[Show abstract][Hide abstract] ABSTRACT: 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.
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.
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.
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.
No preview · Article · Mar 2012 · Sichuan da xue xue bao. Yi xue ban = Journal of Sichuan University. Medical science edition
[Show abstract][Hide abstract] ABSTRACT: 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.
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.
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.
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.
No preview · Article · Mar 2010 · Sichuan da xue xue bao. Yi xue ban = Journal of Sichuan University. Medical science edition