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Effective silencing of Sry gene with RNA interference in developing mouse embryos resulted in feminization of XY gonad.

Wu N, Yu AB, Zhu HB, Lin XK - J. Biomed. Biotechnol. (2012)

Bottom Line: A Sry gene-specific shRNAs expression vector (pSilencer4.1/Sry565) was constructed.Our results showed that Sry gene was downregulated significantly in developing embryos.The transfection efficiency is associated with the amount of plasmid DNA injection, injection time, injection speed, and volume.

View Article: PubMed Central - PubMed

Affiliation: Institute of Oceanology, Chinese Academy of Sciences, Qingdao, China.

ABSTRACT
Delivering siRNA or shRNA into the developing embryos is still a main challenge to use of RNAi in mammalian systems. Here we analyze several factors influencing RNAi-mediated silencing of Sry gene, which is a tightly controlled spatiotemporal expressed gene and only shortly expressed in developing mouse embryo gonad. A Sry gene-specific shRNAs expression vector (pSilencer4.1/Sry565) was constructed. The shRNA constructs were mixed with polyethylenimines (PEIs) to form a complex and then injected into pregnant mice though tail vein. Our results showed that Sry gene was downregulated significantly in developing embryos. Further study revealed that knocking-down of Sry expression resulted in feminization of gonad development in mouse embryos and the expression level of Sox9 and Wt1 gene was also significantly changed by downregulation of Sry. The transfection efficiency is associated with the amount of plasmid DNA injection, injection time, injection speed, and volume. Our studies suggest that transplacental RNAi could be implemented by tail vein injection of plasmid vector into pregnant mice.

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Related in: MedlinePlus

Quantitative RT-PCR and Western-blotting analysis to detect the expression of gonadal GFP and Sry expression in embryonic mouse gonads. Gonads at 11.5 dpc were grouped on the basis of overall GFP expression observed at the macroscopic level. Quantitative RT-PCR was then carried out on grouped gonads. Gene expression was normalized to IL3 (autosomal in mouse) (mean ± SEM; n = 3). (a), Relative expression of GFP in embryos. Relative high level of GFP expression was found in controls, and variable expression levels of GFP in male and female gonads with Sry knockdown. (b) Sry expression in control and Sry knockeddown gonads. (c) Western-blotting analysis of Sry expression. Control males which treated with 5% glucose solution or negative control vector (pSilencer4.1/EGFP), males embryos treated with Sry shRNA (pSilencer4.1/Sry565) and with low, medium, or high GFP expression, and the control females treated with control vector and show high GFP expression were all collected at 11.5 dpc and each gonad were separated for protein isolation. Western-blotting were performed to determine the SRY expression level in each group of embryos and β-actin was also be detected as a loading control. (d) Quantitative analysis of the Sry protein level. The expression of Sry was determined by Western blot. The value of Sry protein expression level was normalized to the β-actin expression level and the relative expression level of Sry in embryos which treated by 5% glucose solution was set at 100%. Results are expressed as the mean ± SEM, (n > 20).
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fig2: Quantitative RT-PCR and Western-blotting analysis to detect the expression of gonadal GFP and Sry expression in embryonic mouse gonads. Gonads at 11.5 dpc were grouped on the basis of overall GFP expression observed at the macroscopic level. Quantitative RT-PCR was then carried out on grouped gonads. Gene expression was normalized to IL3 (autosomal in mouse) (mean ± SEM; n = 3). (a), Relative expression of GFP in embryos. Relative high level of GFP expression was found in controls, and variable expression levels of GFP in male and female gonads with Sry knockdown. (b) Sry expression in control and Sry knockeddown gonads. (c) Western-blotting analysis of Sry expression. Control males which treated with 5% glucose solution or negative control vector (pSilencer4.1/EGFP), males embryos treated with Sry shRNA (pSilencer4.1/Sry565) and with low, medium, or high GFP expression, and the control females treated with control vector and show high GFP expression were all collected at 11.5 dpc and each gonad were separated for protein isolation. Western-blotting were performed to determine the SRY expression level in each group of embryos and β-actin was also be detected as a loading control. (d) Quantitative analysis of the Sry protein level. The expression of Sry was determined by Western blot. The value of Sry protein expression level was normalized to the β-actin expression level and the relative expression level of Sry in embryos which treated by 5% glucose solution was set at 100%. Results are expressed as the mean ± SEM, (n > 20).

