[Establishment and identification of human lung adenocarcinoma cell line stably expressing the alpha1, 3-galaetosyltransferase gene from pig].
OBJECTIVE
To establish a human lung adenocarcinoma cell subline A549 that can stably express the Chinese Banna minipig inbred-line (BMI) alpha1 ,3-galactosyltransferase (alpha1 ,3GT) gene and alpha-galactosyl (Gala1-3Galb1-4GlcNAc-R, alpha-gal) epitopic, providing a cell model which expressed xenotransplantation antigens for the further research on the effect of complement dependent cytotoxic lysis of the tumor cells triggered by human natural serum.


METHODS
The pEGFP-CMV-GT plasmid containing Banna minipig alpha1 ,3-GT gene was ransfected into A549 cells with lipofectin in vitro. After screened with G418,the single clones were got out and then amplified, the stable transfected cells was named A549-GT. The transcription of alpha1, 3-GT gene in A549-GT cells was detected by RT-PCR. Direct immunofluonrescence methods and flow cytometer were performed to observe the expression of alpha-gal and the binding conditions of IgM and complement C3 in human serum on A549-GT cells. The biological characters of A549-GT cells including morphology, proliferation, and tumorigenesis in nude mice were also examined.


RESULTS
After G418 screening, A549-GT that stablely transfected with alpha1, 3-GT gene was obtained and has been passaged for 2 years. The expression of alpha1,3-GT mRNA and alpha-gal was detected continuously and stably in A549-GT. The expression rate of alpha-gal positive cells reached 80.1% +/- 3.2%. The binding of human serum IgM and C3 in human serum on A549-GT cells were founded. Compared with parental A549 cells, its biological characteristics did not change.


CONCLUSIONS
A549-GT cell line stably and continuously expressing alpha1, 3-GT and alpha-gal was established successfully. It provided a useful cell model for the further study of pig alpha1,3-GT gene in tumor immunotherapy.
Qin
Feng
F
Laboratory of Molecular Diagnosis of Cancer and State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu 610041, China.
Zhu
Sheng-ming
SM
Zheng
Hong
H
Wang
Zhu
Z
Wang
Yu
Y
Zuo
Ying
Y
Chen
Juan
J
Sun
Wei-liang
WL
Wang
Yan-ping
YP
D000230:Adenocarcinoma / Q000276:immunology / Q000378:metabolism / Q000473:pathology*; D000818:Animals; D045744:Cell Line, Tumor; D005700:Galactosyltransferases / Q000096:biosynthesis* / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D008175:Lung Neoplasms / Q000276:immunology / Q000378:metabolism / Q000473:pathology*; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D013552:Swine; D013556:Swine, Miniature; D014162:Transfection; D014312:Trisaccharides / Q000096:biosynthesis / Q000235:genetics
D012333:RNA, Messenger; D011994:Recombinant Proteins; D014312:Trisaccharides; C076569:alpha-galactosyl epitope; D005700:Galactosyltransferases; C530267:alpha-1,3-galactosyltransferase 1, porcine