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Angaben zur Quelle [Bearbeiten]

Autor     Hamdy M. Embark
Titel    Regulation of Renal Ion Channels by Serum and Glucocorticoid Inducible Kinase Isoforms, Ubiquitin Ligase Nedd4-2 and NHE3 Regulating Factor 2 in the Xenopus Laevis Oocyte Expression System
Jahr    2004
Anmerkung    Justus-Liebig-Universität Gießen, Dissertation zur Erlangung des Grades eines Dr. med. vet. beim FB Veterinärmedizin
URL    http://geb.uni-giessen.de/geb/volltexte/2004/1459/pdf/EmbarkHamdy-2004-03-22.pdf

Literaturverz.   

no
Fußnoten    no
Fragmente    8


Fragmente der Quelle:
[1.] Sj/Fragment 024 01 - Diskussion
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3.3 Xenopus laevis oocytes

One of the first and still most widely used assay system for quantifying an authentic protein biosynthetic process is the fully grown oocyte of the South African clawed frog, Xenopus laevis. The value of Xenopus laevis first became apparent in 1971, when Gurdon and co-workers discovered that the oocyte constitutes an efficient system for translating foreign messenger RNA.

The Xenopus oocyte is a cell specialized for the production and storage of proteins for later use during embryogenesis and developmentally divided into 6 stages (112). In addition, the complex architecture of the frog oocyte includes the subcellular systems involved in the export and import of proteins. Therefore, the mRNA-microinjected oocyte is an appropriate system to study the synthesis of specific polypeptides, as well as the storage of particular proteins in various subcellular organelles and the export of others into the extracellular space. Moreover, the subcellular compartmentalization, as well as the structure and biochemical, physiological, and biological properties of the synthesized protein, may be examined from exogenous proteins in the injected oocyte. For experimental studies oocytes of stages V-VI are used with a diameter of some 1.3 mm allowing easy preparation. The developmental stages V-VI are characterized by the occurence of 2 poles i.e. the vegetable (light) and the animal (dark) poles.

The main ion conductance in Xenopus oocytes is a Ca2+-dependent Cl- conductance governing the resting membrane potential close to the Cl- reversal potential of -40 mV. Despite their advantages, several precautions should be taken into consideration. First, the expression of endogenous carriers may interfere with the exogenously expressed proteins in various ways. For instance, it has been observed that injection of heterologous membrane proteins at high levels can induce endogenous channels (113). Second, due to the fact that Xenopus laevis is a poikilothermic animal, its oocytes are best kept at lower temperature and most experiments are carried out at room temperature. Hence, temperature sensitive processes i.e. protein trafficking or kinetics may be altered. Finally, since Xenopus oocytes may have different signaling pathways, precaution should be taken when studying the regulation of expressed proteins. It has been revealed that the PTH receptor regulates the internalization of the sodium-phosphate transporter NaPi, mediated by the PKA and PKC pathway.


112. Costa,PF, Emilio,MG, Fernandes,PL, Ferreira,HG, Ferreira,KG: Determination of ionic permeability coefficients of the plasma membrane of Xenopus laevis oocytes under voltage clamp. J.Physiol 413:199-211, 1989

113. Tzounopoulos,T, Maylie,J, Adelman,JP: Induction of endogenous channels by high levels of heterologous membrane proteins in Xenopus oocytes. Biophys.J. 69:904-908, 1995

1.8 Xenopus laevis oocytes and electrophysiological recording

One of the first and still most widely used assay system for quantifying an authentic protein biosynthetic process is the fully grown oocyte of the South African clawed frog, Xenopus laevis. The value of Xenopus laevis first became apparent in 1971, when Gurdon and co-workers discovered that the oocyte constitutes an efficient system for translating foreign messenger RNA (Gurdon et al., 1971).

