[Cloning of splicing variants of alpha1,3-galactosyltransferase cDNA of Chinese Banna Minipig inbred line and its expression in human cells].
Sichuan Da Xue Xue Bao Yi Xue Ban
; 43(2): 145-50, 2012 Mar.
Article
en Zh
| MEDLINE
| ID: mdl-22650019
ABSTRACT
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.CONCLUSION:
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.
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Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
Transfección
/
Galactosiltransferasas
Límite:
Animals
/
Humans
Idioma:
Zh
Revista:
Sichuan Da Xue Xue Bao Yi Xue Ban
Año:
2012
Tipo del documento:
Article
País de afiliación:
China