[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. 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.

Expressed sequence tags analysis of a liver tissue cDNA library from a highly inbred minipig line.

BACKGROUND: Porcine liver performing efficient physiological functions in the human body is prerequisite for successful liver xenotransplantation. However, the protein differences between pig and human remain largely unexplored. Therefore, we investigated the liver expression profile of a highly inbred minipig line. METHODS: A cDNA library was constructed from liver tissue of an inbred Banna minipig. Two hundred randomly selected clones were sequenced then analysed by BLAST programme. RESULTS: Alignments of the sequences showed 44% encoded previously known porcine genes. Among the 56% unknown genes, sequences of 72 clones had high similarities with known genes of other species and the similarities to human were mostly above 0.80. The other 40 clones showing no similarity to genes in National Centre for Biotechnology Information are newly discovered, expressed sequence tags specific to liver of inbred Banna minipig. Twenty-two of the 200 clones had full length encoding regions, 38 complete 5' terminal sequences and 140 complete 3' terminal sequences. CONCLUSION: These newly discovered expression sequences may be an important resource for research involving physiological characteristics and medical usage of inbred pigs and contribute to matching studies in xenotransplantation.

[Quantitative study on copy numbers of porcine endogenous retroviruses in genome of Banna miniature swine inbred line].

OBJECTIVE: To investigate the copy numbers of porcine endogenous retroviruses in genome of Banna miniature swines for screening of donors in xenotransplantation with porcine transplants. METHODS: The genomic DNA of peripheral blood mononuclear cells(PBMCs) of 50 Banna miniature swines in 4 inbred sublines and 5 European Large White Pigs was extracted for the detection to pol, envA, envB, envC and envA/C recombinant in PERVs with real-time quantitative/semi-quantitative PCR and normal PCR. RESULTS: PCR products of pol, envA and envB of PERV could be obtained from all the Banna samples, and the mean amount of these products was 24.2+/-9.4,13.1+/-8.4,16.4+/-9.8 respectively and was significantly different among the inbred herds (P<0.01). The envC and envA/C were not amplified. CONCLUSION: The copy numbers of pol, envA and envB genes of PERV in porcine genome were significantly different among the inbred herds of Banna miniature swines, and envC and envA/C were absent in genome of Banna Miniature Swines.

[Comparison of hepatic protein synthesis function between Banna Minipig Inbred Line and human].

OBJECTIVE: This is a research aimed at comparing the hepatic protein synthesis function of Banna Minipig Inbred Line (BMI) with that of human so as to evaluate the possibility of porcine liver replacement of human counterpart. METHODS: The venous blood samples from BMI and volunteer participants were collected. The albumin and total protein in the separated sera were tested using Beckman delta CX7 autoanalyzer, and serum protein electrophoresis was performed. RESULTS: No significant differences in albumin and in the percentage and concentration of globulins synthesized in liver (alpha1-, alpha2- and beta-globulin) between BMI and human were observed. The percentage and concentration of gamma-globulin synthesized in the lymphocytes of BMI were significantly higher than those of human. CONCLUSION: The above findings suggested the similarity between BMI liver and human liver in respect to albumin and alpha, beta-globulin synthesis.