RESUMO
Xenotransplantation may compensate the limited number of human allografts for transplantation using pigs as organ donors. Porcine endogenous retroviruses inherit infectious potential if pig cells, tissues, or organs were transplanted to immunosuppressed human recipients. Particularly, ecotropic PERV-C that could recombine with PERV-A to highly replication-competent human-tropic PERV-A/C should be excluded from pig breeds designed for xenotransplantation. Because of their low proviral background, SLAD/D (SLA, swine leukocyte antigen) haplotype pigs are potential candidates as organ donors as they do not bear replication-competent PERV-A and -B, even if they carry PERV-C. In this work, we characterized their PERV-C background isolating a full-length PERV-C proviral clone number 561 from a SLAD/D haplotype pig genome displayed in a bacteriophage lambda library. The provirus truncated in env due to cloning in lambda was complemented by PCR, and the recombinants were functionally characterized, confirming an increased infectivity in vitro compared to other PERV-C. Recombinant clone PERV-C(561) was chromosomally mapped by its 5'-proviral flanking sequences. Full-length PCR using 5'-and 3'-flanking primers specific to the PERV-C(561) locus verified that this specific SLAD/D haplotype pig harbors at least one full-length PERV-C provirus. The chromosomal location is different from that of the previously described PERV-C(1312) provirus, which was derived from the porcine cell-line MAX-T. The sequence data presented here provide further knowledge about PERV-C infectivity and contribute to targeted knockout in order to generate PERV-C-free founder animals. IMPORTANCE Yucatan SLAD/D haplotype miniature swine are candidates as organ donors for xenotransplantation. A full-length replication-competent PERV-C provirus was characterized. The provirus was chromosomally mapped in the pig genome. In vitro, the virus showed increased infectivity compared to other functional PERV-C isolates. Data may be used for targeted knockout to generate PERV-C free founder animals.
Assuntos
Retrovirus Endógenos , Suínos , Animais , Humanos , Porco Miniatura/genética , Retrovirus Endógenos/genética , Replicação Viral , México , Provírus/genética , Transplante HeterólogoRESUMO
The swine leukocyte antigen (SLA) complex harbors highly polymorphic gene clusters encoding glycoproteins that are involved in responses to vaccines, infectious disease, and production performance. Pigs with well-defined SLA class II genes are useful for the study of disease, immunology, and vaccines. In this study, we analyzed four SLA class II genes (SLA-DRA, SLA-DRB1, SLA-DQA, SLA-DQB1) in 22 founder Guizhou minipigs using a sequence-based typing method. Twelve alleles were detected, compared with the SLA class II allele sequences in the GenBank, and one of twelve alleles was found to be novel in Guizhou minipigs. There are four SLA II haplotypes, and one of them has been previously reported in Meishan pigs. Furthermore, based on sequence information of these alleles, we developed a simple SLA typing method implemented to SLA-typing for unknown offspring of Guizhou minipigs, relying on designed twelve sequence specific primers that could discriminate between each other. According to the combination of sequence-based typing and PCR-SSP, we were able to rapidly check SLA typing of Guizhou breeding stock and identified four SLA haplotypes in the herd. Therefore, SLA-defined Guizhou minipigs will be useful as animal models for xenotransplantation and immunological research.
Assuntos
Genes MHC da Classe II/genética , Haplótipos/genética , Antígenos de Histocompatibilidade Classe II/genética , Polimorfismo Genético/genética , Porco Miniatura/genética , Alelos , Animais , Cruzamento/métodos , Antígenos de Histocompatibilidade Classe I , SuínosRESUMO
The mini-pig is a useful animal model for human biomedical research due to its physiological similarity to humans and the ease of handling. In order to optimize the efficiency of production of transgenic Bama mini-pigs through somatic cell nuclear transfer (SCNT), we examined the effects of contact inhibition, roscovitine treatment, and serum starvation on the cell cycle synchronization and transgenic cloned embryo development in vivo and in vitro after nuclear transfer. The analysis showed that the rates of G0/G1 stage cells in the contact inhibition (92.11%) and roscovitine treatment groups (89.59%) were significantly higher than in serum starvation group (80.82%). A higher rate of apoptosis was seen in the serum starvation group (14.13%) compared to the contact inhibition and roscovitine treatment groups (6.71 and 2.46% respectively, P < 0.05). There was a significant decrease in blastocyst yield in the serum starvation group (14.19%) compared to the roscovitine treatment and contact inhibition groups (21.31 and 20.32% respectively, P < 0.05). A total of 1070 transgenic cloned embryos derived from the three treatment groups were transferred to surrogate sows; one pregnancy was established and three embryos from the roscovitine treatment group successfully completed gestation. These results indicate that the roscovitine treatment was more effective at synchronizing transgenic kidney cells in Bama mini-pigs and allowed reconstructed embryos to develop to full term.
