Reproductive and Developmental Biology

The Korean Society of Animal Reproduction (한국동물번식학회)
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Effects of the cis-Acting Element in the 3' End of Porcine $\beta$-Casein Gene on the Expression in Mammary Epithelial Cells

돼지 $\beta$-Casein 유전자의 3' 말단 부위의 cis-Acting Element가 유선 상피 세포내의 발현에 미치는 영향

  • Lee, Hwi-Cheul (Animal Biotechnology Division, National Livestock Research Institute, RDA) ;
  • Kim, Byoung-Ju (Animal Biotechnology Division, National Livestock Research Institute, RDA) ;
  • Byun, Sung-June (Animal Biotechnology Division, National Livestock Research Institute, RDA) ;
  • Lee, Seung-Hoon (Animal Biotechnology Division, National Livestock Research Institute, RDA) ;
  • Kim, Min-Ji (Animal Biotechnology Division, National Livestock Research Institute, RDA) ;
  • Chung, Hee Kyoung (Animal Biotechnology Division, National Livestock Research Institute, RDA) ;
  • Lee, Hyun-Gi (Animal Biotechnology Division, National Livestock Research Institute, RDA) ;
  • Jo, Su-Jin (Animal Biotechnology Division, National Livestock Research Institute, RDA) ;
  • Chang, Won-Kyong (Animal Biotechnology Division, National Livestock Research Institute, RDA) ;
  • Park, Jin-Ki (Animal Biotechnology Division, National Livestock Research Institute, RDA) ;
  • Lee, Poong-Yeon (Animal Genetic Resources Station, National Institute of Animal Science, RDA)
  • 이휘철 (농촌진흥청 축산과학원 응용생명공학과) ;
  • 김병주 (농촌진흥청 축산과학원 응용생명공학과) ;
  • 변승준 (농촌진흥청 축산과학원 응용생명공학과) ;
  • 이승훈 (농촌진흥청 축산과학원 응용생명공학과) ;
  • 김민지 (농촌진흥청 축산과학원 응용생명공학과) ;
  • 정희경 (농촌진흥청 축산과학원 응용생명공학과) ;
  • 이현기 (농촌진흥청 축산과학원 응용생명공학과) ;
  • 조수진 (농촌진흥청 축산과학원 응용생명공학과) ;
  • 장원경 (농촌진흥청 축산과학원 응용생명공학과) ;
  • 박진기 (농촌진흥청 축산과학원 응용생명공학과) ;
  • 이풍연 (농촌진흥청 축산과학원 가축유전자원시험장)
  • Published : 2008.09.30
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Abstract

Tissue-specific and temporal regulation of milk protein gene expression is advantageous when creating transgenic animal that produces foreign protein into milk. Gene expression, i.e. protein production, is regulated not only by promoter strength but also mRNA stability. Especially, poly A tail length by polyadenylation affects in vivo and in vitro mRNA stability and translation efficiency of the target gene. In the present study, nucleotide sequence of 3'-UTR was analyzed to evaluate the effects of mRNA stability on the target gene expression. Based on the poly A signal of 3' -untranslated region (UTR), nucleotide sequences of putative cytoplasmic polyadenylation elements (CPEs) and downstream elements (DSEs: U-rich, G-rich, GU-rich) were analyzed and used to construct 15 luciferase reporter vectors. Each vector was transfected to HC11 and porcine mammary gland cell (PMGC) and measured for dual luciferase expression levels after 48 hours of incubation. Luciferase expression was significantly higher in construct #6 (with CPE 2, 3 and DSE 1 of exon 9) and #11 (with CPE 2, 3 and DSE 1, 2 and 3 of exon 9) than construct #1 in the PMGC. These results suggest that expression of target genes in PMGC may be effectively expressed by using the construct #6 and #11 on production of transgenic pig.

형질 전환 동물 생산에는 조직 및 시기 특이적 발현 조절이 가능하다는 장점 때문에 유즙 내로 외부 유전자를 발현시키는 시스템이 널리 이용되고 있다. 유전자 발현 즉, 단백질 생산은 프로모터의 강도뿐만 아니라 mRNA의 안정성에 의해서도 조절된다. 특히, polyadenylation에 의한 poly A의 길이는 in vivo와 올 in vitro에서 mRNA 안정성 및 목적 유전자의 번역효율에 영향을 준다. 본 연구에서는 이러한 mRNA 안정성이 목적 유전자의 발현에 미치는 영향을 알아보기 위해 3'-UTR 염기 서열을 분석하였다. 이 3'-untranslated region(UTR) 내의 poly A signal을 기준으로 putative cytoplasmic polyadenylation element(CPE) 부위와 downstream elements(DSE: U-rich, G-rich, GU-rich)의 염기 서열을 분석하고, 각각의 element를 기준으로 15 종의 luciferase reporter vector를 제작하여, 생쥐 유선 세포주(HC11)와 돼지 유선 세포주(PMGC)에 각각 transfection시킨 후 48시간 동안 배양하고 luciferase 발현량을 분석하였다. PMGC의 경우, luciferase의 발현은 exon 9의 CPE 2,3 및 DSE 1을 포함한 #6 construct에서 유의적으로 높은 발현량을 보였으며, exon 9의 CPE 2, 3과 DSE를 모두 포함하고 있는 #11 construct에서도 유의적으로 높은 발현량을 보였다. 이러한 결과는 형질 전환 돼지 생산에 있어 #6 및 11 construct의 사용은 목적의 유전자를 효과적으로 발현시키는데 기여할 것으로 사료된다.

Keywords

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