Objectives: 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.
Conclusion: 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.