Assuntos
Clonagem de Organismos , Proteína Glial Fibrilar Ácida/genética , Técnicas de Transferência Nuclear , Porco Miniatura/genética , Animais , Animais Geneticamente Modificados , Apoptose/genética , Ciclo Celular/genética , Reprogramação Celular , Desenvolvimento Embrionário/genética , Fibroblastos/citologia , Fibroblastos/metabolismo , Expressão Gênica , Genes bcl-2 , Humanos , Fenótipo , Suínos , Proteína X Associada a bcl-2/genéticaRESUMO
To optimize the combination of microsatellite loci for genetic control of outbred swine stocks, 32 of 100 loci distributed among almost all chromosomes (except 12) were screened out by 1.5% agarose, 8% polyacrylamide gel and capillary electrophoresis scanning among 3 miniature swine outbred stocks, namely Bama (BM), Guizhou (GZ) and Tibeta (TB). The mean total and effective allele numbers among these stocks were 12.1 and 5.9, respectively. The mean heterozygosity for these breeds was 0.5428, 0.6978 and 0.7646, and polymorphism information content was 0.5469, 0.7296 and 0.7663, respectively. Accordingly, hereditary variation from low to high was BM < GZ < TB. This showed that the genetic relationship between BM and GZ pigs was closer, and both were distant from TB. Additionally, the effectiveness of the 32-locus combination for evaluation of genetic quality was demonstrated in Changchun-junmo-1 (CJ-1), a standard outbred Chinese pig stock, in which the mean total and effective allele numbers and mean heterozygosity were 6.1613, 3.8483 and 0.6903, respectively. Since our results were consistent with the breeding pedigrees, the 32 loci could be used for both genetic monitoring within the individual outbred miniature swine stocks and population structure analysis between them.
Assuntos
Genética Populacional , Repetições de Microssatélites/genética , Porco Miniatura/genética , Suínos/genética , Animais , Eletroforese/métodos , Heterozigoto , Polimorfismo GenéticoRESUMO
Tibetan (TB) and Bama (BM) miniature pigs are two popular pig breeds that are used as experimental animals in China due to their small body size. Here, we analyzed single-nucleotide polymorphisms (SNPs) in gene fragments that are closely related to growth traits [growth hormone (GH), growth hormone receptor (GHR), and insulin-like growth factor (IGF)-1)] in these pig breeds and a large white (LW) control pig breed. On the basis of the analysis of 100 BMs, 108 TBs, and 50 LWs, the polymorphic distribution levels of GH, GHR, and IGF-1 were significantly different among these three pig breeds. According to correlation analyses between SNPs and five growth traits - body weight (BW), body length (BL), withers height (WH), chest circumference (CC), and abdomen circumference (AC) - three SNP loci in BMs and four SNP loci in TBs significantly affected growth traits. Three SNP sites in BMs and four SNP sites in TBs significantly affected growth traits. SNPs located in the GH gene fragment significantly affected BL and CC at locus 12 and BL at locus 45 in BMs, and also BW, WH, CC, and AC at locus 45 and WH and CC at locus 93 in TBs. One SNP at locus 85 in the BM GHR gene fragment significantly affected all growth traits. All indices were significantly reduced with a mixture of alleles at locus 85. These results provide more information regarding the genetic background of these minipig species and indicate useful selection markers for pig breeding programs.
