Reproductive and Developmental Biology

The Korean Society of Animal Reproduction (한국동물번식학회)
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Efficient Control of Human G-CSF Gene Expression in the Primary Culture Cell using a FIV-Tet-On Vector System

FIV-Tet-On Vector System을 이용한 hG-CSF 유전자의 효율적인 발현 조절

  • Kwon, Mo-Sun (Department of Physiology, Catholic University of Daegu School of Medicine) ;
  • Koo, Bon-Chul (Department of Physiology, Catholic University of Daegu School of Medicine) ;
  • Kim, Te-Oan (Department of Physiology, Catholic University of Daegu School of Medicine)
  • 권모선 (대구가톨릭대학교 의과대학 생리학교실) ;
  • 구본철 (대구가톨릭대학교 의과대학 생리학교실) ;
  • 김태완 (대구가톨릭대학교 의과대학 생리학교실)
  • Published : 2007.09.30
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Abstract

In this study, using FIV-based lentivirus vector system, we tried to express hG-CSF in tetracycline-controllable manner. hG-CSF influences the proliferation, differentiation, and survival of cells in the neutrophil lineage. To enhance stability and translation of hG-CSF transcript, WPRE sequence was also introduced into FIV-Tet-On vector at downstream region of either the hG-CSF gene or the sequence encoding rtTA. Primary culture cells (CEF, chicken embryonic fibroblast; PFF, procine fetal fibroblast) infected with the recombinant FIV were cultured in the medium supplemented with or without doxycycline for 48 hours, and induction efficiency was measured by comparing the hG-CSF gene expression level using quantitative real-time PCR, Western blot and ELISA. Higher hG-CSF expression and tighter expression control were observed from the vector in which the WPRE sequence was placed at downstream of the hG-CSF (in CEF) or rtTA (in PEE) gene. This FIV-Tet-On vector system may be helpful in solving serious physiological disturbance problems which has continuously hampered successful production of transgenic animals and gene therapy.

본 연구에서: hG-CSF의 발현을 유도적으로 조절하기 위한 FIV-Tet-On lentivirus vector system을 구축하고자 하였다. hG-CSF는 호중성구 계열 세포의 증식과 분화, 생존에 영향을 미치는 물질로서, 이 유전자의 발현을 증가시키기 위하여 FIV-Tet-On vector 상의 hG-CSF나 $rtTA2^SM2$ 서열의 3' 위치에 WPRE 서열을 도입하였다. 구축된 각각의 vector는 293FT 세포에 일시적으로 transfection하여 virus를 생산하였으며, 이 virus를 일차 배양 세포인 CEF와 PFF에 감염시켰다. 각 세포에 전이되 hG-CSF의 발현 양상을 관찰하기 위하여 doxycycline을 첨가하거나 첨가하지 않은 배지에서 배양한 후 quantitative real-time PCR, Western blot과 ELISA를 이용하여 hG-CSF 유전자의 발현 정도를 비교 측정한 결과, CEF에서는 WPRE가 hG-CSF의 3' 위치에 도입된 경우에 발현량과 유도율이 가장 높은 것으로 나타났고, PFF에서는 rtTA 서열의 3'위치에 도입된 경우에 발현량과 유도율이 가장 큰 것으로 확인되었다. 이 FIV-Tet-On vector system은 형질 전환 동물의 생산이나 유전자 치료에서 문제시되는 외래 유전자의 지속적인 과다 발현에 의한 개체의 생리적인 부작용을 최소화하기 위한 해결 방법으로 제시될 수 있을 것이다.

