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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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Primary culture of Sertoli cells from EGFP transgenic mice (FM131) transfected with pGtoR. The analysis was performed at 120 h (A, B and C) and 140 h (D, E and F) after transfection. Green fluorescence (excitation wavelength 488 nm) (A and D). Red fluorescence (B and E). Merge (C and F). Transfected cells (as demonstrated by red fluorescence) are indicated by arrows. Bar represents 10 μm.
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Figure 4: Primary culture of Sertoli cells from EGFP transgenic mice (FM131) transfected with pGtoR. The analysis was performed at 120 h (A, B and C) and 140 h (D, E and F) after transfection. Green fluorescence (excitation wavelength 488 nm) (A and D). Red fluorescence (B and E). Merge (C and F). Transfected cells (as demonstrated by red fluorescence) are indicated by arrows. Bar represents 10 μm.

Mentions: In order to compare gene silencing by RNAi in cultured primary Sertoli cells and in in vivo transfected cells, Sertoli cells isolated from the EGFP transgenic mouse line FM131 were cultured. A reduction in green fluorescence due to EGFP protein was observed in the red fluorescent cells transfected with pGtoR compared to those that were not transfected (Fig. 4).


Gene silencing by RNAi in mouse Sertoli cells.

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

Primary culture of Sertoli cells from EGFP transgenic mice (FM131) transfected with pGtoR. The analysis was performed at 120 h (A, B and C) and 140 h (D, E and F) after transfection. Green fluorescence (excitation wavelength 488 nm) (A and D). Red fluorescence (B and E). Merge (C and F). Transfected cells (as demonstrated by red fluorescence) are indicated by arrows. Bar represents 10 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Primary culture of Sertoli cells from EGFP transgenic mice (FM131) transfected with pGtoR. The analysis was performed at 120 h (A, B and C) and 140 h (D, E and F) after transfection. Green fluorescence (excitation wavelength 488 nm) (A and D). Red fluorescence (B and E). Merge (C and F). Transfected cells (as demonstrated by red fluorescence) are indicated by arrows. Bar represents 10 μm.
Mentions: In order to compare gene silencing by RNAi in cultured primary Sertoli cells and in in vivo transfected cells, Sertoli cells isolated from the EGFP transgenic mouse line FM131 were cultured. A reduction in green fluorescence due to EGFP protein was observed in the red fluorescent cells transfected with pGtoR compared to those that were not transfected (Fig. 4).

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