Genome-wide single nucleotide polymorphism array and whole-genome sequencing reveal the inbreeding progression of Banna minipig inbred line.

Inbred pigs are promising animal models for biomedical research and xenotransplantation. Established in 1980, the Banna minipig inbred (BMI) line originated from a sow and its own male offspring. It was selected from a small backcountry minority Lahu village, where records show that no other pig breed has ever been introduced. During the inbreeding process, we perfomed extreme inbreeding over 23 consecutive generations using full-sibling or parent-offspring mating. In order to investigate the inbreeding effects in BMI pigs across generations over the past 40 years, in this study we conducted a genome-wide SNP genotyping of the last 10 generations, representing generations 14-23. In total, we genotyped 57,746 SNPs, corresponding to an average decrease in heterozygosity rate of 0.0078 per generation. Furthermore, we were only able to identify 18,216 polymorphic loci with a MAF larger than 0.05, which is substantially lower than the values in previous reports on other pig breeds. In addition, we sequenced the genome of the first pig in the twenty-third generation (inbreeding coefficient 99.28%) to an average coverage of 12.4× to evaluate at the genome level the impact of advanced inbreeding. ROH analysis indicates that BMI pigs have longer ROHs than Wuzhishan and Duroc pigs. Those long ROH regions in BMI pigs are enriched for distinct functions compared with the highly polymorphic regions. Our study reveals a genome-wide allele diversity loss during the progress of inbreeding in BMI pigs and characterizes ROH and polymorphic regions as a result of inbreeding. Overall, our results indicate the successful establishment of the BMI line, which paves the way for further in-depth studies.

Cloning, sequence characterization, and expression patterns of members of the porcine TSSK family.

Testis-specific serine kinases (TSSKs) are a family of serine/threonine kinases highly expressed in the testes that are responsible for regulating many spermatogenesis-related protein activities. Mutations in this family have a positive relationship with oligospermia and azoospermia in human and mouse. Here, five members of the TSSK family from a Banna mini-pig inbred line (BMI) were cloned, sequenced, and characterized. The full-length coding sequences of BMI TSSKs varied from 807 (TSSK3) to 1095 bp (TSSK1) and encoded 268 to 364 amino acids with molecular weights in the range 30.11 to 41.34 kDa. Following comparison with TSSK4 genes in other species, BMI TSSK4 was found to contain three alternatively spliced variants, inform1, inform 3, and inform 4. BMI TSSK1 and TSSK2 are co-localized on the Sus scrofa chromosome (SSC) 14, and consist of a single exon; TSSK3, TSSK4, and TSSK6 are on SSC6, SSC7, and SSC2, and consist of two, four, and one exon, respectively. Multiple protein sequence alignment and phylogenetic analysis showed that the regions spanning the S_TKc domains were more conserved between pig and other animals: with TSSK1 and TSSK2 and TSSK3 and TSSK6 displaying the greatest degree of homology across species, and the TSSK4 protein clearly distinct from other members. Multi-tissue RT-PCR showed BMI TSSK1, TSSK3, and TSSK4 were only expressed in the testes and seminal vesicle, TSSK2 was confined to testes only, while TSSK6 was expressed widely in adult tissues but was highest in the testes.

Complementary DNA cloning, sequence analysis, and tissue transcription profile of a novel U2AF2 gene from the Chinese Banna mini-pig inbred line.

U2 small nuclear RNA auxiliary factor 2 (U2AF2) is an important gene for pre-messenger RNA splicing in higher eukaryotes. In this study, the Banna mini-pig inbred line (BMI) U2AF2 coding sequence (CDS) was cloned, sequenced, and characterized. The U2AF2 complete CDS was amplified using the reverse transcription-polymerase chain reaction (RT-PCR) technique based on the conserved sequence information of cattle and known highly homologous swine expressed sequence tags. This novel gene was deposited into the National Center for Biotechnology Information database (Accession No. JQ839267). Sequence analysis revealed that the BMI U2AF2 coding sequence consisted of 1416 bp and encoded 471 amino acids with a molecular weight of 53.12 kDa. The protein sequence has high sequence homology with U2AF65 of 6 species - Homo sapiens (100%), Equus caballus (100%), Canis lupus (100%), Macaca mulatta (99.8%), Bos taurus (74.4%), and Mus musculus (74.4%). The phylogenetic tree analysis revealed that BMI U2AF65 has a closer genetic relationship with B. taurus U2AF65 than with U2AF65 of E. caballus, C. lupus, M. mulatta, H. sapiens, and M. musculus. RT-PCR analysis showed that BMI U2AF2 was most highly expressed in the brain; moderately expressed in the spleen, lung, muscle, and skin; and weakly expressed in the liver, kidney, and ovary. Its expression was nearly silent in the spinal cord, nerve fiber, heart, stomach, pancreas, and intestine. Three microRNA target sites were predicted in the CDS of BMI U2AF2 messenger RNA. Our results establish a foundation for further insight into this swine gene.