Keywords

References

  1. Amado RG, Chen IS (1999): Lentiviral vectors-the promise of gene therapy within reach? Science 285: 674-676 https://doi.org/10.1126/science.285.5428.674
  2. Berkowitz R lives H, Lin WY, Eckert K, Coward A, Tamaki S, Veres G, Plavec I (2001): Construction and molecular analysis of gene transfer systems derived from bovine immunodeficiency virus. J Virol 75: 3371-3382 https://doi.org/10.1128/JVI.75.7.3371-3382.2001
  3. Bober LA, Grace MJ, Puliese-Sivo C, Rojas-Triana A, Waters T, Sullivan LM, Narula SK, (1995): The effect of GM-CSF and G-CSF on human neutrophil function. Immunopharmacology 29:111-119 https://doi.org/10.1016/0162-3109(94)00050-P
  4. Bum S, Faucon-Biguet N, Mallet J (2003): Optimization of transgene expression at the posttranscriptional level in neural cells: implication for gene therapy. Mol Ther 7:782-789 https://doi.org/10.1016/S1525-0016(03)00097-2
  5. Curran MA, Ochoa MS, Molano RD, Pileggi A, Inverardi L, Kenyon NS, Nolan GP, Ricordi C, Fenjves ES (2002): Efficient transduction of pancreatic islets by feline immunodeficiency virus vectors. Transplantation 74:299-306 https://doi.org/10.1097/00007890-200208150-00003
  6. Emerman M (1996): From curse to cure: HIV for gene therapy? Nat Bioteclmol 14:943 https://doi.org/10.1038/nbt0896-943
  7. Fabian I, Kletter Y, Bleiberg I, Gadish M, Naparsteck E, Slavin S (1991): Effect of exogenous recombinant human granulocyte and granulocyte-macrophage colony-stimulating factor on neutrophil function following allogeneic bone marrow transplantation. Exp Hematol 19:868-873
  8. Gossen M, Bujard H (1992): Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc Natl Acad Sci USA 89: 5547-5551
  9. Hlavaty J, Schittmayer M, Stracke A, Jandl G, Knapp E, Felber BK, Salmons B, Gunzburg WH, Renner M (2005): Effect of posttranscriptional regulatory elements on transgene expression and virus production in the context of retrovirus vectors. Virology 341:1-11 https://doi.org/10.1016/j.virol.2005.06.037
  10. Johnston JC, Gasmi M, Lim LE, Elder JH, Yee J-K, Jolly DJ, Campbell KP, Davidson BL, Sauter SL (1999): Minimum requirements for efficient transduction of dividing and nondividing cells by feline immunodeficiency virus vectors. J Virol 73:4991-5000
  11. Kafri T, van Praag H, Gage FH, Verma IM (2000): Lentiviral vectors: regulated gene expression. Mol Ther 1:516-521 https://doi.org/10.1006/mthe.2000.0083
  12. Koponen JK, Kankkonen H, Kannasto J, Wirth T, Hillen W, Bujard H, Yla-Herttuala S (2003): Doxycycline-regulated lentiviral vector system with a novel reverse transactivator $rtTA2^S$-M2 shows a tight control of gene expression in vitro and in vivo. Gene Ther 10:459-466 https://doi.org/10.1038/sj.gt.3301889
  13. Lieschke GJ, Burgess AW (1992): Granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor. N Eng J Med 327:28-35 https://doi.org/10.1056/NEJM199207023270106
  14. Livak KJ, Schmittgen TD (2001): Analysis of relative gene expression data using real-time quantitative PCR and the 2 (-Delta Delta C (T) Method. Methods 25:402-408 https://doi.org/10.1006/meth.2001.1262
  15. Loeb JE, Cordier WS, Harris ME, Weitzman MD, Hope TJ (1999): Enhanced expression of transgenes from adeno-associated virus vectors with the woodchuck hepatitis virus posttranscriptional regulatory element: implications for gene therapy. Hum Gene Ther 10:2295-2305 https://doi.org/10.1089/10430349950016942
  16. Loewen N, Bahler C, Teo WL, Whitwam T, Peretz M, Xu R, Fautsch MP, Johnson DH, Poeschla EM (2002): Preservation of aqueous outflow facility after second-generation FIV vector-mediated expression of marker genes in anterior segments of human eyes. Invest Ophthalmol Vis Sci 43:3686-3690
