[Cloning of splicing variants of alpha1,3-galactosyltransferase cDNA of Chinese Banna Minipig inbred line and its expression in human cells].
OBJECTIVE
To study the transfection and expression of the splicing variants of alpha1, 3-galactosyltransferase cDNA of Chinese Banna Minipig Inbred Line (BMI) in human A549 cells.


METHODS
Full length of alpha1,3-GT gene cDNA was amplified by RT-PCR from total RNA of BMI liver tissue and cloned into T-A cloning vector. Two different splicing variants of BMI alpha1,3-GT cDNA were confirmed by sequencing 15 positive clones and inserted respectively into pEGFP-N1 to construct eukaryotic expression vectors pN-GT1 and pN-GT2. The vectors were transfected into human lung adenocacinoma A549 cells and the expression of alpha1,3-GT gene was detected by RT-PCR. The expression of the a-Gal epitopes on transfected cells was confirmed under fluorescent microscope and by flow cytometry using FITC-BS-IB4 lectin. The binding of IgM and complement C3 in human serum to a-Gal on transfected cells were measured by flow cytometry using FITC-anti-IgM and FITC-anti-C3.


RESULTS
There was no other splicing variants of alpha1,3-GT cDNA found in BMI except GT1 and GT2, which were 1116 bp and 1080 bp in length respectively, the latter lacks exon 5. The expression of BMI alpha1,3-GT mRNA and the synthesis of a-Gal on A549 cells transfected with either pN-GT1 or pN-GT2 were detected, and the binding of IgM nature antibodies and complements C3 in human serum on transfected A549 cells were observed. The expression level of alpha-Gal and the deposits of IgM and C3 on transffected cells showed no significant difference between pN-GT1 and pN-GT2.


CONCLUSIONS
The splicing variants of alpha1,3-GT cDNA of BMI could express in human cells, which provide the basis for genetic manipulation of the alpha1,3-GT of BMI for future xenotransplantation studies.
Laboratory of Molecular Diagnosis of Cancer and State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu 610041, China.
Mei
M
Liu
Sheng-Ming
SM
Zhu
Hong
H
Zheng
Yu
Y
Wang
Zhu
Z
Wang
Ya-Jun
YJ
Yang
Yan-Xia
YX
Wu
Yang-Zhi
YZ
Zeng
Yan-Ping
YP
Wang
D000230:Adenocarcinoma / Q000378:metabolism / Q000473:pathology; D000818:Animals; D017849:Animals, Inbred Strains; D001483:Base Sequence; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D005700:Galactosyltransferases / Q000096:biosynthesis / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D007527:Isoenzymes / Q000096:biosynthesis / Q000235:genetics; D008175:Lung Neoplasms / Q000378:metabolism / Q000473:pathology; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D013552:Swine; D013556:Swine, Miniature; D014162:Transfection
D018076:DNA, Complementary; D007527:Isoenzymes; D012333:RNA, Messenger; D005700:Galactosyltransferases; C530267:alpha-1,3-galactosyltransferase 1, porcine