Mentions: In our next step, we tried to understand if the Sry expression was affected by the shRNA; quantitative RT-PCR and Western blotting analysis were performed to detect the Sry level in certain embryos. The gonads were dissected from selected embryos, and green fluorescence was determined using a microscopic. Gonads were then classified into groups on the basis of their GFP expression level, which based at the macroscopic level: low, medium, or high. Quantitative PCR with reverse transcription (RT-PCR) analysis revealed an inverse correlation between gonadal GFP and endogenous Sry gene expression; Gonads with high levels of GFP showed significantly reduced Sry messenger RNA, whereas those with lower GFP gene expression showed modest Sry mRNA reduction (Figures 2(a) and 2(b)). Since the expression level of GFP represents expression of shRNA, our results confirmed that the shRNA could downregulate the Sry expression efficiently. Of note, male embryos with low or no GFP expression appeared normal (confirmed by histology or immunostaining) and were excluded from further analysis. Each group of embryos tested in the study were provide in Table 1. Western-blot analysis was performed to further confirm the interfering efficiency of shRNA, and the results showed that in the gonads with high level of GFP expression, the Sry was significantly reduced (Figure 2(c)). Treatment of the gonads with pSilencer4.1/Sry565 resulted in significantly inhibition of Sry protein expression in male embryos, and the inhibition rate reached 75% (Figures 2(c) and 2(d)) compared with the male embryos treated with control plasmid, pSilencer4.1/EGFP, or treated with 5% glucose solution. The results revealed that tail vein injection is an effective approach to interfering genes expression in developing embryos.


Effective silencing of Sry gene with RNA interference in developing mouse embryos resulted in feminization of XY gonad.

Wu N, Yu AB, Zhu HB, Lin XK - J. Biomed. Biotechnol. (2012)

