Homozygous Pigs Research Articles

The transcriptomic insight into the differential susceptibility of African Swine Fever in inbred pigs

African swine fever (ASF) is a global threat to animal health and food security. ASF is typically controlled by strict biosecurity, rapid diagnosis, and culling of affected herds. Much progress has been made in developing modified live virus vaccines against ASF. There is host variation in response to ASF infection in the field and under controlled conditions. To better understand the dynamics underlying this host differential morbidity, whole transcriptome profiling was carried out in twelve immunized and five sham immunized pigs. Seventeen MHC homozygous inbred Large white Babraham pigs were sampled at three time points before and after the challenge. The changes in the transcriptome profiles of infected animals were surveyed over time. In addition, the immunization effect on the host response was studied as well among the contrasts of all protection subgroups. The results showed two promising candidate genes to distinguish between recovered and non-recovered pigs after infection with a virulent African swine fever virus (ASFV) pre-infection: HTRA3 and GFPT2 (padj < 0.05). Variant calling on the transcriptome assemblies showed a two-base pair insertion into the ACOX3 gene closely located to HTRA3 that may regulate its expression as a putative genomic variant for ASF. Several significant DGEs, enriched gene ontology (GO) terms, and KEGG pathways at 1 day and 7 days post-infection, compared to the pre-infection, indicate a significant inflammation response immediately after ASF infection. The presence of the virus was confirmed by the mapping of RNA-Seq reads on two whole viral genome sequences. This was concordant with a higher virus load in the non-recovered animals 7 days post-infection. There was no transcriptome signature on the immunization at pre-infection and 1 day post-infection. More samples and data from additional clinical trials may support these findings.

Pilot study on CHCF1 genotype in a pig challenge model for enterotoxigenic Escherichia coli F4ab/ac associated post-weaning diarrhea

Host genotype is important for enterotoxigenic E. coli (ETEC) susceptibility. We conducted two trials to evaluate the effect of CHCF1 genotype on incidence of ETEC diarrhea. In trial 1 (n = 15 pigs), pigs were inoculated with 108 CFU or 1010 CFU doses of an ETEC F4ac strain. In trial 2 (n = 33 pigs), pigs were inoculated with ETEC F4ab or F4ac. Across trials, all inoculated pigs that developed ETEC diarrhea were CHCF1 heterozygous susceptible (6/6). No inoculated CHCF1 homozygous resistant pigs developed ETEC diarrhea (0/26). Susceptibility towards ETEC F4ac/ab infection might correspond with CHCF1 genotype.

The Effect of Point Mutations in the RYR-1 Gene on the Physicochemical Properties of Meat

The impact of gene mutations in the RYR-1 gene on the physicochemical qualities and nutritional value of homozygous and heterozygous stress-resistant NOR carcasses with RSE and DFD defects was studied using molecular genetics methods. NOR, which is meat of homozygous cross-breeding pigs, had a 0.44 higher pH level, 2.62% higher water-holding ability, 2.36 units×103 higher color and 0.37% higher content of organic matter; the tryptophan content and protein-quality index were lower by 0.3 mmol/g and 0.29, respectively. This low acidity caused the denaturation of some sarcoplasmic proteins, which contributed to the loss of the meat’s water-holding ability. A sharp decrease in pH led to partial denaturation of the sarcoplasmic proteins, which determined the pale color of the PSE meat. It was found that, compared to the heterozygous stress-sensitive animals with DFD defects, the NOR pH level was 0.55 lower, the water-holding ability was 4.05% lower, and the color intensity was 4.97 units×103 lower; the protein-quality indicator amounted to 8.24, which was 1.12 higher than that in the heterozygous stress-sensitive animals with DFD defects. The defects had a significant impact on other physicochemical properties of the meat. Defects of PSE meat worsened meat color. The intensity of staining of muscle tissue with PSE defects was lower than in normal pork by 11.5, 8.7 and 6.4 units×103 , respectively. DFD-pork exceeded normal meat in color by 13.7 (P&gt; 0.99), 9.5 (P&gt; 0.99) and 7.4 units×103 .&#x0D; Keywords: stress tolerance, stress sensitivity, genotype, meat defects PSE and DFD-, RYR-1 gene, pH, water-holding ability, meat coloring

Generation of ETV5 Knockout Pigs with CRISPR/Cas9

Background: Ets variant factor 5 (ETV5) plays an important regulatory role in mouse Spermatogonial stem cells (SSCs) self-renewal. ETV5 knockout (KO) mice exhibit a progressive loss of SSCs and resulting in a Sertoli cell-only phenotype. The current study was aimed to use gene editing technology to obtain ETV5-KO pigs as a model for studying the apoptosis mechanism of SSCs and further clarify the function of ETV5 gene in pigs.Methods: A gene editing plasmid for the porcine ETV5 gene was constructed, transfected into porcine fetal fibroblasts by electroporation to obtain ETV5-KO cells. ETV5-KO cells were used as donors to prepare ETV5-KO pigs by somatic cell nuclear transfer (SCNT). Testis tissues were collected for hematoxylin and eosin (HE), immunohistochemistry (IHC), RT-PCR testing and blood for ELISA testing from ETV5-KO pig.Result: In the present study, we used the CRISPR/Cas9 system and SCNT to generate homozygous ETV5-KO pigs. We observed 3 phenotypes in these pigs: normal testis development after birth, the SSCs in the seminiferous tubules did not show obviously extinction at sexual maturity and normal spermatogenesis.

