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| Interference of Polypeptide 1 Gene(CYP19A1) Expression by shRNA Eukaryotic Expression Vector in Female Chick Embryo Gonads |
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2. China Agricultural University
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Abstract 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.
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Received: 05 May 2011
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