Single nucleotide polymorphism analysis of exons 3 and 4 of IGF-1 gene in pigs.

The IGF-1 gene has been implicated as a candidate gene for the regulation of pig growth traits. We analyzed exons 3 and 4 of IGF-1 gene polymorphisms of the Banna mini-pig (28), the Tibetan mini-pig (30), the Junmu pig (55), and L. Yorkshire species (50) using PCR-SSCP. Three genotypes in exon 3 and 6 genotypes in exon 4 were observed, among which, one single nucleotide polymorphism, G201A, on exon 3 and two single nucleotide polymorphisms, A440G and T455C, on exon 4 were found. Statistical analysis of genotype frequencies revealed that the A allele was dominant in the large pig at the G201A locus (PIC = 0.20-0.34), and the AT alleles were dominant in the large pig at the A440G and T455C loci (PIC = 0.30-0.60). The genotype distribution between the various groups was significantly different (P< 0.01), with the highest heterozygosity seen in Junmu pigs at 0.223 and the lowest seen in L. Yorkshire at 0.098. The genetic distance of the Junmu pig from the L. Yorkshire is the smallest, the distance from the Tibetan miniature pigs is larger, and the distance from the Banna mini-pig is the largest. The IGF-1 gene polymorphism and heterozygosity results from various pig breeds indicate that IGF-1 is substantially polymorphic with significant difference of the polymorphic distribution and expression levels among various pig breeds. This information provides a theoretical basis for the genetic background of miniature pigs but also provides means to breed improved pig varieties.

Immunogenicity and immune modulation of osteogenic differentiated mesenchymal stem cells from Banna minipig inbred line.

Mesenchymal stem cells (MSCs) represent a good choice for cell transplantation due to their multilineage differentiation ability and low immunogenicity. Our previous in vitro studies indicated that undifferentiated swine MSCs show low immunogenicity suppressing the proliferative responses of human peripheral blood lymphocyte to several antigens. In this study, we investigated the immunogenicity and immune modulation ability of osteogenic differentiated MSCs. SLA class I (P1, P14) was detectable by reverse-transcriptase polymerase chain reaction on both differentiated and undifferentiated MSCs. SLA class II (SLA-DRA, SLA-DQA) was only detectable on differentiated MSCs mixed lymphocyte reaction assays demonstrated that both differentiated and undifferentiated MSCs failed to stimulate proliferative responses by human peripheral blood lymphocytes (hPBLs). Furthermore, as undifferentiated MSCs, osteogenic differentiated MSCs also suppressed hPBL proliferation to phytohemaglutinin.

Suppression of human peripheral blood lymphocyte proliferation by immortalized mesenchymal stem cells derived from bone marrow of Banna Minipig inbred-line.

This study sought to investigate whether mesenchymal stem cells (MSC) derived from Banna Minipig Inbred-line (BMI-MSC) suppressed human peripheral blood lymphocyte (hPBLs) proliferation in a one-way mixed lymphocyte reaction system. BMI-MSC failed to stimulate proliferative responses by hPBLs, which were activated by allogenic endothelial cells, BMI-PBLs and non-specific mitogenic stimuli. Furthermore, BMI-MSC also suppressed proliferation of hPBLs stimulated by mismatched allogenic, as well as xenogenic PBLs, and the mitogenic stimulus ConA. The suppression occurred in dose-dependent fashion when the ratio of hPBLs to BMI-MSC varied from 1 to 5 fold; fewer, BMI-MSC (0.001 to 0.01 times) showed no obvious suppression. When BMI-MSC were added to hPBLs stimulated for 72 hours, the proliferative suppression was still evident. Addition of anti-FasL or anti-TGF-beta1 antibody attenuated the proliferative suppression, while antibody against IL-10 had no effect on it. Further immunofluorescence analysis demonstrated that FasL and TGF-beta1 constitutively expressed BMI-MSC. These findings suggest that BMI-MSC suppress hPBLs proliferation relying on FasL and TGF-beta1 mediated pathways.

