Journal of Aquaculture (한국양식학회지)

The Korean Society of Fisheries and Aquatic Science (한국양식학회)
권호 표지

Activity Analysis of Misgurnus mizolepis Experssion Vector

미꾸라지 발현백터의 활성도 조사

  • Published : 1998.11.01
Download PDF
⟨ Previous Next ⟩

Abstract

An expression vector, pUC19N6-luc, containing nuclear matrix attachment region(MAR) isolated from Misgurnus mizolepis liver and control expressino vector, pUC19-luc, were constructed. After these vectors were transferred into CHSE-214 cell line by electroporation, the expression rate of luckferase gens, copy number of vectors and chromosome integration of vectors were analyzed by using assay of luciferase activity, PCR and Southern blotting. While the expression pattern of luciferase gene of pUC19-luc was shown in typicla transient ecpression pattern, that of pUC19N6-luc was highly increased at the 5 days after transfectrion. Although the cope number of pUC19N6-luc vector was higher than that of pUC19-luc vector, these vectors were integrated into chromosome at the same time point in the transfected CHSE-214 cells. In conclusion, the increase of luciferase gene expression of pUC19N6-luc was resulted from not the maintaining of the high copy number but the formation of transcription-favorable structure by MAR effect after chromosomal integration.

미꾸라지(Misgrunus mizolepis)의 DNA로부터 클론된 핵기질 부착부위(MAR)를 포함하는 발현벡터인 pUC19N6-luc 벡터를 구성하였다. 이를 물고기 CHSE-214 세포주에 electroporation으로 transfection 시킨 후 유전자의 발현율, 벡터의 copy 수 및 염색체내 삽입 양상을 luciferase 활성도 분석, PCR 및 Southern blotting를 통해 분석하였다. 대조군 발현벡터에 luciferase 유전자는 전형적인 transient 발현양상을 나타내는데 비해, 미꾸라지 MAR가 포함된 pUC19N6-luc 벡터의 luciferase 유전자의 발현은 transfection 후 5일째부터 급격히 증가하는 양상을 보였다. Transfection된 CHSE-214 세포내에서 pUC19N6-luc 벡터는 대조군 벡터에 비해 높은 copy 수를 유지하였으나, 염색체내 삽입은 거의 비슷한 시간에 일어났다. 결론적으로 transfection 후 시간경과에 따른 pUC19N6-luc 벡터내의 luciferase 유전자의 발현 증가에 미치는 MAR의 효과는 벡터 copy수 증가 때문이 아니라, 염색체내 삽입후 형성되는 전사활성구조의 형성에 기인하는 것으로 판단된다.

Keywords

References

  1. Transgenic models in medicine and agriculture Chourrout, D.;R. Guyomard;L. M. Houdebine;R. Church(ed.)
  2. Nature v.371 Extraordinary salmon growth Devlin, R. H.;T. Y. Yesaki;C. A. Biagi;E. M. Donaldson
  3. J. Biol. Chem. v.260 The relation between DNA synthesis and DNA polymerases bound to the nuclear matrix in synchronized HeLa cells Foster, K. A.;J. M. Collins
  4. Experientia v.47 Transgenesis in fish Houdebine, L. M.;D. Chourrout
  5. Kor. J. of Aquaculture v.8 Cloning and Characterization of Replication Origins from Misgurnus mizolepis Lim, H. S.;M. S. Kim;H. H. Lee
  6. Plant. Cell Physiol. v.39 The influences of two plant nuclear matrix attachment regions (MARs) on expression in transgenic plants Liu, J. W.;L. M. Tabe
  7. Science v.250 A view of interphase chromosomes Manuelidis, L.
  8. Exp. Cell Res. v.180 Supercoiled loop organization of genomic DNA : A close relationship between loop domains, expression units and replicon organization in rDNA in Xenopus laevis Marilley, M.;G. Gassend-Bonnet
  9. Biochemistry v.29 Hierarchical binding of DNA fragments derived from scaffold-attached region : Correlations of properties in vitro and function in vivo Mielke, C.;Y. Kohwi;T. Kohwi-Shigematsu;J. Bode, J.
  10. Histochemistry v.98 Nuclear Matrix Bound Replicational Sites Detected In Situ by 5-Bromodeoxyuridine Neri, L. M.;G. Mazzoggi;S. Capitani;N. M. Maraldi;C. Cinti;N. Baldini;R. Rana;A. M. Martelli
  11. Mol. Cell. Biol. v.10 The chicken lysozyme 5' matrix attachment region increases transcription from a heterologous promoter in heterologous cells and dampens position effects on expression of transfected genes Phi-Van, L.;von Kries, J. P.;Ostertag, W.;Str tling, W. H.
  12. Science v.246 Fish as model systems Powers, D.
  13. Ann. Rev. Biochem. v.65 DNA topoisomerase Wang, J. C.
  14. Gene v.139 A 5' beta-globin matrix-attachment region and the polyoma enhancer togather confer position-independent transcription Yu, J.;J. H. Bock;J. L. Slightom;B. Villeponteau
  15. 한국양식학회지 v.9 미꾸라지의 복제원점에 대한 특성 및 구조 분석 임학섭;김무상;석영선;박상대;이형호