摘要
为研究Sry基因的调控网络,采用siRNA表达载体介导的RNAi技术,特异性地抑制睾丸决定因子Sry在小鼠胚胎中的表达,并观察Sry基因沉默后对在两性性腺分化中起重要作用的Wt1,Sf1,Dax1,Gata4,Sox9及Amh基因表达的影响。利用本课题组先前构建的siRNA重组表达载体(pSilencer4.1/Sry217及pSilencer4.1/Sry565),通过尾静脉注射法导入妊娠9.5天(9.5dpc)的母鼠体内,在11.5dpc时取胚胎,对性别鉴定为雄性的胚胎以RT-PCR法和Western-blot检测Sry基因的表达抑制效果,并同时用定量PCR法检测Wt1等上述性别决定相关基因表达变化情况。结果表明,注射干扰质粒后48hSry基因的mRNA和蛋白表达水平均降低,其中siRNA表达质粒pSilencer4.1/Sry565的抑制效果显著,可达到80%的抑制率。Sry基因沉默后,Wt1基因表达量显著升高;Sf1,Dax1,Gata4,Sox9基因表达水平没有明显变化;Amh基因无表达。试验结果表明,Sry基因表达抑制会导致Wt1基因表达升高;另外,Sry基因激活Sox9基因的表达可能需要其他的辅助因子协同作用。
In order to investigate Sry regulation network in the development of male embryo, we inhibited the Sry gene expression by RNAi and then examined expression of other sex-related genes. Six genes （Sox9, Wtl, Sfl, Daxl, Gata4 and Amh）, which are suggested to be closely related to Sry regulation were studied. Two siRNA expression vectors pSilencer4, l/Sry217 and pSilencer4.1/Sry565 were constructed and injected into gestated mouse through tail vein at 9.5 day of conception （dpc）. The inhibition efficiency of Sry and the expression of other six genes were examined in male embryos at 11.5 dpc by RT-PCR and Western-blot. Expressions of the other six genes were analyzed by fluorescence quantity PCR. The results indicated both the pSilencer4.1/Sry217 and pSilencer4.1/Sry565 could inhibit significantly increased after Sry si- lencing. In contrast, no significant changes were observed in the expression of Sfl, Amh, Gata4, Daxl and Sox9 when si- lencing Sry by siRNA. Our results suggested that the Wtl transcription was regulated by Sry, whereas the Sox9 expression is not directly regulated by Sry in the development of genital ridge.
出处
《遗传》
CAS
CSCD
北大核心
2008年第2期195-202,共8页
Hereditas（Beijing)
基金
国家自然科学基金项目资助(编号:30671501)~~
