[The porcine alpha1, 3 galactosyltransferase gene siRNA targeted heterozygous hepatocyte negative express GT].
OBJECTIVE
To study whether the porcine alpha1, 3 galactosyltransferase gene siRNA targeted heterozygous hepatocyte negatively expresses GT mRNA and resists to the cytotoxicity of nature antibody in human serum.


METHODS
The porcine alpha1, 3 galactosyltransferase gene siRNA targeted vector (pPNTloxPGTsiRNA) were construct with pPNTloxPGT and pMXSV/U6 vector. Positive-negative selection was used to produce a heterozygous pPNTloxPGTsiRNA knockout (+/-) clone. The GT mRNA expressions were detected with northern blot. Complement-mediated NAb cytotoxicity after incubation of hepatocytes with NAbs and complement was determined using 3- (4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium(MTS, tetrazolium salt) colorimetric assay.


RESULTS
The pPNTloxPGTsiRNA targeted porcine hepatocyte (+/-) negative express GT mRNA. Only 14% to 18% cytotoxicity can be detected at the highest serum concentration. The pPNTloxPGT targeted porcine hepatocyte (+/-) express GT mRNA just as the wild type porcine cells and the cytotoxicity are 77% to 83%.


CONCLUSIONS
The porcine a1, 3 galactosyltransferase gene siRNA targeted heterozygous hepatocyte (+/-) negative express GT and resisted to nature antibody in human serum.
Yan
Quan-jian
QJ
Urology institute of Fudan University, Urology department of Huashan Hospital, 12 Wulumuqizhonglu, Shanghai 200040, China.
Zhang
Yuan-fang
YF
Yang
Jie
J
Ding
Qiang
Q
Wang
Ji-gong
JG
Jiang
Hao-wen
HW
Zhao
Hong
H
Xu
Ke
K
Gong
Jian
J
Li
Liu-jin
LJ
Liu
Cheng-hai
CH
Guo
Jin-long
JL
Liu
En-jing
EJ
D000818:Animals; D002478:Cells, Cultured; D003001:Cloning, Molecular; D003602:Cytotoxicity, Immunologic / Q000235:genetics; D005700:Galactosyltransferases / Q000096:biosynthesis / Q000235:genetics*; D020868:Gene Silencing; D018390:Gene Targeting / Q000379:methods; D022781:Hepatocytes / Q000166:cytology / Q000378:metabolism*; D006579:Heterozygote; D007108:Immune Tolerance / Q000235:genetics; D007694:Killer Cells, Natural / Q000276:immunology*; D009154:Mutation; D034741:RNA, Small Interfering / Q000096:biosynthesis / Q000235:genetics; D013552:Swine; D014162:Transfection
D034741:RNA, Small Interfering; D005700:Galactosyltransferases; C047006:N-acetyllactosaminide alpha-1,3-galactosyltransferase