The Xenopus oocyte is a cell specialized for the production and storage of proteins for later use during embryogenesis and developmentally divided into 6 stages (Dumont, 1972). In addition, the complex architecture of the frog oocyte includes the subcellular systems involved in the export and import of proteins. Therefore, the mRNA-microinjected oocyte is an appropriate system in which to study the synthesis of specific polypeptides, as well as the storage of particular proteins in various subcellular organelles and the export of others into the extracellular space. Moreover, the subcellular compartmentalization, as well as the structure and biochemical, physiological, and biological properties of the synthesized protein, may be examined from exogenous proteins in the injected oocyte (reviewed in Wagner et al., 2000).

For experimental studies oocytes of stages V-VI are used with a diameter of some 1.3 mm allowing easy preparation. The developmental stages V-VI are characterized by the occurence of 2 poles i.e. the vegetable (light) and the animal (dark) poles. [...] The main ion conductance in Xenopus oocytes is a Ca2+ -dependent Cl-conductance governing the resting membrane potential close to the Cl- reversal potential of -40 mV, (Dascal, 1987).

[page 31]

Despite their advantages, several precautions should be taken into consideration. First, the expression of endogenous carriers may interfere with the exogenously expressed proteins in various ways. For instance, it has been observed that injection of heterologous membrane proteins at high levels can induce endogenous channels (Tzounopoulos et al., 1995). Second, due to the fact that Xenopus laevis is a poikilothermic animal, its oocytes are best kept at lower temperature and most experiments are carried out at room temperature. Hence, temperature sensitive processes i.e. protein trafficking or kinetics may be altered (Wagner et al., 2000).

[page 32]

Finally, since Xenopus oocytes may have different signaling pathways, precaution should be taken when studying the regulation of expressed proteins. It has been revealed that the PTH receptor regulates the internalization of NaPi, mediated by the PKA and PKC pathway.

Anmerkungen

The source is not given.

Parts of this documented passage can also be found in an earlier source: Sj/Dublette/Fragment 024 01

Sichter
(Hindemith) LieschenMueller

[2.] Sj/Fragment 025 01 - Diskussion
Zuletzt bearbeitet: 2016-11-23 20:54:55 WiseWoman
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[However, in NaPi-3 expressing Xenopus oocytes PKC-mediated PTH regulation] can not be observed. Instead, coupling to the PKA pathway leads to the alteration of PKA-regulated ion channels (114). Exposing the Xenopus oocytes to the regulators of intracellular signaling such as PKC activator phorbol esters may unspecifically lead to internalization of the plasma membrane and the expressed proteins (115;116). In summary, the Xenopus oocyte system has the advantage that channels, receptors and transporters can rapidly be expressed and identified by their electrophysiological properties. Once cDNA clones have been isolated, oocytes are an excellent system for correlating structure with function using a combination of molecular biological and electrophysiological techniques and analyzed both biochemically and electro physiologically in an in vivo situation.

114. Waldegger,S, Raber,G, Sussbrich,H, Ruppersberg,JP, Fakler,B, Murer,H, Lang,F, Busch,AE: Coexpression and stimulation of parathyroid hormone receptor positively regulates slowly activating IsK channels expressed in Xenopus oocytes. Kidney Int. 49:112-116, 1996

115. Vasilets,LA, Schwarz,W: Regulation of endogenous and expressed Na+/K+ pumps in Xenopus oocytes by membrane potential and stimulation of protein kinases. J.Membr.Biol. 125:119-132, 1992

116. Loo,DD, Hirsch,JR, Sarkar,HK, Wright,EM: Regulation of the mouse retinal taurine transporter (TAUT) by protein kinases in Xenopus oocytes. FEBS Lett. 392:250-254, 1996

However, in NaPi-3 expressing Xenopus oocytes PKC-mediated PTH regulation can not be observed (Wagner et al., 1996). Instead, coupling to the PKA pathway leads to the alteration of PKA-regulated ion channels (Waldegger et al., 1996). Exposing the Xenopus oocytes to the regulators of intracellular signaling such as PKC activator phorbol esters may unspecifically lead to internalization of the plasma membrane and the expressed proteins (Vasilets and Schwarz, 1992; Loo et al., 1996).

