Identification of the miniature pig inbred line by skin allograft

Skin grafting has been used as one of the most reliable tests to determine the genetic stability of laboratory animal such as mice and rats inbred line, but no identification of swine inbred lines by skin grafting has been reported. At present, Wuzhishan miniature pig （WZSP） inbred line has acquired the F24 individuals in China. In order to verify whether WZSP inbred line had D^en cultivated successfully, allogeneic skin grafts and related research were performed on F20 individuals of WZSP inbreeding population, compared with a control group of autologous transplantation. We observed the transplant recipients＇ wounds, detected peripheral blood-related indicators interleukin-2, 4 and 10, CD4~ and CD8~ lymphocytes, and conducted hematoxylin-eosin （HE） and Masson＇s staining of skin to judge whether the immune rejection reactions occurred within 28 days after transplantation. Chr. 7 genomic heterozygosity of 48 WZSP individuals from F20 to F22 was analyzed by high-density single nucleotide polymorphism （SNP） chips （60 000 SNPs）. The result showed that there were no significant differences in graft skin, the plasma interleukin-2, 4, 10, CD4~ and CD8~, HE and Masson＇s staining results between the allograft and autograft groups, and no immune rejection occurred on the allograft group. We found that 11 genes in Chr. 7 of major histocompatibility complex （MHC） I and MHC II were homozygous which confirmed that immune antibody of the allograft and autograft groups were highly identical and also provided a theoretical basis to no immune rejection occurred on the allograft in the inbred WZSP. The result proved that the WZSP inbred line had been cultivated successfully for the first time in the world. The test methods also provide a scientific basis for the identification of swine and mammal inbred lines.

Study of Swine Leukocyte Antigen Class I-3 (SLA-3) Gene for Inbreeding Wuzhishan Pig

To elucidate the structure of SLA-3 alleles on inbred line of Wuzhishan pig （WZSP） population, we examined the partial exon 1, completed exon 2, and partial exon 3 of SLA-3 loci using the reverse transcription-polymerase chain reaction （RT- PCR） and the sequencing-based method in 32 WZSPs. According to pedigree and amplification results, PCR products of 8 WZSPs were selected to clone and sequence. Nine different nucleotide sequences were obtained. After comparing the DNA and protein sequences of the WZSPs SLA-3 alleles with the published GenBank SLA sequences, it was found that the SLA-3 alleles in WZSPs were all novel, but there were very few variations among them. Comparision of SLA-3 and HLA-A protein sequences indicated that there was more sequence homology. Meanwhile, the construction of a phylogenetic tree using the nucleotide sequences of 23 SLA-3 alleles and 1 HLA-A allele represented that the WZSP population owns its unique genetics resource. In this study, the alleles of SLA-3 on WZSP group were successfully detected and analyzed, which provided the firm basis on the genotype of SLA-3 for breeding specific haplotypes WZSPs.

Analysis of SNPs in the Promoter Region of the Growth Hormone(GH) Gene in Mini-pigs

Single nucleotide polymorphisms(SNPs) of the growth hormone(GH) gene were investigated in six pig breeds,consisting of four mini-pig breeds(Wuzhishan,Bama,Xiang and Tibet pig),and two others(Dahlan and Landrace pig).Three pairs of primers for promoter regions of the GH gene were designed on the basis of the pig genomic sequence and SNPs were detected by the PCR-SSCP method.The results indicated three mutations in the 5’-flanking region.The analysis results showed that the frequencies of allele A and D in four mini-pig breeds were higher than that in other breeds at a locus within the 5’-flanking region(P【0.05).These results suggest that differences in body size may be associated with these SNPs of 5’-flanking region and amino acid mutation of the signal peptide of GH in these pig breeds.