Interference of Polypeptide 1 Gene (CYP19A1) Expression by shRNA Eukaryotic Expression Vector in Female Chick Embryo Gonads | 利用shRNA真核表达载体干涉雌性鸡胚性腺中芳香化酶基因(CYP19A1)的表达

Chinese. 本研究通过把构建的短发夹结构 RNA(shRNA)真核表达载体导入鸡胚，检测鸡胚性腺分化期质粒载体在鸡胚体内代谢情况及雌性鸡胚性腺中芳香化酶基因(CYP19A1) mRNA表达效率，进而探讨利用该方法在鸡胚体内进行特定基因干涉的可行性。实验针对CYP19A1基因构建了4条特异性表达载体，一条非特异性表达载体。每个实验组选取45个新鲜种蛋作为实验材料进行胚盘下腔注射，并设立空白对照组。鸡胚发育12 d时，检测各处理组鸡胚肝脏组织中质粒存在情况，并取其左侧性腺组织进行目标基因的荧光定量分析。研究结果表明，导入组在12日胚龄时所有鸡胚基因组中均可检测到绿色荧光蛋白基因(EGFP)；荧光定量结果显示，导入特异性表达载体cyp-580、cyp-1083和cyp-1295 后，对应雌性鸡胚性腺CYP19A1 mRNA表达效率显著低于空白对照组，干涉效率分别为：73%、52%和85%；特异性表达载体cyp-1403组CYP19A1 mRNA表达效率与空白对照组相比有所降低但无显著性差异。本实验为诱导鸡胚性反转提供了新方法并建立了鸡胚发育期特定基因体内干涉新平台。

English. In this study, we have constructed and injected short hairpin RNA(shRNA) eukaryotic expression vectors into fertilized eggs. Upon incubation, the transferred plasmids were analyzed for their metabolism in the embryo gonads during gonadal differentiation, and their interference on the expression of polypeptide 1(CYP19A1) gene (belonging to cytochrome P450, subfamily A, family 19) was assayed subsequently. The feasibility of the method was then further discussed for gene-specific interference in the chick embryo. In the experiment, four specific shRNA expression vectors and a non-specific expression vector were constructed. For each vector, 45 fresh eggs were injected through the subgerminal cavity. Twenty non-treated eggs served as controls. Embryos were sacrificed after incubation for 12 days (E12, stage 38) and their livers were collected to detect the metabolism of the injected plasmids. Specifically, expression of each vector was investigated in the left gonads by quantitative RT-PCR and the co-produced enhanced green fluorescence protein (EGFP) was detected in all the injected embryos. The quantitative RT-RCR results showed that the specific expression vector cyp-580, cyp-1083 and cyp-1295 significantly suppressed CYP19A1 mRNA levels with a silencing efficiency of 73%、52% and 85%, respectively. However, the vector cyp-1403 could not suppress the expression of the CYP19A1 gene. The present study has provided a method to produce the sex-reversed chicken and has established a platform for gene-specific interference in vivo.