In summary, the Xenopus oocyte system has the advantage that channels, receptors and transporters can rapidly be expressed and analyzed both biochemically and electrophysiologically in an in vivo situation. The system can be used quite effectively as an assay for the functional cloning of channels that have only been identified by their electrophysiological properties. Once cDNA clones have been isolated, oocytes are an excellent system for correlating structure with function using a combination of molecular biological and electrophysiological techniques.

Anmerkungen

The source is not given.

Sichter
(Hindemith) LieschenMueller

[3.] Sj/Fragment 025 18 - Diskussion
Zuletzt bearbeitet: 2016-11-23 20:42:01 WiseWoman
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3.3.1 In vitro RNA transcription

In-vitro cRNA transcription involves 2 consecutive steps i.e. linearization of the plasmid DNA containing the inserted cDNA of interest by the corresponding restriction enzyme and the synthesis of RNA.

a. The inserted DNA should be cut at the 3’ end yielding a 5’ protruding or a blunt end by restriction enzyme. Plasmid DNA (10 μg) was incubated with 20 U restriction enzyme and an 10x buffer (5 μl) in a final volume of 50 μl at 37°C for 2 h or overnight.

b. To ascertain the linearization process, a 5 μl aliquot was taken out and analysed on a 1% agarose.

c. 1 volume isopropanol (50 μl) and 1/10 volume 3 M sodium acetate (5 μl) pH 5.2 was then added and incubated at room temperature for 10 min to precipitate the DNA.

d. The precipitated DNA was recovered by centrifugation at 17,000 rpm for 15 min at 4°C. The DNA pellet was washed by adding 100 μl of cold 70% ethanol to the pellet followed by centrifugation at 17,000 rpm for 5 min at 4°C. This washing stage was repeated. The DNA pellet was air dried and then resuspended in 10 μl of DNase free H2O. The concentration of DNA was determined spectrophotometrically by measuring the absorbance at 260 nm.

2.2.1 In vitro cRNA transcription

As illustrated in Fig. 12, in vitro cRNA transcription involves 2 consecutive steps i.e. linearisation of the plasmid DNA containing the inserted cDNA of interest by the corresponding restriction enzyme and the synthesis of RNA.

a. The inserted DNA should be cut at the 3’ end yielding a 5’ protruding or a blunt end by restriction enzyme. Plasmid DNA (10 μg) was incubated with 20 U restriction enzyme and an 10x buffer (5 μl) in a final volume of 50 μl at 37°C for 2 h or overnight.

b. To ascertain the linearization process, a 5 μl aliquot was taken out and analysed on a 1% agarose.

c. 1 volume isopropanol (50 μl) and 1/10 volume 3 M sodium acetate (5 μl) pH 5.2 was then added and incubated at room temperature for 10 min to precipitate the DNA.

d. The precipitated DNA was recovered by centrifugation at 17,000 rpm for 15 min at 4°C. The DNA pellet was washed by adding 100 μl of cold 70% ethanol to the pellet followed by centrifugation at 17,000 rpm for 5 min at 4°C. This washing stage was repeated. The DNA pellet was air dried and then resuspended in 10 μl of DNase free H2O. The concentration of DNA was determined spectrophotometrically by measuring the absorbance at 260 nm.

Anmerkungen

The source is not mentioned.

It is not surprising that identical procedures are used in different studies and it makes sense to describe those procedures with the same words, but it should be made transparent.

Sichter
(Hindemith) LieschenMueller

[4.] Sj/Fragment 026 01 - Diskussion
Zuletzt bearbeitet: 2016-11-23 21:37:32 WiseWoman
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e. 1 μg of linearised DNA was added to 1 μl rNTPS (20 nM), 2.5 μl Cap analogue (to prevent the degradation of the 5’ end of the synthesized RNA), 1 μl RNAase inhibitor (to protect the RNA from degradation by RNAase) and 2.4 μl 10 x transcription buffer(s). After mixing, 1 μl of T7 polymerase was added and the n incubated at 37°C for 1 hr. 1 μl DNase was added and the mixture was subsequently shaken for 15 min at 37°C. After addition of 100 μl DEPC-water and 125 μl phenol chloroform, the mixture was centrifuged at 13,000 rpm for 2 min. e. 1 μg of linearised DNA was added to 1 μl rNTPS (20 nM), 2.5 μl Cap analogue (to prevent the degradation of the 5’ end of the synthesized RNA), 1 μl RNAase inhibitor (to protect the RNA from degradation by RNAase) and 2.4 μl 10 x transcription buffer(s).

