Whole-genome sequencing study to identify candidate markers indicating susceptibility to type 2 diabetes in Bama miniature pigs

Background:Hundreds of single-nucleotide polymorphism(SNP)sites have been found to be potential genetic markers of type 2 diabetes mellitus(T2DM).However,SNPs related to T2DM in minipigs have been less reported.This study aimed to screen the T2DM-susceptible candidate SNP loci in Bama minipigs so as to improve the success rate of the minipig T2DM model.Methods:The genomic DNAs of three Bama minipigs with T2DM,six sibling lowsusceptibility minipigs with T2DM,and three normal control minipigs were compared by whole-genome sequencing.The T2DM Bama minipig-specific loci were obtained,and their functions were annotated.Meanwhile,the Biomart software was used to perform homology alignment with T2DM-related loci obtained from the human genome-wide association study to screen candidate SNP markers for T2DM in Bama miniature pigs.Results:Whole-genome resequencing detected 6960 specific loci in the minipigs with T2DM,and 13 loci corresponding to 9 diabetes-related genes were selected.Further,a set of 122 specific loci in 69 orthologous genes of human T2DM candidate genes were obtained in the pigs.Collectively,a batch of T2DM-susceptible candidate SNP markers in Bama minipigs,covering 16 genes and 135 loci,was established.Conclusions:Whole-genome sequencing and comparative genomics analysis of the orthologous genes in pigs that corresponded to the human T2DM-related variant loci successfully screened out T2DM-susceptible candidate markers in Bama miniature pigs.Using these loci to predict the susceptibility of the pigs before constructing an animal model of T2DM may help to establish an ideal animal model.

Heat Stress Upregulates the Expression of TLR4 and Its Alternative Splicing Variant in Bama Miniature Pigs

Alternative splicing is a cellular mechanism in eukaryotes that results in considerable diversity ofgene products. It plays an important role in several diseases and cellular signal regulation. Heat stress is a major factor that induces immunosuppression in pigs. Little is known about the correlation between alternative splicing and heat stress in pigs. Therefore, this study aimed to clone, sequence and quantify the alternative splicing variant of toll-like receptor 4 （TLR4） in Bama miniature pigs （Sus scrofa domestica） following exposure to heat stress. The results showed that the second exon of TLR4 was spliced and 167 bp shorter in the alternative splicing variant, and the protein was putatively identified as a type of truncated membrane protein consisting of extramembrane, transmembrane and intramembrane regions lacking a signal peptide. Further, it was not a non- classical secretory protein. Five potential reference genes were screened for their potential as reliable standards to quantify the expression of TLR4 alternative spliced variants by real-time quantitative reverse-transcriptase polymerase chain reaction （qRT-PCR）. The stability of these reference genes was ranked using the geNorm and NormFinder programs, and ribosomal protein L4 （RPL4） and TATA box-binding protein （TBP） were found to be the two genes showing the most stable expression in the in vitro cultured peripheral blood mononuclear ceils （PBMCs） during heat shock. The mRNA level of the TLR4 gene （both classical and spliced） in stressed pigs increased significantly （P〈0.05）. Further, the expression levels of the alternative spliced variant of TLR4 （TLR4-ASV） showed a 2-3 folds increase in heat-stressed PBMCs as compared to control pigs. The results of the present study suggested that heat shock might modulate the host immune response by regulating the expressions of TLR4 and its alternative splicing variant.

Nucleus Transfer Efficiency of Ear Fibroblast Cells Isolated from Bama Miniature Pigs at Various Ages

Summary： Somatic cell nucleus transfer （SCNT） has been considered the most effective method for conserving endangered animals and expanding the quantity of adult animal models. Bama miniature pigs are genetically stable and share similar biological features to humans. These pigs have been used to establish animal models for human diseases, and for many other applications. However, there is a pan- city of studies on the effect of ear fibroblasts derived from different age of adult Bama miniature pigs on nucleus transfer （NT）. The present study examined the NT efficiency of ear fibroblasts from fetal, new- born, 1-, 2-, 4-, 6-, 12-month-old miniature pigs by using trypan blue staining, flow cytometry and NT technique, etc., and the cell biological function and SCNT efficiency were compared between groups. The results showed that ear fibroblasts grew well after passage in each group. Spindle-shaped cells ini- tially predominated, and gradually declined with increase of culture time and replaced by polygonal cells. Irregular cell growth occurred in the 2-month-old group and the elder groups. The growth curves of the ear fibroblasts were ＂S-shaped＂ in different age groups. The cell proliferation of postnatal ear fi- broblasts, especially those from 2-, 4-, 6-, 12-month-old miniature pigs was significantly different from that of fetus ear fibroblasts （P〈0.05 or P〈0.01）. Two-month- and 4-month-old ear fibroblasts had a sig- nificantly higher proportion of G1 stage cells （85% to 91%） than those at 6 and 12 months （66% to 74%, P〈0.01）. The blastocyst rate of reconstructed embryos originating from newborn, 1-, 2-, 4-month-old donor pigs was 6.06% to 7.69% with no significant difference from that in fetus fibroblast group （8.06%）. It was concluded that 〈4-month-old adult Bama miniature pigs represent a better donor cell resource than elder pigs.

Establishment of a model of Mycoplasma hyopneumoniae infection using Bama miniature pigs

Mycoplasma hyopneumoniae(M.hyopneumoniae),is the primary aetiological agent of enzootic pneumonia leading to chronic respiratory disease prevalent worldwide.Conventional pigs are the only animals used for pathogenicity studies and vaccine evaluations of M.hyopneumoniae.Considering that the challenge animals have better genetic stability and a smaller body size to operate with,an alternative experimental animal model of M.hyopneumoniae infection with Bama miniature pigs was established.Nine seven-week-old snatch-farrowed,porcine colostrum-deprived(SF-pCD)Bama miniature pigs and nine conventional pigs were randomly divided into two infected groups(Bama miniatureinfected(BI)and conventional-infected groups(CI),BI and CI,n=6)and two control groups(Bama miniature control(BC)and conventional control(CC)groups,BC and CC,n=3).Every piglet was tracheally inoculated with 5×10^(8) CCU/mL containing 10%suspension of a stock of frozen lung homogenate from SF-pCD pigs infected with virulent strain JS or sterilized KM2 medium.Typical lung lesions appeared in all infected pigs after necropsy,and the mean gross lung lesions was 17.3 and 13.7 in groups of BI and CI.Serum IgG and nasal sIgA antibody titres were increased significantly.Cilia shedding and mucus staining increased greatly in JS-infected bronchi.Obvious reddish gross lesions and M.hyopneumoniae antigen were detected,especially apparently observed in group of BI.Moreover,DNA copies of M.hyopneumoniae from bronchoalveolar lavage fluid(BALF)of each JS-infected piglet reached more than 10^(8),and M.hyopneumoniae could be re-isolated from each infected BALF.These results indicate that Bama miniature pigs could be used as an alternative and more maneuverable experimental infection model for M.hyopneumoniae and display typical clinical and pathological features consistent with those in conventional pigs.