNAi by using the small dsRNA species generated by Dicer as guidance molecules to target the homologous, endogenous mRNA for degradation (Elbashir et al., 2001b,c; Zamore et al., 2000).
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These discoveries led to the rapid improvement of RNAi tools, tailored to the needs of the various experimental systems, and triggered intense genetic and biochemical research into the molecular basis and regulation of RNAi (Hammond et al., 2001b; Tijsterman et al., 2002). It became clear that RNAi is a highly conserved mechanism that functions in many different cellular pathways from regulating gene expression to fighting infection and the dangers of mobile genetic elements.

1.2.1 Mechanism of RNAi

The genetic and biochemical analysis of RNAi has led to a model, in which RNAi can be divided into two distinct phases: an initiation and an execution phase. The initiation phase involves the processing of dsRNA into siRNA. In the execution phase, siRNAs are then incorporated into large ribonucleoprotein complexes. These effector complexes interfere with gene expression by using the small RNA strand to identify their complementary mRNA, which becomes cleaved and degraded. In a related pathway, short non-coding single stranded RNAs, which are derived from partially complementary dsRNA precursor molecules, are used to regulate the translation of mRNAs harbouring complementary sequences in their 3’'UTRs (Fig. 1).

These discoveries led to the rapid improvement of RNAi tools, tailored to the needs of the various experimental systems, and triggered intense genetic and biochemical

[Seite 986]

research into the molecular basis and regulation of RNAi (Hammond et al., 2001b: Tijsterman et al., 2002). It became clear that RNAi is a highly conserved mechanism that functions in many different cellular pathways from regulating gene expression to fighting infection and the dangers of mobile genetic elements.

[...]

2. The RNAi mechanism

The genetic and biochemical analysis of RNAi has led to a model, in which RNAi can be divided into two distinct phases: an initiation and an execution phase. The initiation phase involves processing of dsRNA into small RNA molecules, called small interfering RNAs (siRNA). In the execution phase, siRNAs are then incorporated into large ribonucleoprotein complexes. These effector complexes interfere with gene expression by using the small RNA strand to identify their complementary mRNA, which becomes cleaved and degraded. In a related pathway, short non-coding single stranded RNAs, which are derived from partially complementary dsRNA precursor molecules, are used to regulate the translation of mRNAs harbouring complementary sequences in their 3' UTRs.

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The goal of the initiation step of RNAi is the generation of siRNAs from long dsRNAs or mature miRNAs from their primary transcripts. This is achieved by the action of two families of RNase III-dependent genes, Dicer and Drosha. RNase III enzymes fall into three classes (Nicholson, 2003). Class I enzymes, found in bacteria and yeast, contain a single RNase III domain joined to a dsRBD (dsRNA binding domain proteins). Class II and III enzymes contain two RNase III catalytic domains. Class III enzymes are further characterized [by a helicase domain and a PAZ (Piwi/Argonaute/Zwille) domain.] The goal of the initiator step of RNAi is the generation of siRNAs from long dsRNAs, or mature microRNAs from their primary transcripts. This is achieved by the action of two families of RNase III genes, Dicer and Drosha.

[Seite 5823]

RNaseIII enzymes fall into three classes (see Fig. 1, [11] for a review). Class I enzymes, found in bacteria and yeast, contain a single RNaseIII domain joined to a dsRBD. Class II and III enzymes contain two RNaseIII catalytic domains. Class III enzymes are further characterized by a helicase domain and a PAZ (Piwi/Argonaute/Zwille) domain.


[11] Nicholson, A.W. (2003) The ribonuclease III superfamily: forms and functions in RNA maturation, decay, and gene silencing (Hannon, G.J., Ed.), RNAi: A Guide to Gene Silencing, vol. 8, pp. 149–174, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

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[Class III enzymes are further characterized] by a helicase domain and a PAZ (Piwi/Argonaute/Zwille) domain. This last domain is also present in Argonaute family proteins, already known to be essential for RNAi, which led to the proposal that Class III enzymes are the initiator of RNAi (Bass, 2000).

The generation of a siRNA from dsRNA potentially requires four endonucleolytic reactions. It has been revealed that Dicer acts as a monomer, using two endonucleolytic reactions to generate one new terminus (Zhang et al, 2004). This would occur if Dicer binds to an existing terminus and makes a cut ~21 nucleotides from the end (Schütze, 2004). If however the enzyme can not initiate processing from the end and is forced to cut internally, the reaction becomes significantly delayed. Once binding of Dicer occurred and a single new terminus is created, further processing occurrs at normal rates, since the enzyme now has terminal ends from which to process.

MicroRNAs (miRNAs) are transcribed by RNA polymerase II as long primary transcripts (Kim, 2005). The active miRNA species, termed the mature RNA, is present in a stem–loop structure within the primary transcript. The stem–loop can be located in an exon or in an intron. For example, the miRNAs miR-106b, miR-93, and miR-25 are located within an intron of the protein coding gene mcm-7. After transcription, the miRNAs are processed from the primary transcript, and the spliced mRNA is exported and translated . Sequential processing of the primary transcript by the RNase III enzymes Drosha and Dicer liberates the mature RNA. Drosha cleavage releases the stem–loop, termed the precursor, which is exported from the nucleus in an exportin-5/RAN-GTPase-dependent manner. In the cytoplasm, the precursor is processed into a siRNA-like structure by Dicer. Drosha generates a 2 nt 3’ overhang terminus on the precursor which is recognized by the PAZ domain of Dicer, analogous to the recognition of dsRNA termini. The double stranded miRNA is incorporated into RISC in a similar manner as siRNAs.

Drosha is a Class II enzyme. This enzyme assumes a pseudo-dimer catalytic [core similar to Dicer (Han et al, 2004).]

Class III enzymes are further characterized by a helicase domain and a PAZ (Piwi/Argonaute/Zwille) domain. This last domain is also present in Argonaute family proteins, already known to be essential for RNAi, which led to the proposal that Class III enzymes are the initiator of RNAi [12,13]. [...]

The generation of an siRNA from dsRNA potentially requires four endonucleolytic reactions. How does Dicer achieve this? Early models were based on the prediction that Dicer forms a dimer on the substrate and performs four cleavage reactions [15]. Recent data, however, favors a model whereby Dicer acts as a monomer, using two endonucleolytic reactions to generate one new terminus [16,17]. This would occur if Dicer bound to an existing terminus and made a cut ~21 nucleotides from the end. This was first suggested by studies using dsRNA substrates with blocked termini [16]. If the enzyme could not initiate processing from the end and was forced to process internally, the reaction was significantly delayed. The authors' interpretation was that internal binding was less efficient and caused a lag in processing. Once binding occurred and a single new terminus was created, further processing occurred at normal rates, since the enzyme now had terminal ends from which to process.

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MicroRNAs are transcribed from RNA polymerase II as long primary transcripts (see [26] for a review). The active microRNA species, termed the mature RNA, is present in a stem–loop structure within the primary transcript. The stem–loop can be located in an exon or an intron. For example, the microRNAs miR-106b, miR-93, and miR-25 are located within an intron of the protein coding gene mcm-7. After transcription, the microRNAs are processed out of the primary transcript, and the spliced mRNA is exported and translated. Whether the microRNA is processed before, during, or after splicing is not known. Sequential processing of the primary transcript by the RNaseIII enzymes Drosha and Dicer liberates the mature RNA. Drosha cleavage releases the stem–loop, termed the precursor, which is exported from the nucleus in an Exportin-5/RAN-GTPase-dependent manner. In the cytoplasm, the precursor is processed into a siRNA-like structure by Dicer. Drosha generates a 2 nt 30 overhang terminus on the precursor which is recognized by the PAZ domain of Dicer, analogous to the recognition of dsRNA termini. The doublestranded microRNA is incorporated into RISC in a similar manner as siRNAs.

