Abstract: The study aimed to use Homologous recombination build DKK1 and BMP4 genes expression vector study the interaction between DKK1 and BMP4 genes by change of expression. Firstly,a pair of primers that have homologous arm were designed by RNA extraction and referred DKK1 and BMP4 genes sequence information of Aohan Fine Wool Sheep in GenBank,the DKK1 and BMP4 genes fragment was amplified by PCR. We used SoSo kit connect the target fragment to pcDNA3.1 eukaryotic expression vector. Recombined plasimid pcDNA3.1-DKK1 and pcDNA3.1-BMP4 was constructed and transformed into E. coli DH5α competent cell. Then plasmid pcDNA3.1-DKK1, BMP4 was cotransfected into fibroblasts,and used qRT-PCR to detect the expression level of these genes. The results showed that,pcDNA3.1-DKK1, BMP4 plasmid cotransfected successfully and identified by enzyme and sequencing. The expression level of DKK1 gene in fibroblasts was lower than the control group and the expression level of BMP4 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 DKK1 gene decreased and the expression of BMP4 gene no variation. Therefore, BMP4 gene inhibited the expression of DKK1 gene,and the DKK1 gene had no effect on the expression of BMP4 gene. These results constructed the basis for further research.

Key words: Homologous recombination, Fibroblasts, DKK1,BMP4 cotransfection, RT-PCR, Western Blot

CLC Number: 

• Q78