Prediction of apatite lattice constants from their constituent elemental radii and artificial intelligence methods.

Lattice constants (LCs) of all possible 96 apatite compounds, A(5)(BO(4))(3)C, constituted by A[double bond]Ba(2+), Ca(2+), Cd(2+), Pb(2+), Sr(2+), Mn(2+); B[double bond]As(5+), Cr(5+), P(5+), V(5+); and C[double bond]F(1-), Cl(1-), Br(1-), OH(1-), are predicted from their elemental ionic radii, using pattern recognition (PR) and artificial neural networks (ANN) techniques. In particular, by a PR study it is demonstrated that ionic radii predominantly govern the LCs of apatites. Furthermore, by using ANN techniques, prediction models of LCs a and c are developed, which reproduce well the measured LCs (R(2)=0.98). All the literature reported on 30 pure and 22 mixed apatite compounds are collected and used in the present work. LCs of all possible 66 new apatites (assuming they exist) are estimated by the developed ANN models. These proposed new apatites may be of interest to biomedical research especially in the design of new apatite biomaterials for bone remodeling. Similarly these techniques may also be applied in the study of interface growth behaviors involving other biomaterials.

[Genetic analysis of 35 microsatellite loci in 5 lineages of xishuangbanna miniature pig inbred line].

The polymorphism of 35 microsatellites in 5 lineages of Xishuangbanna small-ear miniature pig inbred line (XMI) was analysed. Nmuber of alleles of each lineage was counted, and rates of homozygote for 35 microsatellite loci in 5 lineages were calculated. According to gene frequencies of 35 microsatellites polymorphism information content (PIC) and mean heterozygosity were calculated for each lineage, and genetic distances between these lineage were estimated. The dendrograms were obtained based on genetic distances. The results suggest that rates of homozygote in these lineage are all high, and that is the highest in lineage 151. The results also suggest that polymorphism information content and mean heterozygosity in all the lineages are low. Composition of alleles of each lineage was quite different and the genetic relationship between lineages accorded with the process of inbred line. So it is suggested that the inbreeding degree of 5 lineages of XMI are all high, and the richness of genetic diversity is lower than general commercial pig breeds. It also shows each lineage has been different groups with individual genes.

[The RAPD analysis of haploid strain of thermotolearnt yeast].

The haploid strains HZ line from the thermololeant strain HU-TY-1 of Saccharomyces cerevisiae have been obtained by sporulation, and the analysis of growth and fermentation test was performed. The strains HZ-21 and HZ-84 were used as the analysis of random amplified polymerphic DNA (RAPD). The result showed that there were some polymorphic DNA fragments of genomic DNA among haploid, the diploid parent strain HU-TY-1 and the original strain LK, some of them may be correlation with thermotolerant property.

Functional consequences of the Ser334-->Pro mutation in a human factor X variant (factor XMarseille).

A factor X molecular variant was identified in a 55-year-old woman at a routine preoperative coagulation screening. Plasma factor X antigen was normal, whereas factor X activity was decreased when factor X was activated by either the extrinsic pathway (21%), the intrinsic pathway (21%) or the factor X activator from Russell viper venom, RVV-X (26%). Factor XMarseille was isolated from plasma by immunoaffinity chromatography and compared with normal factor X purified by the same method. Activation of factor XMarseille by factor IXa or by RVV-X in a purified system showed that the rate of cleavage was decreased, whereas once produced, factor XaMarseille had a normal catalytic efficiency for either the peptide substrate S-2765 (D-Arg-Gly-Arg-NH-Np) or prothrombin. The rate of inhibition of factor XaMarseille by antithrombin III was also normal. Defective proteolysis of factor XMarseille by factor IXa or by RVV-X was the consequence of a threefold decrease in the kcat for the activation of factor XMarseille while the Km of RVV-X or factor IXa for factor X was normal. We have determined the molecular basis of the defect in the factor XMarseille gene by amplification of all eight exons, single-strand conformational polymorphism analysis of the amplified exons and subsequent sequence analysis. The patient was homozygous for a T-->C mutation in exon VIII, resulting in the substitution of Ser334 by proline. From comparison of three-dimensional models of various serine proteases, it appears that Ser334 is located within a surface-exposed variable region of factor X. This observation suggests that the Ser334-->Pro mutation either is responsible for a misalignment of the active sites of specific factor X activators in close proximity to the cleavage site, or that the Ser-->Pro mutation alters the spatial orientation of the cleavage site by nonlocal modifications of factor X structure.