Drosha is a Class II enzyme as shown in Fig. 1. This enzyme assumes a pseudo-dimer catalytic core similar to Dicer [27].


[12] Tabara, H., Sarkissian, M., Kelly, W.G., Fleenor, J., Grishok, A., Timmons, L., Fire, A. and Mello, C.C. (1999) The rde-1 gene, RNA interference, and transposon silencing in C. elegans. Cell 99, 123–132.

[13] Bass, B.L. (2000) Double-stranded RNA as a template for gene silencing. Cell 101, 235–238.

[14] Bernstein, E., Caudy, A.A., Hammond, S.M. and Hannon, G.J. (2001) Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 409, 363–366.

[15] Zamore, P.D. (2001) Thirty-three years later, a glimpse at the ribonuclease III active site. Mol. Cell 8, 1158–1160.

[16] Zhang, H., Kolb, F.A., Brondani, V., Billy, E. and Filipowicz, W. (2002) Human Dicer preferentially cleaves dsRNAs at their termini without a requirement for ATP. EMBO J. 21, 5875–5885.

[17] Zhang, H., Kolb, F.A., Jaskiewicz, L., Westhof, E. and Filipowicz, W. (2004) Single processing center models for human Dicer and bacterial RNase III. Cell 118, 57–68.

[26] Kim, V.N. (2005) MicroRNA biogenesis: coordinated cropping and dicing. Nat. Rev. Mol. Cell. Biol. 6, 376–385.

[27] Han, J., Lee, Y., Yeom, K.H., Kim, Y.K., Jin, H. and Kim, V.N. (2004) The Drosha-DGCR8 complex in primary microRNA processing. Genes Dev. 18, 3016–3027.

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[Drosha is a Class II enzyme. This enzyme assumes a pseudo-dimer catalytic] core similar to Dicer (Han et al, 2004). The substrates of Drosha, miRNA primary transcripts, are structurally distinct from Dicer substrates. Drosha does not process from a dsRNA terminus. Rather, data suggests that primarily the stem–loop structure is recognized. In particular, the loop size appears to be important for recognition (Zeng et al, 2005). In addition, unstructured sequences flanking the stem–loop are essential for processing (Chen et al, 2004; Zeng et al, 2005). It is not evident how Drosha is able to recognize these sequences, as they are outside of the dsRNA stem. Possibly other unidentified cofactors play a role. Conserved sequence elements have been found in flanking regions of C. elegans miRNAs (Ohler et al, 2004). Drosha is a Class II enzyme as shown in Fig. 1. This enzyme assumes a pseudo-dimer catalytic core similar to Dicer [27]. The substrate of Drosha, microRNA primary transcripts, is structurally distinct from Dicer substrates. Drosha does not process from a dsRNA terminus. Rather, data suggests that the stem–loop structure is recognized. In particular, the loop size appears to be important for recognition [28]. In addition, unstructured sequences flanking the stem–loop are essential for processing [29,30]. It is not evident how Drosha would recognize these sequences, as they are outside of the dsRNA stem. Possibly other, unidentified cofactors play a role. Conserved sequence elements have been found in flanking regions of C. elegans microRNAs [31].

[27] Han, J., Lee, Y., Yeom, K.H., Kim, Y.K., Jin, H. and Kim, V.N. (2004) The Drosha-DGCR8 complex in primary microRNA processing. Genes Dev. 18, 3016–3027.

[28] Zeng, Y., Yi, R. and Cullen, B.R. (2005) Recognition and cleavage of primary microRNA precursors by the nuclear processing enzyme Drosha. EMBO J. 24, 138–148.

[29] Chen, C.Z., Li, L., Lodish, H.F. and Bartel, D.P. (2004) MicroRNAs modulate hematopoietic lineage differentiation. Science 303, 83–86.

[30] Zeng, Y. and Cullen, B.R. (2005) Efficient processing of primary microRNA hairpins by Drosha requires flanking non-structured RNA sequences. J. Biol. Chem..

[31] Ohler, U., Yekta, S., Lim, L.P., Bartel, D.P. and Burge, C.B. (2004) Patterns of flanking sequence conservation and a characteristic upstream motif for microRNA gene identification. RNA 10, 1309–1322

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1.2.1.2 Execution phase: assembly of siRNA containing silencing complexes

Dicer-generated siRNAs are then incorporated into a large multiprotein complex, which is involved in various gene-silencing modes, and is called the RNA induced silencing complex, or RISC (Hammond et al, 2000; Nykanen et al, 2001). Processing of dsRNA and assembly of a functional RISC likely occurs in the cytoplasm, as Dicer is a cytosolic enzyme and RISC activity can be purified from the cytosol (Billy et al, 2001). R2D2, a Drosophila gene related to the C. elegans RNAi gene RDE-4, has been implicated in the transfer of siRNAs into the RISC (Liu et al, 2003). Generation of siRNAs from dsRNA in Drosophila embryo extracts, unwinding of the siRNA duplex, and incorporation into the RISC requires ATP (Nykanen et al, 2001). In contrast, human Dicer does not seem to rely on ATP for processing of dsRNA into siRNA molecules (Zhang et al., 2002).

2.2. The execution phase: assembly of siRNA containing silencing complexes

Dicer-generated siRNAs are then incorporated into a large multiprotein complex, which is involved in various gene-silencing modes, and is called the RNA induced silencing complex, or RISC (Hammond et al., 2000; Nykanen et al., 2001). Processing of dsRNA and assembly of a functional RISC likely occurs in the cytoplasm, as Dicer is a cytosolic enzyme and RISC activity can be purified from cytosol (Billy et al., 2001). R2D2, a Drosophila gene related to the C. elegans RNAi gene RDE-4, has been implicated in the transfer of siRNAs into the RISC (Liu et al., 2003). Generation of siRNAs from dsRNA in Drosophila embryo extracts, unwinding of the siRNA duplex, and incorporation into the RISC require ATP (Nykanen et al., 2001). In contrast, human Dicer does not seem to require ATP for processing of dsRNA into siRNA molecules (Zhang et al., 2002).

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Chromatographic purification of RISC nuclease activity from Drosophila cells revealed several RISC components. The first identified component was Argonaute2 (Ago2) (Hammond et al, 2001). This protein is a member of a gene family conserved in most eukaryotic and several prokaryotic genomes. The C. elegans homolog, rde-1, was previously identified in a genetic screen for RNAi-deficient mutants, reinforcing its connection with RNAi (Tabara et al, [1999).] Chromatographic purification of RISC nuclease activity from Drosophila cells revealed several RISC components. The first identified component was Argonaute2 (Ago2) [43]. This protein is a member of a gene family conserved in most eukaryotic and several prokaryotic genomes. The C. elegans homolog, rde-1, was previously identified in a genetic screen for RNAi-deficient mutants, reinforcing its connection with RNAi [12].

[12] Tabara, H., Sarkissian, M., Kelly, W.G., Fleenor, J., Grishok, A., Timmons, L., Fire, A. and Mello, C.C. (1999) The rde-1 gene, RNA interference, and transposon silencing in C. elegans. Cell 99, 123–132.

