为了研究 Sry 基因的调控网络,采用 siRNA 技术使 Sry 基因沉默,探讨了有效沉默 Sry 基因的途径和最佳条件. 设计、合成针对小鼠 Sry 基因的发夹状寡核苷酸链,退火后连入真核表达载体pSilencer 4.1-CMV neo vector,构建以小鼠 Sry 基因为靶点的 siRNA 干涉载体 pSilencer 4.1/Sry217及 pSilencer 4.1/Sry565,通过尾静脉注射法将载体质粒导入妊娠小鼠体内,于小鼠妊娠第 11.5 天,即 11.5 dpc (days post coitum,性交后天数) 取出胚胎,采用双重 PCR 法对胚胎进行性别鉴定,鉴定为雄性的胚胎采用半定量 RT-PCR 法检测Sry 基因的表达量,研究不同干扰序列、不同注射时间及注射剂量对 Sry 基因表达量的影响. 研究结果,确定了质粒的最佳注射时间为 9.5 dpc,注射剂量为 20 μg,注射干扰质粒 pSilencer 4.1/Sry565 对 Sry基因的抑制效率达 85% 左右. 结果表明,siRNA 可以显著抑制雄性胚胎 Sry 基因的表达.
In order to study the regulation mechanism of Sry in sex determination, the siRNA technique was used to silence the Sry expression in mouse embryos. Two siRNA expression vectors were synthesized, pSilencer4.1/Sry217 and pSilencer 4.1/Sry565. The siRNA expression vectors were injected into gestated mouse through tail vein. The sex was identified by PCR, and the expression level of Sry gene was determined using RT-PCR technique in the male mouse embryos at 11.5 dpc(days post coitum). The time- and dose -dependent effects was studied. The results confirmed that one of the siRNA expression plasmids, pSilencer4.1/Sry565, could inhibit the Sry gene expression significantly. Eighty-five percent inhibition was obtained when injecting 20 μg siRNA vector at 9.5 dpc.
赵金红,杜卫华,道尔吉,裴 杰,张 丽,林秀坤.小鼠胚胎Sry基因的RNA干涉研究[J].生物化学与生物物理进展,2006,33(9):890-894
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