Quantitative RT-PCR and Western-blotting analysis to detect the expression of gonadal GFP and Sry expression in embryonic mouse gonads. Gonads at 11.5 dpc were grouped on the basis of overall GFP expression observed at the macroscopic level. Quantitative RT-PCR was then carried out on grouped gonads. Gene expression was normalized to IL3 (autosomal in mouse) (mean ± SEM; n = 3). (a), Relative expression of GFP in embryos. Relative high level of GFP expression was found in controls, and variable expression levels of GFP in male and female gonads with Sry knockdown. (b) Sry expression in control and Sry knockeddown gonads. (c) Western-blotting analysis of Sry expression. Control males which treated with 5% glucose solution or negative control vector (pSilencer4.1/EGFP), males embryos treated with Sry shRNA (pSilencer4.1/Sry565) and with low, medium, or high GFP expression, and the control females treated with control vector and show high GFP expression were all collected at 11.5 dpc and each gonad were separated for protein isolation. Western-blotting were performed to determine the SRY expression level in each group of embryos and β-actin was also be detected as a loading control. (d) Quantitative analysis of the Sry protein level. The expression of Sry was determined by Western blot. The value of Sry protein expression level was normalized to the β-actin expression level and the relative expression level of Sry in embryos which treated by 5% glucose solution was set at 100%. Results are expressed as the mean ± SEM, (n > 20).
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig2: Quantitative RT-PCR and Western-blotting analysis to detect the expression of gonadal GFP and Sry expression in embryonic mouse gonads. Gonads at 11.5 dpc were grouped on the basis of overall GFP expression observed at the macroscopic level. Quantitative RT-PCR was then carried out on grouped gonads. Gene expression was normalized to IL3 (autosomal in mouse) (mean ± SEM; n = 3). (a), Relative expression of GFP in embryos. Relative high level of GFP expression was found in controls, and variable expression levels of GFP in male and female gonads with Sry knockdown. (b) Sry expression in control and Sry knockeddown gonads. (c) Western-blotting analysis of Sry expression. Control males which treated with 5% glucose solution or negative control vector (pSilencer4.1/EGFP), males embryos treated with Sry shRNA (pSilencer4.1/Sry565) and with low, medium, or high GFP expression, and the control females treated with control vector and show high GFP expression were all collected at 11.5 dpc and each gonad were separated for protein isolation. Western-blotting were performed to determine the SRY expression level in each group of embryos and β-actin was also be detected as a loading control. (d) Quantitative analysis of the Sry protein level. The expression of Sry was determined by Western blot. The value of Sry protein expression level was normalized to the β-actin expression level and the relative expression level of Sry in embryos which treated by 5% glucose solution was set at 100%. Results are expressed as the mean ± SEM, (n > 20).
Mentions: In our next step, we tried to understand if the Sry expression was affected by the shRNA; quantitative RT-PCR and Western blotting analysis were performed to detect the Sry level in certain embryos. The gonads were dissected from selected embryos, and green fluorescence was determined using a microscopic. Gonads were then classified into groups on the basis of their GFP expression level, which based at the macroscopic level: low, medium, or high. Quantitative PCR with reverse transcription (RT-PCR) analysis revealed an inverse correlation between gonadal GFP and endogenous Sry gene expression; Gonads with high levels of GFP showed significantly reduced Sry messenger RNA, whereas those with lower GFP gene expression showed modest Sry mRNA reduction (Figures 2(a) and 2(b)). Since the expression level of GFP represents expression of shRNA, our results confirmed that the shRNA could downregulate the Sry expression efficiently. Of note, male embryos with low or no GFP expression appeared normal (confirmed by histology or immunostaining) and were excluded from further analysis. Each group of embryos tested in the study were provide in Table 1. Western-blot analysis was performed to further confirm the interfering efficiency of shRNA, and the results showed that in the gonads with high level of GFP expression, the Sry was significantly reduced (Figure 2(c)). Treatment of the gonads with pSilencer4.1/Sry565 resulted in significantly inhibition of Sry protein expression in male embryos, and the inhibition rate reached 75% (Figures 2(c) and 2(d)) compared with the male embryos treated with control plasmid, pSilencer4.1/EGFP, or treated with 5% glucose solution. The results revealed that tail vein injection is an effective approach to interfering genes expression in developing embryos.

Bottom Line: A Sry gene-specific shRNAs expression vector (pSilencer4.1/Sry565) was constructed.Our results showed that Sry gene was downregulated significantly in developing embryos.The transfection efficiency is associated with the amount of plasmid DNA injection, injection time, injection speed, and volume.

View Article: PubMed Central - PubMed

Affiliation: Institute of Oceanology, Chinese Academy of Sciences, Qingdao, China.

ABSTRACT
Delivering siRNA or shRNA into the developing embryos is still a main challenge to use of RNAi in mammalian systems. Here we analyze several factors influencing RNAi-mediated silencing of Sry gene, which is a tightly controlled spatiotemporal expressed gene and only shortly expressed in developing mouse embryo gonad. A Sry gene-specific shRNAs expression vector (pSilencer4.1/Sry565) was constructed. The shRNA constructs were mixed with polyethylenimines (PEIs) to form a complex and then injected into pregnant mice though tail vein. Our results showed that Sry gene was downregulated significantly in developing embryos. Further study revealed that knocking-down of Sry expression resulted in feminization of gonad development in mouse embryos and the expression level of Sox9 and Wt1 gene was also significantly changed by downregulation of Sry. The transfection efficiency is associated with the amount of plasmid DNA injection, injection time, injection speed, and volume. Our studies suggest that transplacental RNAi could be implemented by tail vein injection of plasmid vector into pregnant mice.

Show MeSH
Related in: MedlinePlus