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Angaben zur Quelle [Bearbeiten]

Autor     Peer Wulff, Volker Vallon, Dan Yang Huang, Harald Völkl, Fang Yu, Kerstin Richter, Martina Jansen, Michaela Schlünz, Karin Klingel, Johannes Loffing, Gunther Kauselmann, Michael R. Bösl, Florian Lang, Dietmar Kuhl
Titel    Impaired renal Na+ retention in the sgk1-knockout mouse
Zeitschrift    Journal of Clinical Investigation
Ausgabe    110
Jahr    2002
Seiten    1263-1268
DOI    10.1172/JCI200215696.
URL    http://www.jci.org/articles/view/15696/version/1/pdf/render

Literaturverz.   

yes
Fußnoten    yes
Fragmente    2


Fragmente der Quelle:
[1.] Dsa/Fragment 055 05 - Diskussion
Zuletzt bearbeitet: 2015-05-30 10:24:56 Kybot
Dsa, Fragment, KeineWertung, SMWFragment, Schutzlevel, Wulff et al 2002, ZuSichten

Typus
KeineWertung
Bearbeiter
Hindemith
Gesichtet
No.png
Untersuchte Arbeit:
Seite: 55, Zeilen: 5-16
Quelle: Wulff et al 2002
Seite(n): 1264, Zeilen: l.col: 1ff
In brief, a conditional targeting vector was generated from a 7-kb fragment encompassing the entire transcribed region on 12 exons. The neomycin resistance cassette was flanked by two loxP sites and inserted into intron 11. Exons 4–11, which code for the sgk1 kinase domain, were “floxed” by inserting a third loxP site into intron 3. A clone with a recombination between the first and third loxP site (type I recombination) was injected into C57BL/6 blastocytes. Male chimeras were bred to C57BL/6 and 129/SvJ females. Heterozygous SGK1-deficient mice were backcrossed to 129/SvJ wild-type mice (Charles River, Sulzfeld, Germany) for ten generations and then intercrossed to generate homozygous SGK1 knockout mice (sgk1−/−) and their wild type littermates (sgk1+/+). Male and female sgk1−/− mice were used in the studies and compared with littermate sgk1+/+ mice of the respective gender. Mice were genotyped by PCR (Huang Y. et al., (2004) J Am Soc Nephol; Wulff P. et al., (2002) J Clin Invest). A 7-kb fragment encompassing the entire transcribed region on 12 exons was subcloned into pBluescript (Stratagene, La Jolla, California, USA). This plasmid was used for generation of a conditional targeting vector (Figure 1a). The vector was linearized at a unique NotI site and electroporated into R1 ES cells. Positive clones were identified by Southern blot analysis. A targeted ES cell clone was transiently transfected with Cre recombinase. The resulting recombination types were identified by Southern blot analysis. One clone with a recombination between the first and third loxP site (type I recombination) was injected into C57BL/6 blastocytes. Male chimeras were bred to C57BL/6 and 129/SvJ females. Heterozygous sgk1-deficient mice were either inbred or backcrossed to 129/SvJ wild-type mice for two generations and then intercrossed to generate homozygous sgk1–/– and sgk1+/+ mice. Male and female sgk1–/– mice were used in the studies and compared with littermate sgk1+/+ mice of the respective gender. Mice were genotyped by PCR.
Anmerkungen

The source is given before the documented passage as well as at the end of it, but it is not clear that the description of the method is taken from it mostly verbatim.

Sichter
(Hindemith)

[2.] Dsa/Fragment 056 00 - Diskussion
Zuletzt bearbeitet: 2016-08-20 19:30:29 WiseWoman
Dsa, Fragment, Gesichtet, KomplettPlagiat, SMWFragment, Schutzlevel sysop, Wulff et al 2002

Typus
KomplettPlagiat
Bearbeiter
Hindemith
Gesichtet
Yes.png
Untersuchte Arbeit:
Seite: 56, Zeilen: figure
Quelle: Wulff et al 2002
Seite(n): 1264, Zeilen: figure
Dsa 56a diss.png

Figure nr. 13 - Generation of sgk1–/– mice.

(a) Targeting strategy. The neomycin resistance cassette (gray box) was flanked by two loxP sites (ovals) and inserted into intron 11. Exons 4–11, which code for the Sgk1 kinase domain (open boxes), were “floxed” by inserting a third loxP site into intron 3. N indicates NheI restriction sites, and the small black bar indicates the external 5′ probe used for Southern blot analysis. Expected fragment sizes of the wild-type and targeted sgk1 locus are also indicated. One homologously recombined ES cell clone was transiently transfected with Cre recombinase, and a clone that had undergone recombination between the first and the third loxP site (type I recombination) was chosen for injection. Arrows below the gene indicate PCR primers used for genotyping. Numbers between the arrows indicate the size of the amplified fragments. Crossed bars below (a) indicate homologous recombination. (b) Southern blot of NheI-digested genomic DNA from ES cell clones after gene targeting hybridized with a 5′ external probe (black bar in a). Lane 5 shows a targeted ES cell line. (c) Genotyping by PCR of genomic tail DNA of homozygous (–/–) and heterozygous (-/+) sgk1-deficient mice and wild-type mice (+/+) using a mix of three specific primers (arrows in a). (d) Autoradiograph of Northern blot analysis of Sgk1-specific transcripts in +/+ and –/– mice. The deletion of the kinase domain from the genome results in a size reduction of 0.9 kb at the mRNA level in sgk1–/– mice.

Dsa 56a source.png

Figure 1

Generation of sgk1–/– mice. (a) Targeting strategy. The neomycin resistance cassette (gray box) was flanked by two loxP sites (ovals) and inserted into intron 11. Exons 4–11, which code for the Sgk1 kinase domain (open boxes), were “floxed” by inserting a third loxP site into intron 3. N indicates NheI restriction sites, and the small black bar indicates the external 5′ probe used for Southern blot analysis. Expected fragment sizes of the wild-type and targeted sgk1 locus are also indicated. One homologously recombined ES cell clone was transiently transfected with Cre recombinase, and a clone that had undergone recombination between the first and the third loxP site (type I recombination) was chosen for injection. Arrows below the gene indicate PCR primers used for genotyping. Numbers between the arrows indicate the size of the amplified fragments. Crossed bars below a indicate homologous recombination. (b) Southern blot of NheI-digested genomic DNA from ES cell clones after gene targeting hybridized with a 5′ external probe (black bar in a). Lane 5 shows a targeted ES cell line. (c) Genotyping by PCR of genomic tail DNA of homozygous (–/–) and heterozygous (–/+) sgk1-deficient mice and wild-type mice (+/+) using a mix of three specific primers (arrows in a). (d) Autoradiograph of Northern blot analysis of Sgk1-specific transcripts in +/+ and –/– mice. The deletion of the kinase domain from the genome results in a size reduction of 0.9 kb at the mRNA level in sgk1–/– mice.

Anmerkungen

The source is not mentioned here.

Sichter
(Hindemith), WiseWoman

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