α1,3-Galactosyltransferase 유전자 좌위에서 Membrane Cofactor Protein을 효과적으로 발현하는 자성 돼지 섬유아세포의 생산

다국어 초록 (Multilingual Abstract)

Xenotransplantation of pig organs into primates results in fatal damage, referred as hyperacute rejection (HAR), and acute humoral xenograft rejection (AHXR), to the organ graft mediated by antibodies pre-existing and newly-producing in primates against their cognate pig antigens. Functional ablation of α1,3-galactosyltransferase (Gal-T KO) of pig which is an enzyme involved in synthesis of Gala1-3Galb1-4GlcNAc-R antigen is essentially required to prevent HAR. Moreover, additional genetic modification under Gal-T KO background for enforced expression of human complement regulatory proteins which can inhibits complement activation is known to effectively imped HAR and AHXR. In this study, we constructed a membrane cofactor protein (MCP) expression cassette under control of human EF1α promoter. This cassette was inserted between homologous recombination regions corresponding to Gal-T locus. Subsequently this vector was introduced into ear skin fibroblasts of female pig by nucleofection. We were able to obtained 40 clones by neomycin selection and 4 clones among them were identified as clones targeted into Gal-T locus of MCP expression cassette by long-range PCR. Real time RT-PCR was shown to down-regulation of Gal-T expression. From these results, we demonstrated human EF1α promoter could induce efficient expression of MCP on cell surface of fibroblasts of female pig.

참고문헌 (Reference)

1 Yang YG, "Xenotransplantation: current status and a perspective on the future" 7 : 519-531, 2007

2 Ekser B, "Xenotransplantation of solid organs in the pig-to-primate model" 21 : 87-92, 2009

3 White DJ, "The control of hyperacute rejection by genetic engineering of the donor species" 9 (9): 185-189, 1995

4 Macher BA, "The Galalpha1,3Galbeta1,4Glc- NAc-R (alpha-Gal) epitope: a carbohydrate of unique evolution and clinical relevance" 1780 : 75-88, 2008

5 Dai Y, "Targeted disruption of the alpha 1,3-galactosyltransferase gene in cloned pigs" 20 : 251-255, 2002

6 Davila E, "T-cell responses during pig-toprimate xenotransplantation" 13 : 31-40, 2006

7 Ahn KS, "Resurrection of an alpha-1,3-galactosyltransferase gene-targeted miniature pig by recloning using postmortem ear skin fibroblasts" 75 : 933-939, 2011

8 Lin CC, "Recipient tissue factor expression is associated with consumptive coagulopathy in pig-to-primate kidney xenotransplantation" 10 : 1556-1568, 2010

9 Nottle MB, "Production of homozygous alpha-1,3-galactosyltransferase knockout pigs by breeding and somatic cell nuclear transfer" 14 : 339-344, 2007

10 McCreath KJ, "Production of gene-targeted sheep by nuclear transfer from cultured somatic cells" 405 : 1066-1069, 2000

11 Kolber-Simonds D, "Production of alpha-1,3-galactosyltransferase null pigs by means of nuclear transfer with fibroblasts bearing loss of heterozygosity mutations" 101 : 7335-7340, 2004

12 Lai L, "Production of alpha-1,3-galactosyltransferase knockout pigs by nuclear transfer cloning" 295 : 1089-1092, 2002

13 Phelps CJ, "Production of alpha 1,3-galactosyltransferase-deficient pigs" 299 : 411-414, 2003

14 Ramsoondar JJ, "Production of alpha 1,3-galactosyltransferase- knockout cloned pigs expressing human alpha 1,2-fucosylosyltransferase" 69 : 437-445, 2003

15 Takahagi Y, "Production of alpha 1,3-galactosyltransferase gene knockout pigs expressing both human decay-accelerating factor and N-acetylglucosaminyltransferase III" 71 : 331-338, 2005

16 Phelps CJ, "Production and characterization of transgenic pigs expressing porcine CTLA4-Ig" 16 : 477-485, 2009

17 Zhou CY, "Production and characterization of a pig line transgenic for human membrane cofactor protein" 9 : 183-190, 2002

18 Ramirez P, "Prevention of hyperacute rejection in a model of orthotopic liver xenotransplantation from pig to baboon using polytransgenic pig livers (CD55, CD59, and H-transferase)" 37 : 4103-4106, 2005

19 Petersen B, "Pigs transgenic for human thrombomodulin have elevated production of activated protein C" 16 : 486-495, 2009

20 Ko N, "Nucleofection- mediated alpha 1,3-galactosyltransferase gene inactivation and membrane cofactor protein expression for pigto- primate xenotransplantation" 24 : 253-267, 2013

21 Sheyn D, "Nonvirally engineered porcine adipose tissue-derived stem cells: use in posterior spinal fusion" 26 : 1056-1064, 2008

22 Dickens S, "Nonviral transfection strategies for keratinocytes, fibroblasts, and endothelial progenitor cells for ex vivo gene transfer to skin wounds" 16 : 1601-1608, 2010

23 Gresch O, "New non-viral method for gene transfer into primary cells" 33 : 151-163, 2004

24 Ekser B, "Impact of thrombocytopenia on survival of baboons with genetically modified pig liver transplants: clinical relevance" 10 : 273-285, 2010

25 Adams DH, "Human membrane cofactor protein (MCP, CD 46) protects transgenic pig hearts from hyperacute rejection in primates" 8 : 36-40, 2001

26 Ekser B, "Genetically-engineered pig-tobaboon liver xenotransplantation: histopathology of xenografts and native organs" 7 : 29720-, 2012

27 Klymiuk N, "Genetic modification of pigs as organ donors for xenotransplantation" 77 : 209-221, 2010

28 Fodor WL, "Expression of a functional human complement inhibitor in a transgenic pig as a model for the prevention of xenogeneic hyperacute organ rejection" 91 : 11153-11157, 1994

29 Macchiarini P, "Evidence of human non-alpha-galactosyl antibodies involved in the hyperacute rejection of pig lungs and their removal by pig organ perfusion" 116 : 831-843, 1998

30 Londrigan SL, "Evaluation of promoters for driving efficient transgene expression in neonatal porcine islets" 14 : 119-125, 2007

31 Liszewski MK, "Emerging roles and new functions of CD46" 27 : 345-358, 2005

32 Nakayama A, "Efficient transfection of primarily cultured porcine embryonic fibroblasts using the Amaxa nucleofection system" 9 : 523-534, 2007

33 Lyons I, "Efficient generation of alpha(1,3) galactosyltransferase knockout porcine fetal fibroblasts for nuclear transfer" 11 : 143-150, 2002

34 Hauschild J, "Efficient generation of a biallelic knockout in pigs using zinc-finger nucleases" 108 : 12013-12017, 2011

35 Skrzyszowska M, "Development of porcine transgenic nuclear-transferred embryos derived from fibroblast cells transfected by the novel technique of nucleofection or standard lipofection" 70 : 248-259, 2008

36 Niemann H, "Cytomegalovirus early promoter induced expression of hCD59 in porcine organs provides protection against hyperacute rejection" 72 : 1898-1906, 2001

37 Miyagawa S, "Complement regulation in the Gal-T KO era" 17 : 11-25, 2010

38 Chen G, "Acute rejection is associated with antibodies to non-Gal antigens in baboons using Galknockout pig kidneys" 11 : 1295-1298, 2005