Construction of Eukaryotic Expression Vector of Hoxa5,BMP6 Gene by Homologous Recombination and Its Expression in Fibroblasts

doi:10.7668/hbnxb.201750906

Abstract

Abstract: The study aimed to use homologous recombination build Hoxa5 and BMP6 genes expression vector study the interaction between Hoxa5 and BMP6 genes by change of expression.Firstly,a pair of primers that have homologous arm were desigened by RNA extraction and referred Hoxa5 and BMP6 genes sequence information of Aohan Fine Wool Sheep in GenBank,the Hoxa5 and BMP6 genes fragment were amplified by PCR. We used SoSo kit connect the target fragment to pcDNA3.1 eukaryotic expression vector.Recombined plasimd pcDNA3.1-Hoxa5 and pcDNA3.1-BMP6 were constructed and transformed into E.coli DH5α competent cell. Then plasmid pcDNA3.1-Hoxa5, pcDNA3.1-BMP6 was cotransfected into fibroblasts,and used RT-PCR to detect the expression level of these genes. As the results showed that,pcDNA3.1-Hoxa5, pcDNA 3.1-BMP6 plasmid cotransfected successfully and identified by enzyme and sequencing. The expression level of Hoxa5 gene in fibroblasts was lower than the control group and the expression level of BMP6 gene in fibroblasts was higher than the control group. The plasmid was constructed and cotransfected into fibroblasts successfully by homologous recombination technique.The expression level of BMP6 gene increased significantly,and the expression of Hoxa5 gene decreased. Therefore,we can concluded that BMP6 gene inhibited the expression of Hoxa5 gene,and Hoxa5 gene promoted the expression of BMP6 gene.These results would lay a foundation for the further research.

Key words: Homologous recombination, Fibroblats, Hoxa5,BMP6 cotransfection, RT-PCR, Western Blot

CLC Number:

S826
Q78

LI Jing, ZHANG Mengyao, LIU Kaidong, LIU Nan, HE Jianning. Construction of Eukaryotic Expression Vector of Hoxa5,BMP6 Gene by Homologous Recombination and Its Expression in Fibroblasts[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2020, 35(4): 187-194. doi: 10.7668/hbnxb.201750906.

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