  17. Minvielle-Sebastia L, Keller W (1999): mRNA polyadenylation and its coupling to other RNA processing reactions and to transcription. Curr Opin Cell BioI 11:352-357 https://doi.org/10.1016/S0955-0674(99)80049-0
  18. Mitrophanous K, Yoon S, Rohll J, Patil D, Wilkes F, Kim V, Kingsman S, Kingsman A, Mazarakis N (1999): Stable gene transfer to the nervous system using a non-primate lentiviral vector. Gene Ther 6: 1808-1818 https://doi.org/10.1038/sj.gt.3301023
  19. Miyoshi H, Blomer U, Takahashi M, Gage FH, Verma IM (1998): Development of a self-inactivating lentivirus vector. J Virol 72:8150-5157
  20. Naldini L, Blomer U, Gage FH, Trono D, Verma IM (1999a): Efficient tansfer, integration, and sustained long-term expression of the transgene in adult rat brains injected with a lentiviral vector. Proc Natl Acad Sci USA 93:11382-11388
  21. Naldini L, Blomer U, Gsllay P, Ory D, Mulligan R, Gage FH, Verma IM, Trono D (1996b): In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science 272:263-267 https://doi.org/10.1126/science.272.5259.263
  22. Poeschla EM, Wong-Staal F, Looney DJ (1998): Efficient transduction of nondividing human cells by feline immunodeficiency virus lentiviral vectors. Nat Med 4:354-357 https://doi.org/10.1038/nm0398-354
  23. Price MA, Case SS, Carbonaro DA, Yu XJ, Petersen D, Sabo KM, Curran MA, Engel BC, Margarian H, Abkowitz JL, Nolan GP, Kohn DB, Crooks GM (2002): Expression from second-generation feline immunodeficiency virus vectors is impaired in human hematopoietic cells. Mol Ther 6:645-652 https://doi.org/10.1016/S1525-0016(02)90725-2
  24. Ramenzani A, Hawley TS, Hawley RG (2000): Lentiviral vectors for enhanced gene expression in human hematopoietic cells. Mol Ther 2:458-467 https://doi.org/10.1006/mthe.2000.0190
  25. Reiser J, Harmison G, Kluepfel-Stahl S, Brady RO, Karlsson S, Schubert M (1996): Transduction of nondividing cells using pseudotyped defective hightiter HIV type 1 particles. Proc Natl Acad Sci USA 93: 15266-15271
  26. Romano G (2005): Current development of lentiviral-mediated Gene Transfer. Drug News & Perspectives. 18: 128-134 https://doi.org/10.1358/dnp.2005.18.2.886481
  27. Saenz DT, Poeschla EM (2004): FIV: from lentivirus to lentivector. J Gene Med 6:595-5104 https://doi.org/10.1002/jgm.627
  28. Sauter SL, Gasmi M (2001): FIV vector systems. Somat Cell Mol Genet 26:99-129 https://doi.org/10.1023/A:1021078714105
  29. Song JJ, Lee B, Chang JW, Kim J-H, Kim Kwon Y, Lee H (2003): Optimization of vesicular stomatits-G pseudotyped feline immunodeficiency virus vector for minimized cytotoxicity with efficient gene transfer. Virus Res 93:25-30 https://doi.org/10.1016/S0168-1702(03)00047-9
  30. Soriano P, Friedrich G, Lawinger P (1991): Promoter interactions in retrovirus vectors introduced into fibroblasts and embryonic stem cells. J Virol 65:2314-2319
  31. Stein CS, Davidson BL (2002): Gene transfer to the brain using feline-immunodeficiency virus-based lentivirus vectors. Methods Enzymol 346:433-454 https://doi.org/10.1016/S0076-6879(02)46070-3
  32. Urlinger S, Baron U, Thellmann M, Hasan MT, Bujard H, Hillen W (2000): Exploring the sequence space for tetracycline-dependent transcriptional activators: novel mutations yield expanded range and sensitivity. Proc Natl Acad Sci USA 97:7963-7968
  33. Wang G, Slepushkin V, Zabner J, Keshavjee S, Johnston JC, Sauter SL, Jolly DJ, Dubensky TW, Davidson BL, McCray PB (1999): Feline immunodeficiency virus vectors persistently transduce nondividing airway epithelia and correct the cystic fibrosis defect. J Clin Invest 104:R55-R62 https://doi.org/10.1172/JCI8390
  34. Zufferey R Donello JE, Trono D, Hope TJ (1999): Woodchuck hepatitis virus posttranscriptional regulatory element enhances expression of transgenes delivered by retroviral vectors. J Virol 73:2886-2892