[43] Hammond, S.M., Boettcher, S., Caudy, A.A., Kobayashi, R. and Hannon, G.J. (2001) Argonaute2, a link between genetic and biochemical analyses of RNAi. Science 293, 1146–1150

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Structurally, this protein family is characterized by two domains, the PAZ domain and the PIWI domain. Structures for both domains have been solved. Additional RISC components with unknown roles in RNAi have also been identified. These include the RNA binding protein VIG, the Drosophila homolog of the Fragile X protein, dFXR, helicase proteins, and Tudor-SN (Ishizuka et al, 2002; Caudy et al, 2003). This last protein has five staphylococcal nuclease (SNase) domains and a Tudor domain. In humans, there are four closely related Argonaute family members, named Ago1-4. All four bind siRNAs and miRNAs at similar levels, and are widely expressed. Only Ago2, however, is present in a cleavage-competent RISC-complex (Meister et al, 2004). Similarly, siRNA-mediated knockdown, or targeted knockout, of Ago2 impaired RNAi of a reporter, while knockdown of Ago1, 3, 4 had no effect. The crystal structure of an Argonaute family member from Pyrococcus furiosus has been revealed (Song et al, 2004). The structure displayed an RNaseH fold for the signature PIWI domain. The crystal structure of a second archaean argonaute, Archaeoglobus fulgidus Piwi (AfPiwi), confirmed the RNaseH fold (Parker et al, 2004). The final demonstration that Slicer activity was contained within Ago2 was the reconstitution of minimal RISC with bacterially expressed, purified Ago2 and a single-stranded siRNA (Rivas et al, 2005). Structurally, this protein family is characterized by two domains, the PAZ domain and the PIWI domain. Structures for both domains have been solved (see below). Additional RISC components with unknown roles in RNAi

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have also been identified. These include the RNA binding protein VIG, the Drosophila homolog of the Fragile X protein, dFXR, helicase proteins, and Tudor-SN [46–48]. This last protein has five staphylococcal nuclease (SNase) domains and a Tudor domain. [...]

[...]

[...] In humans, there are four closely related Argonaute family members, named Ago1-4. All four bind siRNAs and microRNAs at similar levels, and are widely expressed. Only Ago2, however, is present in a cleavage- competent RISC [52,53]. Similarly, siRNA-mediated knockdown, or targeted knockout, of Ago2 impairs RNAi of a reporter, while knockdown of Ago1, 3, 4 had no effect. These data can be interpreted in two ways: Ago2 alone is capable of interacting with Slicer, or Ago2 itself is Slicer. The answer was provided by the crystal structure of an Argonaute family member from Pyrococcus furiosus [54]. The structure revealed an RNaseH fold for the signature PIWI domain. The crystal structure of a second archaean Argonaute, Archaeoglobus fulgidus Piwi (AfPiwi), confirmed the RNaseH fold [55]. The final demonstration that Slicer activity was contained within Ago2 was the reconstitution of minimal RISC with bacterially expressed, purified Ago2 and a single-stranded siRNA [45].


[46] Caudy, A.A., Myers, M., Hannon, G.J. and Hammond, S.M. (2002) Fragile X-related protein and VIG associate with the RNA interference machinery. Genes Dev. 16, 2491–2496.

[47] Caudy, A.A. et al. (2003) A micrococcal nuclease homologue in RNAi effector complexes. Nature 425, 411–414.

[48] Ishizuka, A., Siomi, M.C. and Siomi, H. (2002) A Drosophila fragile X protein interacts with components of RNAi and ribosomal proteins. Genes Dev. 16, 2497–2508.

[52] Meister, G., Landthaler, M., Patkaniowska, A., Dorsett, Y., Teng, G. and Tuschl, T. (2004) Human Argonaute2 mediates RNA cleavage targeted by miRNAs and siRNAs. Mol. Cell 15, 185–197.

[53] Liu, J. et al. (2004) Argonaute2 is the catalytic engine of mammalian RNAi. Science 305, 1437–1441.

[54] Song, J.J., Smith, S.K., Hannon, G.J. and Joshua-Tor, L. (2004) Crystal structure of Argonaute and its implications for RISC slicer activity. Science 305, 1434–1437.

[55] Parker, J.S., Roe, S.M. and Barford, D. (2004) Crystal structure of a PIWI protein suggests mechanisms for siRNA recognition and slicer activity. EMBO J. 23, 4727–4737.

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1.2.2 Therapeutic applications of RNAi

The therapeutic applications of RNAi are potentially enormous. The genetic etiology of many disorders has now been defined and, in some cases, has been targeted by RNAi in in vitro and in vivo model systems. Because the specificity of RNAi is governed by sequence complementarity between the siRNA and the target RNA, the most obvious application would be to treat diseases in which genetic polymorphisms within the disease-inducing gene in a particular lesion or tumor can be targeted for degradation without affecting RNA from wild-type alleles.

Therapeutic Applications

The therapeutic applications of RNAi are potentially enormous. The genetic etiology of many disorders has now been defined and, in some cases, has been targeted by RNAi in in vitro and in vivo model systems. Because the specificity of RNAi is governed by sequence complementarity between the siRNA and the target RNA, the most obvious application would be to treat diseases in which genetic polymorphisms within the disease-inducing gene in a particular lesion or tumor can be targeted for degradation without affecting RNA from wild-type alleles.

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1.2.2.1 Cancer

There are two general abnormalities in cancer cells that exhibit a dysregulation of the cell cycle resulting in uncontrolled growth and are resistant to death as a result of abnormalities in one or more proteins that mediate apoptosis (Nam and Parang, 2003). The goals for RNAi approaches for cancer therapy are therefore to silence the expression of a cell cycle gene and/or an anti-apoptotic gene in the cancer cells thereby stopping tumor growth and killing the cancer cells. To selectively eliminate cancer cells without damaging normal cells, the RNAi needs to be targeted to a gene specifically involved in the growth or survival of the cancer cell, or the siRNAs would be selectively delivered into the cancer cells.


Nam NH, Parang K. Current targets for anticancer drug discovery. Curr Drug Targets. 2003 Feb;4(2):159-179.

A. Cancer

There are two general abnormalities in cancer cells — they exhibit dysregulation of the cell cycle resulting in uncontrolled growth and they are resistant to death as a result of abnormalities in one or more proteins that mediate apoptosis (Nam and Parang, 2003). The goals for RNAi approaches for cancer therapy are therefore to knock out the expression of a cell cycle gene and/or an anti-apoptotic gene in the cancer cells thereby stopping tumor growth and killing the cancer cells. To selectively eliminate cancer cells without damaging normal cells, the RNAi would be targeted to a gene specifically involved in the growth or survival of the cancer cell, or the siRNAs would be selectively delivered into the cancer cells.


Nam NH and Parang K (2003) Current targets for anticancer drug discovery. Curr Drug Targets 4:159–179.

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The translocation of the Philadelphia chromosome (Ph) generates a fusion gene called BCR-ABL. The translation product of this gene creates a constitutively active protein tyrosine kinase that induces and maintains leukemic transformation in chronic myelogenous leukemia and Ph-positive acute lymphoblastic leukemia. The siRNAs specific for the BCR-ABL transcript have been shown to silence the oncogenic fusion transcripts without affecting expression levels of normal c-ABL and c-BCR transcripts (Scherr et al, 2003; Wohlbold et al, 2003).

Pancreatic and colon carcinomas, in which RAS genes are often mutated, provide another example for potential RNAi applications. In many cases, the RAS oncogenes contain point mutations that differ by a single-base mutation from their normal counterparts. The use of retroviral vectors to introduce interfering RNAs specific for an oncogenic variant of KRAS (called K-RASV12) reduced the level of K-RASV12 transcripts and resulted in a loss of anchorage-independent growth and tumorigenicity (Brummelkamp et al, 2002). Studies of this kind provided proof-of-concept data for RNAi-based strategies aiming to reverse tumorigenesis.