f. After mixing, 1 μl of T7 polymerase was added and the n incubated at 37°C for 1 h.

g. 1 μl DNase was added and the mixture was subsequently shaken for 15 min at 37°C.

h. After addition of 100 μl DEPC-water and 125 μl phenolchloroform, the mixture was centrifuged at 13,000 rpm for 2 min.

Anmerkungen

The source is not given.

Sichter
(Hindemith) LieschenMueller

[5.] Sj/Fragment 026 18 - Diskussion
Zuletzt bearbeitet: 2016-11-23 21:39:53 WiseWoman
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3.3.2 Preparation of Xenopus oocytes

An adult female Xenopus laevis frog was submersed in one liter of 3- aminobenzoic acid ethyl ester (0.1%) for about 15-30 min (Figure 6A). After the frog was fully anesthetized it was placed on ice for surgery.

A small abdominal incision (1 cm) was carried out and a segment of ovary was removed (Figure 6B,C). Subsequently the wound was closed with a reabsorbable suture (Figure 6D). The frog was then kept wet and warm by placing it in a cavity filled by a small amount of warm water to avoid drowning and hypothermia.

The ovarial sacs were manually separated into groups of 10-20 oocytes, put into a 15 ml tube and then enzymatically defolliculated by treatment with an OR- 2 (Oocytes-Ringer) (Table 1) solution containing 1-2 mg/ml collagenase A for 2-2.5 h at room temperature (Figure 6E) with gentle agitation. Defolliculation of the oocytes was stopped by washing several times with ND96 (Table 1).This step also removes all detritus permitting oocyte sorting. Oocytes were then sorted using a self-made apparatus (Figure 6F). Only large oocytes (stage V or VI) were selected and stored overnight in a ND96 storage solution at 16°C.

2.2.2 Preparation of oocytes

An adult female Xenopus laevis frog was submersed in one liter of 3-aminobenzoic acid ethyl ester (0.1%) for about 15-30 min (Fig. 13A). After the frog was fully anesthetized it was placed on ice for surgery. A small abdominal incision (1 cm) was carried out and a segment of ovary was removed (Fig. 13B, C). Subsequently the wound was closed with a reabsorbable suture (Fig. 13D). The frog was then kept wet and warm by placing it in a cavity filled by a small amount of warm water to avoid drowning and hypothermia.

The ovarial sacs were manually separated into groups of 10-20 oocytes, put into a 15 ml tube and then enzymatically defolliculated by treatment with an OR-2 (Oocytes-Ringer) solution containing 1-2 mg/ml collagenase A for 2-2.5 h at room temperature (Fig. 13E) with gentle agitation.

Defolliculation of the oocytes was stopped by washing several times with ND96. This step also removes all detritus permitting oocyte sorting. Oocytes were then sorted using a self-made apparatus (Fig. 13F). Only large oocytes (stage V or VI) were selected and stored overnight in a ND96 storage solution at 16°C.

Anmerkungen

Nothing has been marked as a citation.

Sichter
(Graf Isolan) LieschenMueller

[6.] Sj/Fragment 027 01 - Diskussion
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Sj 27 a diss.png

Figure 6. Oocytes preparation and injection. A The frog is anesthetized in 1 liter of 3-aminobenzoic acid ethyl ester (1%) in tap water at near room temperature. B The frog is placed on its back during operation. An incision about 1 cm long is made in the skin. C A small portion of the ovary is pulled out with forceps and removed with a pair of scissors. D The peritoneum and the muscle tissue are sewn up and then the skin closed off using cat gut. E The clump of oocytes is immediately transferred to a petri dish containing modified Barth medium with antibiotic. F Oocytes of stage V and VI are separated with a platinum wire loop. G For injection, the oocytes are aligned relative to the tip of the needle.

