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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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Comparative results from flow cytometry analysis of EGFP-expression in seminiferous epithelium cells transfected in vivo with pRed or pGtoR. (A) Representative traces of flow cytometry using pGtoR vector. 1) R2 corresponds to non-transfected cell population; R3 represent the transfected cells (red fluorescent). 2) M1 represents the level of green fluorescent (EGFP) of R2 cell population 3) represents the level of EGFP in transfected cells (R3). B) The histogram shows the mean (Mean ± SE) intensity of green fluorescence of the cells (EGFP) transfected in vivo with pRed or pGtoR, as determined by flow cytometry four days after transfections. Asterisk shows statistically significant differences as measured by p < 0.05.
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Figure 3: Comparative results from flow cytometry analysis of EGFP-expression in seminiferous epithelium cells transfected in vivo with pRed or pGtoR. (A) Representative traces of flow cytometry using pGtoR vector. 1) R2 corresponds to non-transfected cell population; R3 represent the transfected cells (red fluorescent). 2) M1 represents the level of green fluorescent (EGFP) of R2 cell population 3) represents the level of EGFP in transfected cells (R3). B) The histogram shows the mean (Mean ± SE) intensity of green fluorescence of the cells (EGFP) transfected in vivo with pRed or pGtoR, as determined by flow cytometry four days after transfections. Asterisk shows statistically significant differences as measured by p < 0.05.

Mentions: The vector pHcRed1-shRNA-EGFP (pGtoR) [50] allows detection of transfected cells expressing shRNA-EGFP, based on the co-expression of the red fluorescence protein HcRed1. Nevertheless, in tissue sections, an accurate measure of the fading of green fluorescence at the cellular level is difficult to detect due to the frequent superposition of adjacent cells and to the variability of EGFP expression between different cell types of the seminiferous epithelium [57]. To quantify the level of post-transcriptional silencing of EGFP in transfected cells, the reduction in green fluorescence from monocellular dispersions of seminiferous tubule cells from in vivo transfected testis was measured by fluorescence activated cell sorting (FACS). Red fluorescent cells were selected four days after transfection, and a reduction of 41.94% of green fluorescence was detected in the cells transfected with pGtoR compared to those transfected with pRed (used as a control). A significant difference in green fluorescence (t test; p = 0.047) was observed between red fluorescent cells depending on the vector used (pGtoR or pRed) (Fig. 3). This difference can only be interpreted as a specific silencing of EGFP mediated by shRNA-EGFP.


Gene silencing by RNAi in mouse Sertoli cells.

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

Comparative results from flow cytometry analysis of EGFP-expression in seminiferous epithelium cells transfected in vivo with pRed or pGtoR. (A) Representative traces of flow cytometry using pGtoR vector. 1) R2 corresponds to non-transfected cell population; R3 represent the transfected cells (red fluorescent). 2) M1 represents the level of green fluorescent (EGFP) of R2 cell population 3) represents the level of EGFP in transfected cells (R3). B) The histogram shows the mean (Mean ± SE) intensity of green fluorescence of the cells (EGFP) transfected in vivo with pRed or pGtoR, as determined by flow cytometry four days after transfections. Asterisk shows statistically significant differences as measured by p < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC2483279&req=5

Figure 3: Comparative results from flow cytometry analysis of EGFP-expression in seminiferous epithelium cells transfected in vivo with pRed or pGtoR. (A) Representative traces of flow cytometry using pGtoR vector. 1) R2 corresponds to non-transfected cell population; R3 represent the transfected cells (red fluorescent). 2) M1 represents the level of green fluorescent (EGFP) of R2 cell population 3) represents the level of EGFP in transfected cells (R3). B) The histogram shows the mean (Mean ± SE) intensity of green fluorescence of the cells (EGFP) transfected in vivo with pRed or pGtoR, as determined by flow cytometry four days after transfections. Asterisk shows statistically significant differences as measured by p < 0.05.
Mentions: The vector pHcRed1-shRNA-EGFP (pGtoR) [50] allows detection of transfected cells expressing shRNA-EGFP, based on the co-expression of the red fluorescence protein HcRed1. Nevertheless, in tissue sections, an accurate measure of the fading of green fluorescence at the cellular level is difficult to detect due to the frequent superposition of adjacent cells and to the variability of EGFP expression between different cell types of the seminiferous epithelium [57]. To quantify the level of post-transcriptional silencing of EGFP in transfected cells, the reduction in green fluorescence from monocellular dispersions of seminiferous tubule cells from in vivo transfected testis was measured by fluorescence activated cell sorting (FACS). Red fluorescent cells were selected four days after transfection, and a reduction of 41.94% of green fluorescence was detected in the cells transfected with pGtoR compared to those transfected with pRed (used as a control). A significant difference in green fluorescence (t test; p = 0.047) was observed between red fluorescent cells depending on the vector used (pGtoR or pRed) (Fig. 3). This difference can only be interpreted as a specific silencing of EGFP mediated by shRNA-EGFP.

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