1.2.2.2 Infectious diseases

The translocation of the Philadelphia chromosome (Ph) generates a fusion gene called BCR-ABL. The translation product of this gene creates a constitutively active protein tyrosine kinase that induces and maintains leukemic transformation in chronic myelogenous leukemia and Ph-positive acute lymphoblastic leukemia. The siRNAs specific for the BCR-ABL transcript have been shown to silence the oncogenic fusion transcripts without affecting expression levels of normal c-ABL and c-BCR transcripts.20,21

Pancreatic and colon carcinomas, in which RAS

[Seite 1775]

genes are often mutated, provide another example. In many cases, the RAS oncogenes contain point mutations that differ by a single-base mutation from their normal counterparts. The use of retroviral vectors to introduce interfering RNAs specific for an oncogenic variant of K-RAS (called K-RASV12) reduces the level of K-RASV12 transcripts and effects a loss of anchorage-independent growth and tumorigenicity.22,23 Studies of this kind provide proof-of-concept for RNAi-based strategies aimed at reversing tumorigenesis. [...]

[...]

Infectious Diseases


20. Scherr M, Battmer K, Winkler T, Heidenreich O, Ganser A, Eder M. Specific inhibition of bcr-abl gene expression by small interfering RNA. Blood 2003;101: 1566-9.

21. Wohlbold L, van der Kuip H, Miething C, et al. Inhibition of bcr-abl gene expression by small interfering RNA sensitizes for imatinib mesylate (STI571). Blood 2003; 102:2236-9.

22. Brummelkamp TR, Bernards R, Agami R. Stable suppression of tumorigenicity by virus-mediated RNA interference. Cancer Cell 2002;2:243-7.

23. Wilda M, Fuchs U, Wossmann W, Borkhardt A. Killing of leukemic cells with a BCR/ABL fusion gene by RNA interference (RNAi). Oncogene 2002;21:5716-24.

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The ability of RNAi to inhibit the replication or cellular uptake of viruses and other infectious agents has been clearly demonstrated in cell culture studies and, therefore, holds promise for the treatment of human patients. The ability of HIV-1 to infect cells and replicate can be severely compromised by targeting viral genes using siRNAs. Examples include the suppression of HIV-1 replication in human cells transfected with siRNA directed against the tat and the rev gene (Capodici et al, 2002; Jacque et al, 2002; Lee et al, 2002a; Novina et al, 2002). Transfection of human cells with siRNAs directed against different genes in the poliovirus genome resulted in resistance of the cells to infection with poliovirus (Gitlin et al., 2002). The ability of siRNAs targeting the gene encoding the death receptor Fas to protect mice from liver failure and fibrosis in two models of autoimmune hepatitis was tested by Song and colleagues (Song et al., 2003). Intravenous injection of Fas siRNA specifically reduced Fas protein levels in the livers of mice during a 10-day period. Fas siRNA treatment abrogated hepatocyte necrosis and inflammatory infiltration and markedly reduced serum concentrations of transaminases demonstrating a clear hepatoprotective effect of the siRNA therapy. [Seite 643]

The ability of RNAi to inhibit the replication or cellular uptake of viruses and other infectious agents has been clearly demonstrated in cell culture studies and, therefore, holds promise for the treatment of human patients. The ability of HIV-1 to infect cells and replicate can be severely compromised by targeting of

[Seite 644]

viral genes using siRNAs. Examples include the suppression of HIV-1 replication in human cells transfected with siRNA directed against tat and the rev gene (Capodici et al., 2002; Jacque et al., 2002; Lee et al., 2002a; Novina et al., 2002). Transfection of human cells with siRNAs directed against different genes in the poliovirus genome resulted in resistance of the cells to infection with poliovirus (Gitlin et al., 2002). The ability of siRNAs targeting the gene encoding the death receptor Fas to protect mice from liver failure and fibrosis in two models of autoimmune hepatitis was tested by Song and colleagues (Song et al., 2003). Intravenous injection of Fas siRNA specifically reduced Fas protein levels in the livers of mice during a 10-day period. Fas siRNA treatment abrogated hepatocyte necrosis and inflammatory infiltration and markedly reduced serum concentrations of transaminases demonstrating a clear hepatoprotective effect of the siRNA therapy.

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Delivery is probably the single biggest obstacle to the development of RNAi-based therapeutic agents. Trigger RNAs (dsRNAs from which siRNAs are derived by the action of Dicer) can be expressed from vectors or delivered as artificial siRNAs. A variety of strategies to express interfering RNAs with the use of plasmid and virus vector-based cassettes have been explored (Li et al, 2002; Dykxhoorn et al, 2003). Well-documented hazards of inserting foreign vector sequences into chromosomal DNA include insertional activation and inactivation of cellular genes. Direct (intravenous) administration of siRNAs would require siRNAs that are modified to be resistant to nucleases and perhaps conjugated with a ligand to target the siRNA to specific tissues. In mice, intravenous introduction of Fas siRNAs leads to specific silencing of Fas mRNA in the liver (Song et al, 2003), so in principle, unmodified siRNAs can be taken up by the liver and perhaps other tissues. It is not clear, however, whether there are selective tissue sites for the uptake of siRNAs and whether the lymphoid system or the brain, for instance, is accessible by this route. Furthermore, the silencing effect of siRNAs is short-lived, because the siRNAs eventually decay within the cell. In addition to the danger of using vectors that integrate into the genome, the expression or injection of siRNAs may also have unwanted biologic side effects. Researchers are continually finding new cellular processes in which RNAi is involved. Therefore, a stoichiometric excess of a virus-specific siRNA, for [example, could saturate RNAi and interrupt the pathway’s normal functions in the cell. Interferons, which form part of the host’s defense against viral infection, are activated by long dsRNA (more than 500 bp).] Delivery is probably the single biggest obstacle to the development of RNAi-based therapeutic agents. Trigger RNAs (dsRNAs from which siRNAs are derived by the action of Dicer) can be expressed from vectors or delivered as artificial siRNAs. A variety of strategies to express interfering RNAs with the use of plasmid and virus vector-based cassettes have been explored.7,37 Well-documented hazards of inserting foreign vector sequences into chromosomal DNA include insertional activation and inactivation of cellular genes.

Direct (e.g., intravenous) administration of siRNAs would require siRNAs that are modified to be resistant to nucleases and perhaps conjugated with a ligand to target the siRNA to specific tissues. In mice, intravenous introduction of Fas siRNAs leads to specific silencing of Fas mRNA in the liver,25 so in principle, unmodified siRNAs can be taken up by the liver and perhaps other tissues. It is not clear, however, whether there are selective tissue sites for the uptake of siRNAs and whether the lymphoid system or the brain, for instance, is accessible by this route. Furthermore, the silencing effect of siRNAs is short-lived, because the siRNAs eventually decay within the cell.

In addition to the danger of using vectors that integrate into the genome, the expression or injection of siRNAs may also have untoward biologic effects. Researchers are continually finding new cellular processes in which RNAi is involved. Therefore, a stoichiometric excess of a virus-specific siRNA, for example, could saturate RNAi and interrupt the pathway's normal functions in the cell.

Interferons, which form part of the host's defense against viral infection, are activated by long dsRNA (more than 500 bp).


7. Dykxhoorn DM, Novina CD, Sharp PA. Killing the messenger: short RNAs that silence gene expression. Nat Rev Mol Cell Biol 2003;4:457-67.

25. Song E, Lee SK, Wang J, et al. RNA interference targeting Fas protects mice from fulminant hepatitis. Nat Med 2003;9:347- 51.