Sj 27 a source.png

Fig. 13. Steps of the oocytes preparation and injection. (A) The frog is anesthetized in 1 liter of 3-aminobenzoic acid ethyl ester (1%) in tap water at near room temperature. (B) The frog is placed on its back during operation. An incision about 1 cm long is made in the skin. (C) A small portion of the ovary is pulled out with forceps and removed with a pair of scissors. (D) The peritoneum and the muscle tissue are sewn up and then the skin closed off using cat gut. (E) The clump of oocytes is immediately transferred to a petri dish containing modified Barth medium with antibiotic. (F) Oocytes of stage V and VI are separated with a platinum wire loop. (G) For injection, the oocytes are aligned relative to the tip of the needle.

Anmerkungen

The source is not given.

The reader is given the impression that the figure shows photos of the lab work of the author.

Sichter
(Hindemith) LieschenMueller

[7.] Sj/Fragment 028 01 - Diskussion
Zuletzt bearbeitet: 2016-11-23 22:33:42 WiseWoman
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3.3.3 cRNA injection

After storing overnight, oocytes were injected using glass microcapillaries (filled with the required cRNA) mounted in a micromanipulator-controlled microinjector (Figure 7). Precaution was taken so that cRNA was not degraded by RNAases and that the injection capillary was not clogged with small particles. To avoid those problems several procedures were carried out such as using only sterile pipettes, gloves and DEPC treated water for dilution of cRNA. Glass capillaries were pulled using a normal puller. The tip was manually broken under the microscope (diameter of about 10-20 μm), backfilled with paraffin oil to seal the pipette from air and loaded with cRNA by suction (usually 1-2 μl).

2.2.3 cRNA injection

After storing overnight, oocytes were injected using glass microcapillaries (filled with the required cRNA) mounted in a micromanipulator-controlled microinjector (Fig. 13G). Precaution should be taken that cRNA was not contaminated with RNAases and that the injection capillary was not clogged with small particles. To avoid those problems several procedures were carried out such as using only sterile pipettes, gloves and DEPC treated water for dilution of cRNA.

Glass capillaries were pulled using a normal puller. The tip was manually broken under the microscope (diameter of about 10-20 μm), backfilled with paraffin oil to seal the pipette from air and loaded with cRNA by suction (usually 1-2 μl).

Anmerkungen

The source is not given.

Sichter
(Hindemith) LieschenMueller

[8.] Sj/Fragment 029 01 - Diskussion
Zuletzt bearbeitet: 2016-11-23 20:23:35 WiseWoman
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[Oocytes were] then placed into a 35 mm petri dish with a polypropylene mesh glued to the bottom to fix the oocytes and injected with a given volume of cRNA (usually 27.6 nl). The oocytes injected are listed in the Table 2.

After injection, oocytes were kept in storage solution at 15°C. To avoid sticking of oocytes to the petri dish or to other oocytes, the dish was gently shaken. At least every two days the storage solution was exchanged and damaged oocytes were removed to maximise the survival of the oocytes.

[page 48]

Ooytes were then placed into a 35 mm petri dish with a polypropylene mesh glued to the bottom to fix the oocytes and injected with a given volume of cRNA (usually 25 nl).

[page 49]

After injection, oocytes were kept in storage solution at 15°C. To avoid sticking of oocytes to the petri dish or to other oocytes, the dish was gently shaken. At least every two days the storage solution was exchanged and damaged oocytes were removed to maximise the survival of the oocytes.

Anmerkungen

The source is not given.

Note that after the documented text in the dissertation a figure follows (Figure 7) that is referenced on page 028. A source for that figure, datiert 15 Januar 2010, can also be found here: [1]. It has not been documented in this fragment, because it was not possible to prove that the image was on the site before the submission of the thesis.

Sichter
(Hindemith), WiseWoman

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