Abstract: Bmp4 regulates the formation of primordial germ cells (PGCs) through BMP signaling pathway in mammals, but its function and mechanism in the formation of chicken PGCs is still unclear. In this study, overexpression and knockout vectors of chicken bmp4 were constructed and transfected into DF-1 cells. The recombinant vector pCDH-CMV-bmp4-EF1-copGFP was constructed by homologous recombination technique, transfected into DF-1 cells, and the expression of bmp4 was detected by RT-qPCR. Three knockout target sites (gRNAs) were designed based on the bmp4 CDS sequence, and PGMLV-GM1 was inserted to construct the recombinant vectors bmp4-sgRNA1, bmp4-sgRNA2 and bmp4-sgRNA3. Cas9 vector was co-transfected into DF-1 cells with bmp4-sgRNA vector, and knockout vector activity and knockout efficiency were detected by T7E1 digestion and TA cloning assay. The full length of bmp4 CDS was 1546 bp, and the pCDH-CMV-bmp4-EF1-copGFP vector was successfully constructed. After transfected into DF-1 cells, it showed green fluorescence expression (25.17%±0.007). The results of RT-qPCR showed that bmp4 was significantly up-regulated in DF-1, after being transfected with pCDH-CMV-bmp4-EF1-copGFP compared with the control group (P<0.01). Three gRNA vectors were successfully constructed. The knockdown activity was detected by T7E1 digestion after transfection of DF-1 cells. The results of TA cloning showed that the knockout efficiencies were 6.25%, 12.5% and 18.75%, respectively. The results indicated that chicken bmp4 overexpression and knockout vector were constructed successfully, which could support the research of bmp4 function verification and mechanism analysis.

Key words: chicken, bmp4, overexpression vector, knockout vector