Abstract:

【Objective】 The objective of this study is to get MSTN knock-out porcine fetal fibroblast. 【Method】 Construction of targeting vector: Neomycin phosphotransferase gene was used as positive-selecting gene, herpes simplex thymidine kinase gene was used as negative-selecting gene. Homologous arms were respectively inserted into the two sides of Neo gene. The 5.3 kb homologous left arm contains partial 5' MSTN gene, the whole exon1 and exon2, and part of intron2, while the 0.8 kb homologous right arm contains part of exon3 and partial 3' MSTN gene. Taking the tissue of 35 days Yorkshire as materials, fibroblast lines were successfully established by trypsin digestion. Targeting vector was introduced into the fetal fibroblast of Yorkshire by lipofectamin-mediated DNA transfection methods, after transfection, selected with G418 (250 μg?ml-1) for 7 days, then continuous selected with 200 μg?ml-1 G418+2 μmol?L-1GANC. Semi-quantitative RT-PCR analysis of MSTN mRNA expression was done using the total RNAs from pig fetal fibroblast before and after transfection. 【Result】 The replacement vector was successfully constructed, which can knock out partial intron2 and exon3 of myostatin. Five resistance cell strains were gotten, through PCR identification, one strain took place correct homologous recombination. The amount of MSTN mRNA expression of transgenic cells is obviously declined. 【Conclusion】 MSTN knock-out porcine fetal fibroblast was obtained.

Key words: swine, fetal fibroblast, gene targeting, MSTN