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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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Seminiferous tubules of testis from C57BL/6J wild type mice after in vivo transfection with the pEGFP-N1 vector. A and C) Merge image of partial view of tubule sections showing EGFP-positive Sertoli cells (with the well-known arborescent-like cytoplasm) as preferentially transfected cells. Nuclei were stained with Hoechst 33258 dye. Spermatocytes (Sp) round spermatid (Rd) are indicated (C). The red lines indicate the basement membranes of seminiferous tubules. B and D show enlarged images of EGFP fluorescence taken 24 hours after transfection. Bars represent 50 μm.
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Figure 2: Seminiferous tubules of testis from C57BL/6J wild type mice after in vivo transfection with the pEGFP-N1 vector. A and C) Merge image of partial view of tubule sections showing EGFP-positive Sertoli cells (with the well-known arborescent-like cytoplasm) as preferentially transfected cells. Nuclei were stained with Hoechst 33258 dye. Spermatocytes (Sp) round spermatid (Rd) are indicated (C). The red lines indicate the basement membranes of seminiferous tubules. B and D show enlarged images of EGFP fluorescence taken 24 hours after transfection. Bars represent 50 μm.

Mentions: To evaluate the efficiency of in vivo gene silencing in mouse testis, after completing the first wave of spermatogenesis, we first characterized the efficiency to deliver plasmid DNA into the cells of the seminiferous epithelium. To determine which cell types were preferentially transfected either pEGFP-N1 or pGtoR was used. The cytological detection of green or red fluorescent proteins indicated that Sertoli cells (Fig. 2) were the cell type most commonly transfected (less than 1% of germ cells were also transfected). However, the efficiency of transfection of Sertoli cells was always less than 10% although no differences were found between the plasmids used. As previously described, altering the experimental conditions, i.e., increasing voltage and/or the number of electrical pulses, was found to damage the seminiferous epithelium as assessed by histopathological analysis (data not shown) [51].


Gene silencing by RNAi in mouse Sertoli cells.

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

Seminiferous tubules of testis from C57BL/6J wild type mice after in vivo transfection with the pEGFP-N1 vector. A and C) Merge image of partial view of tubule sections showing EGFP-positive Sertoli cells (with the well-known arborescent-like cytoplasm) as preferentially transfected cells. Nuclei were stained with Hoechst 33258 dye. Spermatocytes (Sp) round spermatid (Rd) are indicated (C). The red lines indicate the basement membranes of seminiferous tubules. B and D show enlarged images of EGFP fluorescence taken 24 hours after transfection. Bars represent 50 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Seminiferous tubules of testis from C57BL/6J wild type mice after in vivo transfection with the pEGFP-N1 vector. A and C) Merge image of partial view of tubule sections showing EGFP-positive Sertoli cells (with the well-known arborescent-like cytoplasm) as preferentially transfected cells. Nuclei were stained with Hoechst 33258 dye. Spermatocytes (Sp) round spermatid (Rd) are indicated (C). The red lines indicate the basement membranes of seminiferous tubules. B and D show enlarged images of EGFP fluorescence taken 24 hours after transfection. Bars represent 50 μm.
Mentions: To evaluate the efficiency of in vivo gene silencing in mouse testis, after completing the first wave of spermatogenesis, we first characterized the efficiency to deliver plasmid DNA into the cells of the seminiferous epithelium. To determine which cell types were preferentially transfected either pEGFP-N1 or pGtoR was used. The cytological detection of green or red fluorescent proteins indicated that Sertoli cells (Fig. 2) were the cell type most commonly transfected (less than 1% of germ cells were also transfected). However, the efficiency of transfection of Sertoli cells was always less than 10% although no differences were found between the plasmids used. As previously described, altering the experimental conditions, i.e., increasing voltage and/or the number of electrical pulses, was found to damage the seminiferous epithelium as assessed by histopathological analysis (data not shown) [51].

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