Assuntos
Animais , Hormônio do Crescimento/genética , Fator de Crescimento Insulin-Like I/genética , Polimorfismo de Nucleotídeo Único/fisiologia , Receptores da Somatotropina/genética , Porco Miniatura/genética , Alelos , Tamanho Corporal , DNA , Nanismo/genética , Loci Gênicos , Genótipo , Reação em Cadeia da Polimerase , Análise de Sequência de DNA , SuínosRESUMO
Tibetan (TB) and Bama (BM) miniature pigs are two popular pig breeds that are used as experimental animals in China due to their small body size. Here, we analyzed single-nucleotide polymorphisms (SNPs) in gene fragments that are closely related to growth traits [growth hormone (GH), growth hormone receptor (GHR), and insulin-like growth factor (IGF)-1)] in these pig breeds and a large white (LW) control pig breed. On the basis of the analysis of 100 BMs, 108 TBs, and 50 LWs, the polymorphic distribution levels of GH, GHR, and IGF-1 were significantly different among these three pig breeds. According to correlation analyses between SNPs and five growth traits--body weight (BW), body length (BL), withers height (WH), chest circumference (CC), and abdomen circumference (AC)--three SNP loci in BMs and four SNP loci in TBs significantly affected growth traits. Three SNP sites in BMs and four SNP sites in TBs significantly affected growth traits. SNPs located in the GH gene fragment significantly affected BL and CC at locus 12 and BL at locus 45 in BMs, and also BW, WH, CC, and AC at locus 45 and WH and CC at locus 93 in TBs. One SNP at locus 85 in the BM GHR gene fragment significantly affected all growth traits. All indices were significantly reduced with a mixture of alleles at locus 85. These results provide more information regarding the genetic background of these minipig species and indicate useful selection markers for pig breeding programs.
Assuntos
Hormônio do Crescimento/genética , Fator de Crescimento Insulin-Like I/genética , Polimorfismo de Nucleotídeo Único/fisiologia , Receptores da Somatotropina/genética , Porco Miniatura/genética , Alelos , Animais , Tamanho Corporal , DNA/isolamento & purificação , Nanismo/genética , Loci Gênicos , Genótipo , Reação em Cadeia da Polimerase , Análise de Sequência de DNA , SuínosRESUMO
The genetic resources and the mechanism of miniaturization in the Tibet Mini-pig have not been comprehensively studied. Polymorphisms in genes related to the insulin-like growth factor (IGF) axis have been investigated for years, but few on the polymorphism of IGF-binding protein-5 (IGFBP-5) in the Tibetan pig. In this study, allele-specific polymerase chain reaction (AS-PCR) was used to analyze polymorphisms in exon 1 of the IGFBP-5 gene in two pig breeds, Tibet Mini-pigs and Junmu No. 1 White pigs. A BLAST analysis of the expressed sequence tags in the porcine IGFBP-5 gene revealed that exon 1 of this gene has two single nucleotide polymorphisms (SNPs), G188T and G503A. The AS-PCR results demonstrated that in both pig breeds examined, the TT, GT, and GG genotypes existed at the G188T locus, with GT as the most common genotype. At the G503A locus, GG, GA, and AA genotypes existed in Junmu No. 1 White pigs, with the GA genotype as the most frequently occurring. By contrast, at this locus, only the GA and AA genotypes were observed in the Tibetan pigs, and AA was more common than GA. There was a significant difference (P < 0.01) in allele distribution between the two breeds at the G503A locus but not the G188T locus, and there was a lower polymorphism information content for the two polymorphic loci in Tibet Mini-pigs than in Junmu No. 1 White pigs. The present study revealed SNPs in exon 1 of IGFBP-5 gene in the Tibet Mini-pig, possibly providing more understanding of the mechanism of miniaturization.
Assuntos
Proteína 5 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Polimorfismo Genético , Porco Miniatura/genética , Alelos , Animais , Cruzamento , Éxons/genética , Genótipo , Polimorfismo de Nucleotídeo Único , Suínos , Porco Miniatura/crescimento & desenvolvimento , TibetRESUMO
The IGF-1 gene is an important regulating factor that has a growth-promoting effect on growth hormone. The IGF-1 gene promotes muscle cell differentiation in the muscle cell formation process. The IGF-1 gene also regulates the growth of skeletal muscle during skeletal muscle growth. In addition, the IGF-1 gene plays an important role in the formation of mammals and poultry embryos, and the process of postnatal growth. The IGF-1 gene has been implicated as a candidate gene for the regulation of pig growth traits. We analyzed exon 3 of the IGF-1 gene polymorphism in Tibetan miniature pigs (N = 128) by polymerase chain reaction-single-strand conformation polymorphism and DNA sequencing. One single nucleotide polymorphism (T40C) was found on exon 3 of the IGF-1 gene. Statistical analysis of genotype frequencies revealed that the T allele was dominant in Tibetan miniature pigs at the T40C locus. The association analysis showed that the IGF-1 mutation had an effect on the body weight, body length, and chest circumference of pigs aged 6-8 months. In addition, the IGF-1 mutation had an effect on body weight in pigs aged 9-11 months (P < 0.05). We speculated that the pigs with the TT genotype grow more rapidly compared to those with the TC genotype. The TC genotype of the Tibetan miniature pig has a smaller body type. This information provides a theoretical basis for the genetic background of Tibetan miniature pigs.