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http://dx.doi.org/10.12749/RDB.2017.41.1.7

Knock-in Efficiency Depending on Homologous Arm Structure of the Knock-in Vector in the Bovine Fibroblasts  

Kim, Se Eun (Department of Aniaml Science, College of Agriculture and Life Science, Chonnam National University)
Park, Da Som (Department of Aniaml Science, College of Agriculture and Life Science, Chonnam National University)
Koo, Deog-Bon (Department of Biotechnology, College of Engineering, Daegu University)
Kang, Man-Jong (Department of Aniaml Science, College of Agriculture and Life Science, Chonnam National University)
Publication Information
Reproductive and Developmental Biology / v.41, no.1, 2017 , pp. 7-16 More about this Journal
Abstract
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 ${\beta}-casein$ 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 ${\beta}-casein$ 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 ${\beta}-casein$ gene in the mammary gland.
Keywords
Knock-in; ${\beta}-Casein$ gene; Somatic cell; Homologous recombination; Bovine;
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