37. Li H, Li WX, Ding SW. Induction and suppression of RNA silencing by an animal virus. Science 2002;296:1319-21.

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[Therefore, a stoichiometric excess of a virus-specific siRNA, for] example, could saturate RNAi and interrupt the pathway’s normal functions in the cell. Interferons, which form part of the host’s defense against viral infection, are activated by long dsRNA (more than 500 bp). It is now apparent that siRNAs (Sledz et al, 2003) as well as shRNAs expressed from DNA vectors (Bridge et al, 2003) can trigger the activation of interferons. However, there is no evidence that the activation of interferons by short RNAs influences the degree or specificity of RNA silencing. In addition, these effects have to be reconciled with the manufacturing in cells of many thousands of copies of pre-miRNAs (Lagos et al, 2001) that do not appear to activate interferons. Therefore, a stoichiometric excess of a virus-specific siRNA, for example, could saturate RNAi and interrupt the pathway's normal functions in the cell.

Interferons, which form part of the host's defense against viral infection, are activated by long dsRNA (more than 500 bp). It is now apparent that siRNAs45 as well as short hairpin RNAs (short sequences of RNA that make tight hairpin turns and can be used to silence gene expression) expressed from DNA vectors46 can trigger the activation of interferons. However, there is no evidence that the activation of interferons by short RNAs influences the degree or specificity of RNA silencing. In addition, these effects have to be reconciled with the manufacturing in cells of many thousands of copies of pre-micro RNAs47,48,49 that do not appear to activate interferons.


45. Sledz CA, Holko M, de Veer MJ, Silverman RH, Williams BR. Activation of the interferon system by short-interfering RNAs. Nat Cell Biol 2003;5:834-9.

46. Bridge AJ, Pebernard S, Ducraux A, Nicoulaz AL, Iggo R. Induction of an interferon response by RNAi vectors in mammalian cells. Nat Genet 2003;34:263-4.

47. Lau NC, Lim LP, Weinstein EG, Bartel DP. An abundant class of tiny RNAs with probable regulatory roles in Caenorhabditis elegans. Science 2001;294:858-62.

48. Lee RC, Ambros V. An extensive class of small RNAs in Caenorhabditis elegans. Science 2001;294:862-4.

49. Lagos-Quintana M, Rauhut R, Lendeckel W, Tuschl T. Identification of novel genes coding for small expressed RNAs. Science 2001;294:853-8.

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1.4 293FT cell lines

Human Embryonic Kidney cells, also known as HEK cells, HEK 293 or just 293 cells, are a cell line originally derived, as their name indicates, from embryonic human kidney. HEK cells are very easy to grow and transfect very readily and so are widely-used in cell biology research. They are also used by biotechnology industry to produce therapeutic proteins and viruses for gene therapy.

1.4.1 Origins of 293 cells

293 cells were generated by transformation of cultures of normal human embryonic kidney cells with sheared adenovirus 5 DNA in the laboratory of Alex Van der Eb in Leiden, Holland in the early 1970s. They are called HEK for human embryonic kidney, while the number 293 roots from numbering of experiments

Human Embryonic Kidney cells, also known as HEK cells, HEK 293 or just 293 cells, are a cell line originally derived, as their name indicates, from embryonic human kidney. HEK cells are not themselves particularly interesting, but are very easy to work with, and so are a widely-used cell line in cell biology research. [...]

[...]

HEK 293 cells were generated by transformation of cultures of normal human embryonic kidney cells with sheared adenovirus 5 DNA in the laboratory of Alex Van der Eb in Leiden, Holland in the early 70s.

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Subsequent analysis has shown that the transformation was brought about by an insert consisting of -4.5 kilo bases from the left arm of the viral genome, which became incorporated into human chromosome 19 (Louis et al, 1997).

For many years it was assumed that HEK 293 cells were generated by transformation of either a fibroblastic, endothelial or epithelial cell all of which are abundant in kidney. However the fact that the cells originated from [cultured kidney cells does not clearly indicate the exact cellular origin of the HEK 293, as embryonic kidney cultures may contain small numbers of almost all cell types of the body.]

Subsequent analysis has shown that the transformation was brought about by an insert consisting of ~4.5 kilobases from the left arm of the viral genome, which became incorporated into human chromosome 19 (Louis 1997[2]).

For many years it was assumed that HEK 293 cells were generated by transformation of either a fibroblastic, endothelial or epithelial cell all of which are abundant in kidney. However the fact that the cells originated from cultured kidney cells does not say much about the exact cellular origin of the HEK 293, as embryonic kidney cultures may contain small numbers of almost all cell types of the body.

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[However the fact that the cells originated from] cultured kidney cells does not clearly indicate the exact cellular origin of the HEK 293, as embryonic kidney cultures may contain small numbers of almost all cell types of the body. In fact Graham and coworkers more recently provided evidence that HEK 293 cells and several other human cell lines generated by adenovirus transformation of human embryonic kidney cells have many properties of immature neurons, suggesting that the adenovirus was taken up and transformed a neuronal lineage cell in the original kidney culture (Shaw et al, 2002).

1.4.2 Applications of 293 cells

As an experimentally transformed cell line, HEK cells are not a particularly good model for normal cells, cancer cells, or any other kind of cell that is a fundamental object of research. However, they are extremely easy to work with, being straightforward to culture and to transfect, and so can be used in experiments in which the behaviour of the cell itself is not of interest. Typically, these experiments involve transfection in a gene (or combination of genes) of interest, and then analyzing the expressed protein; essentially, the cell is used simply as a test tube with a membrane. The widespread use of this cell line is due to its extreme transfectability by the calcium phosphate method, achieving efficiencies approaching 100% as determined by FACS using a 2 x PBS buffer. A lower efficiency might be achievable with an HBS buffer.

An important variant of this cell line is the 293T cell line that contains, in addition, the SV40 large T antigen, that allows for episomal replication of transfected plasmids containing the SV40 origin of replication. This allows for amplification of transfected plasmids and extended temporal expression of the desired gene products. Note that any similarly domesticated cell line can be used for this fort of work; Hela, COS and Chinese Hamster Ovary cell are common alternatives. Examples of such experiments include: A study of the effects of drug on sodium channels; testing of an inducible RNAi system; testing of an isoform-selective protein kinase C agonist; investigation of the interaction between two proteins; analysis of a nuclear export signal in a [protein (He et al, 1998).]

However the fact that the cells originated from cultured kidney cells does not say much about the exact cellular origin of the HEK 293, as embryonic kidney cultures may contain small numbers of almost all cell types of the body. In fact Graham and coworkers more recently provided evidence that HEK 293 cells and several other human cell lines generated by adenovirus transformation of human embryonic kidney cells have many properties of immature neurons, suggesting that the adenovirus was taken up and transformed a neuronal lineage cell in the original kidney culture (Shaw et al. 2002[3]).

Uses of HEK 293 Cells

As an experimentally transformed cell line, HEK cells are not a particularly good model for normal cells, cancer cells, or any other kind of cell that is a fundamental object of research. However, they are extremely easy to work with, being straightforward to culture and to transfect, and so can be used in experiments in which the behaviour of the cell itself is not of interest. Typically, these experiments involve transfecting in a gene (or combination of genes) of interest, and then analysing the expressed protein; essentially, the cell is used simply as a test tube with a membrane. The widespread use of this cell line is due to its extreme transfectability by the calcium phosphate method, achieving efficiencies approaching 100% as determined by FACS using a 2XPBS buffer. A lower efficiency might be achievable with an HBS buffer.

An important variant of this cell line is the 293T cell line that contains, in addition, the SV40 large T antigen, that allows for episomal replication of transfected plasmids containing the SV40 origin of replication. This allows for amplification of transfected plasmids and extended temporal expression of the desired gene products. Note that any similarly domesticated cell line can be used for this sort of work; HeLa, COS and Chinese Hamster Ovary cell are common alternatives.

