Reproduction Abstracts

Introduction: The production of recombinant protein in transgenic domestic animal is one of the major successes of biotechnology. Knock-in system is a more powerful method to produce mammary gland bioreactor. This study is conducted to development of knock-in somatic cells using bovine β-casein genome for produce of biological substance.

Material and methods: The knock-in vector was constructed by using 5.9 kb fragment of upstream of bovine β-casein genome containing exons 1, 2, and 3 as the left arm and using ~2.08 kb of 3′ region containing bovine β-casein exons 4, 5, and 6 as the right arm. Human FGF2 gene was subcloned in the bovine β-casein gene exon 3 and the gene was linked with F2A self-processing sequence. Neo gene was used as positive selection marker and diphtheria toxin A (DT-A) gene used as negative selection marker. For identification of human FGF2 expression in the knock-in vector, human FGF2 knock-in vector was transfected into HC11 cell line and the mRNA of bovine β-casein-hFGF2 fusion gene was analyzed by RT-PCR. For transfection, linearzed knock-in vectors were introduced into bovine ear fibroblasts by electroporation. After 72 h, the cells were selected with G418 during 11 days. The G418-resistant colonies were picked and analyzed by PCR.

Result and discussion: The hFGF2 mRNA was detected in the HC11 cell transfected with knock-in vector by RT-PCR. The 504 G418 resistant colonies were screened by PCR and four colonies were occurred homologous recombination. These cells may be used to production of knock-in transgenic bovine by somatic cell nuclear transfer.