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KMID : 1023620170410010007
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Reproductive and Developmental Biology
2017 Volume.41 No. 1 p.7 ~ p.16
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Knock-in Efficiency Depending on Homologous Arm Structure of the Knock-in Vector in the Bovine Fibroblasts
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Kim Se-Eun
Park Da-Som
Koo Deog-Bon
Kang Man-Jong
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Abstract
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The knock-in efficiency in the fibroblast is very important to produce transgenic domestic animal using nuclear
transfer. In this research, we constructed three kinds of different knock-in vectors to study the efficiency of knock-in
depending on structure of knock-in vector with different size of homologous arm on the β-casein gene locus in the
somatic cells; DT-A_cEndo Knock-in vector, DT-A_tEndo Knock-in vector I, and DT-A_tEndo Knock-in vector II. The
knock-in vector consists of 4.8 kb or 1.06 kb of 5’ arm region and 1.8 kb or 0.64 kb of 3’ arm region, and neomycin
resistance gene(neor) as a positive selection marker gene. The cEndo Knock-in vector had 4.8 kb and 1.8 kb homologous
arm. The tEndo Knock-in vector I had 1.06 kb and 0.64 kb homologous arm and tEndo Knock-in vector II
had 1.06 kb and 1.8 kb homologous arm. To express endostatin gene as transgene, the F2A sequence was fused to the
5’ terminal of endostatin gene and inserted into exon 7 of the β-casein gene. The knock-in vector and TALEN were
introduced into the bovine fibroblast by electroporation. The knock-in efficiencies of cEndo, tEndo I, and tEndo II
vector were 4.6%, 2.2% and 4.8%, respectively. These results indicated that size of 3’ arm in the knock-in vector is
important for TALEN-mediated homologous recombination in the fibroblast. In conclusion, our knock-in system may
help to create transgenic dairy cattle expressing human endostatin protein via the endogenous expression system of
the bovine β-casein gene in the mammary gland.
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KEYWORD
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Knock-in, β-Casein gene, Somatic cell, Homologous recombination, Bovine
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