Examples of such experiments include:

  • A study of the effects of a drug on sodium channels [4]
  • Testing of an inducible RNA interference system [5]
  • Testing of an isoform-selective protein kinase C agonist [6]
  • Investigation of the interaction between two proteins [7]
  • Analysis of a nuclear export signal in a protein [8]
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A more specific use of HEK cells is in the propagation of adenoviral vectors.

Viruses offer an extremely efficient means of delivering genes into cells, since this is what they have evolved to do, and are thus of great use as experimental tools. However, as pathogens, they also present a degree of danger to the experimenter. This danger can be avoided by the use of viruses which lack key genes, and which are thus unable to replicate after entering a cell. In order to propagate such viral vectors, a cell line that expresses the missing genes is required. Since HEK cells express a number of adenoviral genes, they can be used to propagate adenoviral vectors in which these genes (typically, E1 and E3) are deleted, such as AdEasy. Another application of 293, especially 293T, cells is commonly used for the production of lentiviral and retroviral vectors. Various retroviral and lentiviral packaging cell lines are based on these cells.

A more specific use of HEK cells is in the propagation of adenoviral vectors. Viruses offer an extremely efficient means of delivering genes into cells, since this is what they have evolved to do, and are thus of great use as experimental tools. However, as pathogens, they also present a degree of danger to the experimenter. This danger can be avoided by the use of viruses which lack key genes, and which are thus unable to replicate after entering a cell. In order to propagate such viral vectors, a cell line that expresses the missing genes is required. Since HEK cells express a number of adenoviral genes, they can be used to propagate adenoviral vectors in which these genes (typically, E1 and E3) are deleted, such as AdEasy (He 1998).

293, and especially 293T, cells are commonly used for the production of lentiviral and retroviral vectors. Various retroviral and lentiviral packaging cell lines are based on these cells.

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Fig. 2. Map for the pLKO.1 lentiviral vector. It derives from pRRLSIN.cPPT.PGK/GFP/WRPE and contains elements for efficient viral packaging and shRNA expression. These include Ψ, the lentiviral packaging site; RRE, the Rev-responsive element; cPPT. RSV/5’LTR is a hybrid of the Rous Sarcoma virus promoter and the HIV 5’LTR. SIN 3’LTR is the HIV 3’LTR with a self-inactivating U3 deletion. Expression of the shRNA is driven by the human U6 promoter (hU6). The lentiviral vector also contains the mammalian selection marker puromycin resistance gene (PAC) under the control of the PGK promoter as a mammalian selection marker. pUCori is the bacterial origin of replication of the plasmid, F1 ori is the single-stranded phage F1 origin of replication, and AmpR is the ampicillin resistance gene.

Ww 29a source

Figure 1 Vector map for the pLKO.1 lentiviral vector. The self-inactivating lentiviral vector backbone contains elements for efficient viral packaging and shRNA expression. These include Ψ, the lentiviral packaging site; RRE, the Rev-responsive element; cPPT. RSV/ 5′ LTR is a hybrid of the Rous Sarcoma virus promoter and the HIV 5′ LTR. SIN 3′ LTR is the HIV 3′ LTR with a self-inactivating U3 deletion. Expression of the shRNA is driven by the human U6 promoter (hU6). The lentiviral vector also contains the mammalian selection marker puromycin resistance gene (PAC) under the control of the PGK promoter as a mammalian selection marker. pUCori is the bacterial origin of replication of the plasmid, F1 ori is the single-stranded phage F1 origin of replication, and AmpR is the ampicillin resistance gene.

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The Midi plasmid purification protocols were based on a modified alkaline lysis procedure, followed by binding of plasmid DNA to Anion-Exchange Resin under appropriate low-salt and pH conditions. RNA, proteins, dyes, and low-molecular-weight impurities were removed by a medium-salt wash. Plasmid DNA was eluted in a high salt buffer and then concentrated and desalted by isopropanol precipitation. This plasmid preparation protocol used in this embodiment is based on a modified alkaline lysis procedure, followed by binding of plasmid DNA to anion-exchange resin under appropriate low-salt and pH conditions. RNA, proteins, dyes, and low-molecular-weight impurities in the plasmid are removed by a medium-salt wash. Plasmid DNA is eluted in a high-salt buffer and then concentrated and desalted by isopropanol precipitation.
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Phospholipase C (PLC) constitutes a large family of mammalian hydrolytic phosphodiesterase enzymes that participate in phosphatidylinositol (PIP2) metabolism and lipid signaling pathways in a calcium dependent manner. Currently, the superfamily consists of six sub-families comprising a total of 13 separate isoforms that differ in their mode of activation, catalytic regulation, cellular localization, membrane binding avidity, and tissue distribution. All are capable of catalyzing the hydrolysis of PIP2 into two important second messenger molecules, which go on to alter cell responses such as proliferation, differentiation, apoptosis, cytoskeleton remodeling, vesicular trafficking, ion channel conductance, endocrine function and neurotransmission (Wu et al., 2000; Rhee, 2001). Phospholipase C (PLC) constitutes a large family of mammalian hydrolytic phosphodiesterase enzymes that participate in phosphatidylinositol (PIP2) metabolism and lipid signaling pathways in a calcium dependent manner. Currently, the superfamily consists of six sub-families comprising a total of 13 separate isoforms that differ in their mode of activation, catalytic regulation, cellular localization, membrane binding avidity, and tissue distribution. All are capable of catalyzing the hydrolysis of PIP2 into two important second messenger molecules, which go on to alter cell responses such as proliferation, differentiation, apoptosis, cytoskeleton remodeling, vesicular trafficking, ion channel conductance, endocrine function, and neurotransmission.
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PLC-β4 function as effector enzymes for receptors belonging to the rhodopsin superfamily of transmembrane proteins that contain seven transmembrane spanning (heptahelical) segments (Ji et al, 1998). They are activated by a wide range of stimuli, from photons and tiny odorant molecules, to full-sized proteins and require specific combinations of Gɑ subunits to couple to their effectors. In the standard G protein model of PLC-β4 activation, binding of agonist triggers receptor-catalyzed exchange of GTP for bound GDP on the ɑ-component of the heterotrimer. The GTP-charged subunit then dissociates in the plane of the membrane, increasing its catalytic activity and thereby amplifying the initial receptor stimulus.

PLC-β4 was first isolated from cerebellum and retina (Min et al, 1993; Jiang et al, 1994). Its mRNA is highly concentrated in cerebellar Purkinje and granule cells, the median geniculate body, whose axons terminate in the auditory cortex, and the lateral geniculate nucleus, where most retinal axons terminate [in a visuotopic representation of each half of the visual field.]

PLC-β4 was first isolated from cerebellum (244, 245) and retina (173, 210). Its mRNA is highly concentrated in cerebellar Purkinje and granule cells (308, 362), the median geniculate body (308), whose axons terminate in the auditory cortex, and the lateral geniculate nucleus, where most retinal axons terminate in a visuotopic representation of each half of the visual field.

PLC-β isoforms function as effector enzymes for receptors belonging to the rhodopsin superfamily of trans-

[Seite 1299]

membrane proteins that contain seven transmembrane spanning (heptahelical) segments (169). They are activated by a wide range of stimuli, from photons and tiny odorant molecules, to full-sized proteins and require specific combinations of Gɑ and Gβγ subunits to couple to their effectors. In the standard G protein model of PLC activation, binding of agonist triggers receptor-catalyzed exchange of GTP for bound GDP on the ɑ-component of the heterotrimer. The GTP-charged subunit then dissociates in the plane of the membrane, and either the ɑ-subunit monomer, the βγ-heterodimer, or both bind to PLC-β, increasing its catalytic activity and thereby amplifying the initial receptor stimulus (Fig. 2).


