Earticle

초록

영어

The gene targeting efficiency in the fibroblast is very important to produce transgenic domestic animal using nuclear transfer. However, the efficiency of gene targeting in somatic cells is lower than that in mouse embryonic stem cells. The purpose of this study was to determine whether structure of knock-in vector with different size of homologous arm influence the efficiency of knock-in on the β-casein gene locus in the somatic cells. Three kinds of knock-in vectors were constructed; 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. And then, Antibiotics selection was performed 10～12 days using 500 ug/mL G418. After selection, G418-resistance colonies were analyzed by 3'arm, 5'arm and targeted or non-targeted PCR. 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.

키워드

저자

• Se Eun Kim [ Department of Animal Science, Chonnam National University, Republic of Korea ]
• Da Som Park [ Department of Animal Science, Chonnam National University, Republic of Korea ]
• Deog-Bon Koo [ Department of Biotechnology, Daegu University, Republic of Korea ]
• Man-Jong Kang [ Department of Animal Science, Chonnam National University, Republic of Korea ]

참고문헌

발행기관

간행물

표지보기 관심저널 등록

• 간행물명

  Reproductive & Developmental Biology(Supplement)

• 간기

  연간

• 수록기간

  2001~2017

• 십진분류

  KDC 527 DDC 636