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Gene silencing by RNAi in mouse Sertoli cells.

González-González E, López-Casas PP, Del Mazo J - Reprod. Biol. Endocrinol. (2008)

Bottom Line: A reduction of about 40% in the level of EGFP protein was detected in cells successfully transfected both in vivo and in vitro.Similar levels of RNAi were detected both in vivo and in vitro.This also indicates that Sertoli cells have the necessary silencing machinery to repress the expression of endogenous genes via RNAi.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cell and Developmental Biology, Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, 28040 Madrid, Spain. emilio76@stanford.edu

ABSTRACT

Background: RNA interference (RNAi) is a valuable tool in the investigation of gene function. The purpose of this study was to examine the availability, target cell types and efficiency of RNAi in the mouse seminiferous epithelium.

Methods: The experimental model was based on transgenic mice expressing EGFP (enhanced green fluorescent protein). RNAi was induced by in vivo transfection of plasmid vectors encoding for short hairpin RNAs (shRNAs) targeting EGFP. shRNAs were transfected in vivo by microinjection into the seminiferous tubules via the rete testis followed by square wave electroporation. As a transfection reporter, expression of red fluorescent protein (HcRed 1) was used. Cell types, the efficiency of both transfections and RNAi were all evaluated.

Results: Sertoli cells were the main transfected cells. A reduction of about 40% in the level of EGFP protein was detected in cells successfully transfected both in vivo and in vitro. However, the efficiency of in vivo transfection was low.

Conclusion: In adult seminiferous epithelial cells, in vivo post-transcriptional gene silencing mediated by RNAi via shRNA is efficient in Sertoli cells. Similar levels of RNAi were detected both in vivo and in vitro. This also indicates that Sertoli cells have the necessary silencing machinery to repress the expression of endogenous genes via RNAi.

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Different phases of in vivo microinjection of vectors into testis tubules through the rete testis. Nigrosin was used as tracer.
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Figure 1: Different phases of in vivo microinjection of vectors into testis tubules through the rete testis. Nigrosin was used as tracer.

Mentions: Male mice of 30–45 days post-natal (dpn) were anaesthetized by an intraperitoneal injection of Rompun (Bayer, Kiel Germany)/Ketolar (Pfizer, Dublin Ireland) solution (315 μl/Kg; 84 mg/Kg respectively). After opening the abdominal cavity, the testes were exposed under a binocular microscope as previously described [47]. Approximately 20 μl of plasmid DNA in TE buffer (10 mM Tris, and 1 mM EDTA, pH adjusted to 7.5) (3 μg/μl) containing nigrosine (1 mg/ml) as a tracer was slowly microinjected into the rete testis using a 40–70 μm in diameter glass micropipette (Fig 1). Trypan blue, the standard tracer for procedures of this kind, was ruled out due to its autofluorescence. For in vivo electroporation, each testis was held between tweezer-type electrodes (model 520, 7 mm diameter, BTX, San Diego, CA) briefly soaked in PBS, and two sets of four electric pulses of square wave were applied (using an electric pulse generator ECM 830 [BTX]). Each pulse provided 50 V for 50 ms; the interval between the pulses was 950 ms [51]. The testes were then returned to the abdominal cavity and the skin stitched closed. Four days later the mice were sacrificed and the testes removed for analysis.


Gene silencing by RNAi in mouse Sertoli cells.

González-González E, López-Casas PP, Del Mazo J - Reprod. Biol. Endocrinol. (2008)

Different phases of in vivo microinjection of vectors into testis tubules through the rete testis. Nigrosin was used as tracer.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2483279&req=5

Figure 1: Different phases of in vivo microinjection of vectors into testis tubules through the rete testis. Nigrosin was used as tracer.
Mentions: Male mice of 30–45 days post-natal (dpn) were anaesthetized by an intraperitoneal injection of Rompun (Bayer, Kiel Germany)/Ketolar (Pfizer, Dublin Ireland) solution (315 μl/Kg; 84 mg/Kg respectively). After opening the abdominal cavity, the testes were exposed under a binocular microscope as previously described [47]. Approximately 20 μl of plasmid DNA in TE buffer (10 mM Tris, and 1 mM EDTA, pH adjusted to 7.5) (3 μg/μl) containing nigrosine (1 mg/ml) as a tracer was slowly microinjected into the rete testis using a 40–70 μm in diameter glass micropipette (Fig 1). Trypan blue, the standard tracer for procedures of this kind, was ruled out due to its autofluorescence. For in vivo electroporation, each testis was held between tweezer-type electrodes (model 520, 7 mm diameter, BTX, San Diego, CA) briefly soaked in PBS, and two sets of four electric pulses of square wave were applied (using an electric pulse generator ECM 830 [BTX]). Each pulse provided 50 V for 50 ms; the interval between the pulses was 950 ms [51]. The testes were then returned to the abdominal cavity and the skin stitched closed. Four days later the mice were sacrificed and the testes removed for analysis.

Bottom Line: A reduction of about 40% in the level of EGFP protein was detected in cells successfully transfected both in vivo and in vitro.Similar levels of RNAi were detected both in vivo and in vitro.This also indicates that Sertoli cells have the necessary silencing machinery to repress the expression of endogenous genes via RNAi.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cell and Developmental Biology, Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, 28040 Madrid, Spain. emilio76@stanford.edu

ABSTRACT

Background: RNA interference (RNAi) is a valuable tool in the investigation of gene function. The purpose of this study was to examine the availability, target cell types and efficiency of RNAi in the mouse seminiferous epithelium.

Methods: The experimental model was based on transgenic mice expressing EGFP (enhanced green fluorescent protein). RNAi was induced by in vivo transfection of plasmid vectors encoding for short hairpin RNAs (shRNAs) targeting EGFP. shRNAs were transfected in vivo by microinjection into the seminiferous tubules via the rete testis followed by square wave electroporation. As a transfection reporter, expression of red fluorescent protein (HcRed 1) was used. Cell types, the efficiency of both transfections and RNAi were all evaluated.

Results: Sertoli cells were the main transfected cells. A reduction of about 40% in the level of EGFP protein was detected in cells successfully transfected both in vivo and in vitro. However, the efficiency of in vivo transfection was low.

Conclusion: In adult seminiferous epithelial cells, in vivo post-transcriptional gene silencing mediated by RNAi via shRNA is efficient in Sertoli cells. Similar levels of RNAi were detected both in vivo and in vitro. This also indicates that Sertoli cells have the necessary silencing machinery to repress the expression of endogenous genes via RNAi.

Show MeSH
Related in: MedlinePlus