169. JI TH, GROSSMANN M, AND JI I. G-protein coupled receptors. J Biol Chem 273: 17299–17302, 1998.

173. JIANG H, WU D, AND SIMON MI. Activation of phospholipase C beta 4 by heterotrimeric GTP-binding proteins. J Biol Chem 269: 7593– 7596, 1994

210. LEE CW, LEE KH, AND RHEE SG. Characterization of phospholipase C isozymes in bovine retina: purification of phospholipase C-beta 4. Methods Enzymol 238: 227–237, 1994

244. MIN DS, KIM DM, LEE YH, SEO J, SUH PG, AND RYU SH. Purification of a novel phospholipase C isozyme from bovine cerebellum. J Biol Chem 268: 12207–12212, 1993.

245. MIN DS, KIM Y, LEE YH, SUH PG, AND RYU SH. A G-protein-coupled 130 kDa phospholipase C isozyme, PLC-beta 4, from the particulate fraction of bovine cerebellum. FEBS Lett 331: 38–42, 1993.

308. ROUSTAN P, ABITBOL M, MENINI C, RIBEAUDEAU F, GERARD M, VEKEMANS M, MALLET J, AND DUFIER JL. The rat phospholipase C beta 4 gene is expressed at high abundance in cerebellar Purkinje cells. Neuroreport 6: 1837–1841, 1995.

362. TANAKA O AND KONDO H. Localization of mRNAs for three novel members (beta 3, beta 4 and gamma 2) of phospholipase C family in mature rat brain. Neurosci Lett 182: 17–20, 1994.

Anmerkungen

Ein Verweis auf die Quelle fehlt.


[32.] Ww/Fragment 064 15

BauernOpfer
Untersuchte Arbeit:
Seite: 64, Zeilen: 15-22
Quelle: Paddison et al 2002
Seite(n): 951, Zeilen: l..col: 38ff
Paddison et al. (2002) showed that shRNA were somewhat less potent silencing triggers than that were siRNAs. siRNAs homologous yielded 90-95% suppression of gene expression, whereas suppression levels achieved with shRNAs ranged from 80-90% on average because of mammalian cells contain several endogenous systems that were predicted to hamper the application of RNAi. Chief among these is a dsRNA-activated protein kinase, PKR, which effects a general suppression of translation via phosphorylation of EIF-2α. However, mammalian cells contain several endogenous systems that were predicted to hamper the application of RNAi. Chief among these is a dsRNA-activated protein kinase, PKR, which effects a general suppression of translation via phosphorylation of EIF-2α (Williams 1997; Gil and Esteban 2000). [...]

[...]

[...] Overall, shRNAs were somewhat less potent silencing triggers than were siRNAs. Whereas siRNAs homologous to firefly luciferase routinely yielded ∼90%–95% suppression of gene expression, suppression levels achieved with shRNAs ranged from 80%–90% on average.

Anmerkungen

Die Quelle wird zwar zu Beginn genannt, doch wird nicht klar, dass der gesamte Absatz nahezu wörtlich daraus entnommen wurde. Insbesondere wird der letzte Satz nicht von der Quellenangabe abgedeckt. Der letzte Satz wird in Quelle mit Autoren belegt, die in der untersuchten Arbeit nicht genannt werden.


[33.] Ww/Fragment 065 08

BauernOpfer
Untersuchte Arbeit:
Seite: 65, Zeilen: 8-31
Quelle: McIntyre and Fanning 2006
Seite(n): 1, 2, 3, 4, Zeilen: 1: abstract: 2ff, r.col: 5ff; 2: l.col: 1ff; 3: l.col: letzte Zeile; 4: r.col: 21ff
Moreover, McIntyre and Fanning (2006) revealed that shRNA vector construction can be disturbed by high mutation rates and ensuing sequencing is often problematic. shRNA expression vectors are constructed by one of three different methods. The most common method requires the synthesis, annealing and ligation of two complementary oligonucleotides into an expression vector. The frequency of false positives determined by sequencing is high, about 20-40% high (Miyagishi et al, 2004). The unreliability of this method is in part due to the difficulty in synthesizing long oligonucleotides (> 35 bases) and this method requires two long oligonucleotides then the chance of mutation is doubled. The second strategy is a PCR approach in which a promoter sequence serves as the template. Although it is advantageous that only single long oligonucleotides is required, the strong secondary structure predicted to form within this primer can lead to the amplification of false products (Castanotto et al., 2005). The third method comprises several techniques relating to primer extension. Each is based on the principle of a polymerase extending the 3’ end of overlapping oligonucleotides. Nevertheless, this method reduces the cost of oligonucleotides and does not need purification but may cause off-set by a high rate of polymerase-induced mutations in both the initial extension and repeated cycling steps. In order to reduce mutations, conducting all reactions as single-step extensions and replacing Taq polymerase with an enzyme better able to counter the secondary structure of the hairpin template were adopted. Another reported strategy to alleviate sequencing difficulties is to include mismatched bases within the shRNA stem (Yu et al., 2003). A survey of the literature revealed that shRNA vector construction can be hindered by high mutation rates and the ensuing sequencing is often problematic. [...]

[...]

[...] In a survey of more than 100 papers applying expressed shRNA in mammalian systems we determined that shRNA expression vectors are constructed by one of three different methods (see Additional file 1).

[Seite 2]

The most common method for making shRNA constructs (74 % of surveyed studies) requires the synthesis, annealing and ligation of two complementary oligonucleotides (oligos) into an expression vector (Fig. 1b and Additional file 2). While this cloning method is quick, the oligo synthesis cost is nearly double that of other methods and the frequency of false positives determined by sequencing is high (typically 20 – 40 %) [3]. The unreliability of this method is in part due to the difficulty in the synthesis of long oligos (length > 35 bases) [4]. As this method requires two long oligos the chance of mutation due to synthesis error is doubled.

The second strategy (employed in 22 % of studies) is a PCR approach in which a promoter sequence serves as the template (Fig. 1c). [...] Although it is advantageous that only a single long oligo is required, the strong secondary structure predicted to form within this primer can lead to the amplification of false products. [...] [6]

The third method (applied in 4 % of studies) encompasses several techniques relating to primer extension. Each is based on the principle of a polymerase extending the 3' end of overlapping oligos [7]. [...] This technique is the cheapest of all the construction methods discussed as it both halves the cost of unique oligos (compared to the annealed oligo method) and does not need costly oligo purification (compared to the promoter based PCR method). However, this saving may be off-set by a high rate of polymerase-induced mutation in either the initial extension step or by repeated cycling [4].

[Seite 3]

Our first step to reduce mutations was to remove the possibility of cycling-induced errors by conducting all reactions as single-step extensions. [...]

To improve upon these results, we substituted Taq polymerase with an enzyme better able to counter the secondary structure of the hairpin template.

[Seite 4]

Another reported strategy to alleviate sequencing difficulties is to include mismatched bases within the shRNA stem [3,11].


3. Miyagishi M, Sumimoto H, Miyoshi H, Kawakami Y, Taira K: Optimization of an siRNA-expression system with an improved hairpin and its significant suppressive effects in mammalian cells. J Gene Med 2004, 6:715-723.

4. Paddison PJ, Cleary M, Silva JM, Chang K, Sheth N, Sachidanandam R, Hannon GJ: Cloning of short hairpin RNAs for gene knockdown in mammalian cells. Nat Methods 2004, 1:163-167.

