Reproductive and Developmental Biology

The Korean Society of Animal Reproduction (한국동물번식학회)
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Construction and Expression Analysis of Knock-in Vector for EGFP Expression in the Porcine $\beta$-Casein Gene Locus

돼지 $\beta$-Casein을 이용한 EGFP 발현 Knock-in 벡터의 구축 및 발현 검증

  • Lee, Sang-Mi (Department of Animal Science and Institute of Agricultural Science and Technology, College of Agriculture & Life Science, Chonnam National University) ;
  • Kim, Hey-Min (Department of Animal Science and Institute of Agricultural Science and Technology, College of Agriculture & Life Science, Chonnam National University) ;
  • Moon, Seung-Ju (Department of Animal Science and Institute of Agricultural Science and Technology, College of Agriculture & Life Science, Chonnam National University) ;
  • Kang, Man-Jong (Department of Animal Science and Institute of Agricultural Science and Technology, College of Agriculture & Life Science, Chonnam National University)
  • 이상미 (전남대학교 농업생명과학대학 농업과학기술연구소 동물자원학부) ;
  • 김혜민 (전남대학교 농업생명과학대학 농업과학기술연구소 동물자원학부) ;
  • 문승주 (전남대학교 농업생명과학대학 농업과학기술연구소 동물자원학부) ;
  • 강만종 (전남대학교 농업생명과학대학 농업과학기술연구소 동물자원학부)
  • Published : 2008.09.30
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Abstract

This study was carried out to develop knock-in vector for EGFP (enhanced green fluorescent protein) expression in porcine $\beta$-casein locus. For construction of knock-in vector using porcine $\beta$-casein gene, we cloned the $\beta$-casein genome DNA from porcine fetal fibroblast cells, EGFP and SV40 polyA signal using PCR. The knock-in vectors consisted of a 5-kb fragment as the 5' recombination arm and a 2.7-kb fragment as the 3' recombination arm. We used the neomycin resistance gene ($neo^{r}$) as a positive selectable marker and the diphtheria toxin A (DT-A) gene as a negative selectable marker. To demonstrate EGFP expression from knock-in vector, we are transfected knock-in vector that has EGFP gene in murine mammary epithelial cell line HC11 cells with pSV2 neo plasmid. The EGFP expression was detected in HC11 cells transfected knock-in vector. This result demonstrates that this knock-in vector may be used for the development of knock-in transgenic pig.

본 연구는 돼지 $\beta$-casein 유전자 위치에서 EGFP가 발현될 수 있는 knock-in 벡터를 구축하기 위하여 실시되었다. 돼지의 $\beta$-casein 유전자를 이용하여 knock-in 벡터를 구축하기 위해 돼지의 태아 섬유아세포로부터 $\beta$-casein 유전자를 동정하였고 EGFP, SV4O polyA signal을 동정하였다. Knock-in 벡터는 5' 상동 영역 약 5 kb와 3' 상동 영역 약 2.7 kb로 구성되어있으며, positive selection marker로 $neo^{r}$ 유전자를, negative selection marker로 DT-A 유전자를 사용하였다. 구축된 knock-in 벡터로부터 EGFP의 발현을 확인하기 위하여 생쥐 유선 세포인 HC11 세포에 knock-in 벡터를 도입하였다. 그 결과 EGFP의 발현을 HC11 세포에서 확인하였다. 이와 같은 결과로서 이 block-in 벡터는 knock-in 형질전환 돼지를 생산하는데 사용될 수 있을 것으로 생각된다.

Keywords

References

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