Reproductive and Developmental Biology

The Korean Society of Animal Reproduction (한국동물번식학회)
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Production and Characterization of Porcine Cell Lines Overexpressing Human H-Transferase

사람 H-Transferase 유전자 과발현 형질전환 체세포주 확립 및 검증

  • Lee, Sang-Mi (Department of Animal Science and Institute of Agricultural Science and Technology, College of Agriculture & Life Science, Chonnam National University) ;
  • Park, Hyo-Young (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) ;
  • 박효영 (전남대학교 농업생명과학대학, 농업과학기술연구소, 동물자원학부) ;
  • 김혜민 (전남대학교 농업생명과학대학, 농업과학기술연구소, 동물자원학부) ;
  • 문승주 (전남대학교 농업생명과학대학, 농업과학기술연구소, 동물자원학부) ;
  • 강만종 (전남대학교 농업생명과학대학, 농업과학기술연구소, 동물자원학부)
  • Published : 2007.09.30
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Abstract

This study was canted out to develop cell lines overexpressing human H-transferase (HT). One of the approaches to prevent hyperacute rejection in xenotransplantation might be the expression of human HT in porcine cells. In this study, we cloned human HT gene from HepG2 cells using RT-PCR to establish HT-overexpressing vector. The full-length cDNA of human HT was inserted into the 3' end of CMV promoter for construction of the overexpression vector pRc/CMV-hHT. Using ietPEI DNA transfection reagent, the vector was introduced into porcine ear skin fibroblasts from newborn piglets. Transfected cells were selected by treatment of $300{\mu}g/ml$ G418 for 12 days. After antibiotic selection, survived colonies with approximately 5mm in diameter were picked and analysed for transgene human HT by PCR. The colonies proven to be human HT transfectants were analysed by RT-PCR to determine their expressions or human HT. In all colonies tested, human HT mRNA was detected. This result demonstrates the establishment of porcine cell lines overexpressing human HT, and these cell lines may be used for the development of transgenic pigs for xenotransplantation.

본 연구는 사람 H-transferase가 과발현하는 돼지 체세포주를 개발하는데 있다. 돼지 세포에 사람 H-transferase 유전자를 발현시키는 것은 이종간 장기 이식에 있어서 초급성 거부 반응을 방지하기 위한 한 가지 방법이다. 본 연구에서는 과발현 벡터를 구축하기 위하여 사람 H-transferase을 HepG2 세포로부터 동정하였으며, 이 유전자를 CMV promoter를 이용하여 발현할 수 있도록 포유동물 발현 벡터인 pRc/CMV벡터에 삽입하였다. 또한, 돼지 산자의 귀 세포를 이용하여 체세포를 수립한 후 jetPEI DNA transfection reagent를 이용하여 벡터를 도입하였고, $300{\mu}g/ml$의 G418로 12일간 선별하였다. PCR을 이용하여 선별된 colony들을 분석한 결과, 벡터가 도입되었음을 확인하였고, RT-PCR을 이용하여 사람 H-transferase mRNA가 발현하는 것을 확인하였다. 본 연구에서 확립된 세포주는 사람 H-transferase가 과발현하는 형질 전환돼지의 생산에 이용될 수 있을 것으로 생각된다.

Keywords

References

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