6. Castanotto D, Scherer L: Targeting Cellular Genes with PCR Cassettes Expressing Short Interfering RNAs. Methods Enzymol 2005, 392:173-185.

7. Rossi JJ, Kierzek R, Huang T, Walker PA, Itakura K: An alternate method for synthesis of double-stranded DNA segments. J Biol Chem 1982, 257:9226-9229.

11. Yu JY, Taylor J, DeRuiter SL, Vojtek AB, Turner DL: Simultaneous inhibition of GSK3alpha and GSK3beta using hairpin siRNA expression vectors. Mol Ther 2003, 7:228-236.

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Die Quelle ist am Anfang angegeben, aber es wird nicht klar, dass der Rest der Seite aus ihr stammt. Bemerkenswert ist auch, dass die Quelle von einem "survey of the literature" spricht, W. W. aber nicht.


Quellen

[1.] Quelle:Ww/Wikipedia Small interfering RNA 2007

Titel    Small interfering RNA
Verlag    (Wikipedia)
Jahr    2007
URL    http://en.wikipedia.org/w/index.php?title=Small_interfering_RNA&oldid=147644067

Literaturverz.   

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[2.] Quelle:Ww/Wikipedia HEK 293 cells 2007

Titel    HEK 293 cells
Verlag    (Wikipedia)
Jahr    2007
URL    http://en.wikipedia.org/w/index.php?title=HEK_293_cells&oldid=154716929

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[3.] Quelle:Ww/Milhavet et al 2003

Autor     Ollivier Milhavet, Devin S. Gary, and Mark P. Mattson
Titel    RNA Interference in Biology and Medicine
Zeitschrift    Pharmacological Reviews
Ausgabe    55
Datum    Dezember 2003
Nummer    4
Seiten    629-648
DOI    10.1124/pr.55.4.1
URL    http://pharmrev.aspetjournals.org/content/55/4/629.full

Literaturverz.   

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[4.] Quelle:Ww/Wikipedia Phosphoinositide phospholipase C 2007

Titel    Phosphoinositide phospholipase C
Verlag    (Wikipedia)
Datum    23. Juni 2007
URL    https://en.wikipedia.org/w/index.php?title=Phosphoinositide_phospholipase_C&oldid=140137634

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[5.] Quelle:Ww/Rebecchi Pentyala 2000

Autor     Mario J. Rebecchi, Srinivas N. Pentyala
Titel    Structure, Function, and Control of Phosphoinositide-Specific Phospholipase C
Zeitschrift    Physiological Reviews
Herausgeber    American Physiological Society
Ausgabe    80
Datum    October 2000
Nummer    4
Seiten    1291-1335
ISSN    0031-9333
URL    http://physrev.physiology.org/content/physrev/80/4/1291.full.pdf

Literaturverz.   

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[6.] Quelle:Ww/Wikipedia microRNA 2007

Titel    microRNA
Verlag    (Wikipedia)
Jahr    2007
URL    http://en.wikipedia.org/w/index.php?title=MicroRNA&oldid=157563157

Literaturverz.   

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[7.] Quelle:Ww/Stevenson 2004

Autor     Mario Stevenson
Titel    Therapeutic Potential of RNA Interference
Zeitschrift    The New England Journal of Medicine
Ausgabe    351
Datum    October 2004
Seiten    1772-1777
DOI    10.1056/NEJMra045004
URL    http://www.nejm.org/doi/full/10.1056/NEJMra045004

Literaturverz.   

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[8.] Quelle:Ww/Hammond 2005

Autor     Scott M. Hammond
Titel    Dicing and slicing - The core machinery of the RNA interference pathway
Zeitschrift    FEBS Letters
Ausgabe    579
Jahr    2005
Seiten    5822–5829
URL    http://www.sciencedirect.com/science/article/pii/S0014579305010884

Literaturverz.   

ja
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[9.] Quelle:Ww/Wikipedia Small hairpin RNA 2007

Titel    Small hairpin RNA
Verlag    (Wikipedia)
Datum    25. Juli 2007
URL    http://en.wikipedia.org/w/index.php?title=Small_hairpin_RNA&oldid=146938414

Literaturverz.   

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[10.] Quelle:Ww/Zou and Yoder 2005

Autor     Gang-Ming Zou, Mervin C. Yoder
Titel    Application of RNA interference to study stem cell function: current status and future perspectives
Zeitschrift    Biology of the cell
Verlag    Wiley
Ausgabe    97
Jahr    2005
Seiten    211-219
DOI    10.1042/BC20040084
URL    http://onlinelibrary.wiley.com/doi/10.1042/BC20040084/abstract

Literaturverz.   

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[11.] Quelle:Ww/Geley and Mueller 2004

Autor     Stephan Geley, Christiane Müller
Titel    RNAi: ancient mechanism with a promising future
Zeitschrift    Experimental Gerontology
Verlag    Elsevier
Ausgabe    39
Jahr    2004
Seiten    985-998
DOI    10.1016/j.exger.2004.03.040
URL    http://www.sciencedirect.com/science/article/pii/S0531556504001445

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[12.] Quelle:Ww/Root et al 2006

Autor     David E Root, Nir Hacohen, William C Hahn, Eric S Lander, David M Sabatini
Titel    Genome-scale loss-of-function screening with a lentiviral RNAi library
Zeitschrift    Nature methods
Ausgabe    3
Jahr    2006
Seiten    715 - 719
DOI    10.1038/nmeth924
URL    http://www.nature.com/nmeth/journal/v3/n9/full/nmeth924.html , Abbildung: [1]

Literaturverz.   

ja
Fußnoten    ja


[13.] Quelle:Ww/McIntyre and Fanning 2006

Autor     Glen J McIntyre, Gregory C Fanning
Titel    Design and cloning strategies for constructing shRNA expression vectors
Zeitschrift    BMC Biotechnology
Ausgabe    6
Jahr    2006
Nummer    1
Anmerkung    Zur Dokumentation werden die Seitennummern der verlinkten PDF-Datei verwendet.
DOI    10.1186/1472-6750-6-1
URL    http://www.biomedcentral.com/content/pdf/1472-6750-6-1.pdf

Literaturverz.   

ja
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[14.] Quelle:Ww/Krueger 2007

Autor     Krueger U, Bergauer T, Kaufmann B, Wolter I, Pilk S, Heider-Fabian M, Kirch S, Artz-Oppitz C, Isselhorst M, Konrad J.
Titel    Insights into Effective RNAi Gained from Large-Scale siRNA Validation Screening
Zeitschrift    Oligonucleotides
Verlag    Mary Ann Liebert
Ausgabe    17
Jahr    2007
Nummer    2
Seiten    237-250
DOI    10.1089/oli.2006.0065
URL    http://online.liebertpub.com/doi/abs/10.1089/oli.2006.0065

Literaturverz.   

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[15.] Quelle:Ww/Paddison et al 2002

Autor     Patrick J. Paddison, Amy A. Caudy, Emily Bernstein, Gregory J. Hannon, Douglas S. Conklin
Titel    Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells
Zeitschrift    Genes & Development
Ausgabe    16
Datum    15. April 2002
Nummer    8
Seiten    948-958
DOI    10.1101/gad.981002
URL    http://www.ncbi.nlm.nih.gov/pmc/articles/PMC152352/

Literaturverz.   

ja
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[16.] Quelle:Ww/Kilic and Yuan 2007

Autor     Ali O. Kilic, James T.C. Yuan
Titel    METHOD AND PROCESS OF GENETIC TRANSFORMATION USING SUPERCRITICAL FLUIDS
Datum    22. February 2007
Anmerkung    United States Patent Application 20070042493, June 14, 2006
URL    http://patents.com/us-20070042493.html

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