Vplyv suplementácie betaínu a kreatínmonohydrátu na chemické zloženie,oxidačnú stabilitu a kvalitu mäsa ošípaných heterozygótnych v géne RYR1

The aim of this study was to evaluate an effect of betaine and creatine monohydrate supplementation on chemical composition, meat quality, and oxidative stability of pork in pigs genotyped on mutation in RYR1 gene. Totally, sixty pigs – crosses of Lx(HAxPN) were involved in the experiment. Pigs were divided to one homozygous (NN) – control (C1), and three heterozygous (Nn) groups, each group including 15 animals. One group of Nn pigs was the control (C2) and two Nn groups were experimental. Control groups received a standard diet without supplement. Experimental groups were fed the same composition as controls but with supplement of betaine – BET (1.25 g/kg of feed) or creatine monohydrate - CMH (2.0 g/kg of feed) for thirty days prior to slaughter. Pigs C2 had worse meat quality, especially pH45, colour L* and drip loss (P<0.05), than homozygous C1 pigs. Dietary supplementation with betaine increased initial pH and decreased drip loss in Nn group (P<0.05) compared to C2 group. Supplementary CMH also increased pH45 and had a tendency to lowering the drip loss (P=0.058). Both feed additives substantially improved oxidative stability of pork, especially after 120 min incubation (P<0.01), compared to control C2 pigs.

Effect of MSTN Mutation on Growth and Carcass Performance in Duroc x Meishan Hybrid Population.

Simple SummaryMyostatin (MSTN) is a transcriptional growth factor that inhibits the development and growth of skeletal muscle. The MSTN-deficient animals display an increase in skeletal muscle mass known as double-muscling. Therefore, MSTN becomes an important target for improving lean meat production in livestock husbandry. There are many local pig breeds in China, but because of the slow growth, poor feed conversion, and low lean meat percentage and other unsatisfactory qualities, pure local breeds are rarely used on commercial farms. The objective of this study is to evaluate the effects of MSTN single allele mutation on carcass composition in Meishan crossbred pigs and demonstrate a way to increase lean meat yield while maintaining prolificacy and good meat quality of local pig crossbreeds. This has significant implications for the widespread use and conservation of local pig breeds in China.The Meishan pig is a traditional Chinese native breed, known for its excellent reproduction performance that is widely used in commercial pig production through two-way or three-way crossbreeding systems. However, the lean meat yield of Meishan crossbred pigs is still very low and cannot meet the market demand. To evaluate the lean meat yield of Meishan crossbred pigs, six wild-type Meishan sows were artificially inseminated by using the MSTN+/− Duroc boar semen in this experiment. Some reproductive performance-related traits of Meishan sows were recorded to ensure that semen from MSTN knockout Duroc boar did not affect offspring production, including total births, live births, sex, and litter weight. In total, 73 piglets were obtained and 63 were alive. Male to female ratio was close to 1: 1. because of factors such as disease, only 43 pigs were utilized, including 28 MSTN mutant pigs (MSTN+/−) and 15 MSTN homozygous pigs (MSTN+/+). We compared the growth performance and carcass performance of these full or half-sib populations and found that there were no differences between MSTN+/− and MSTN+/+ genotypes for live animal measures including average daily gain (ADG), body dimensions, or ultrasonic measurement of fat thickness when pigs were harvested after 120 days of feeding. Conversely, the MSTN+/− pigs had higher dressing percentage and lean meat percentage, lower level of carcass fat, larger longissimus muscle area, less percentage of skin and skeleton, thinner average backfat thickness, and lower intramuscular fat (IMF) content than MSTN+/+ pigs. In conclusion, the production of MSTN+/− mutant progeny from Meishan females resulted in improved carcass composition, providing a feasible solution to improve the lean meat yield of Chinese local fat-type pig breeds.

Effect of polymorphisms in porcine guanylate-binding proteins on host resistance to PRRSV infection in experimentally challenged pigs

Guanylate-binding proteins (GBP1 and GBP5) are known to be important for host resistance against porcine reproductive and respiratory syndrome virus (PRRSV) infection. In this study, the effects of polymorphisms in GBP1 (GBP1E2 and WUR) and GBP5 on host immune responses against PRRSV were investigated to elucidate the mechanisms governing increased resistance to this disease. Seventy-one pigs [pre-genotyped based on three SNP markers (GBP1E2, WUR, and GBP5)] were assigned to homozygous (n = 36) and heterozygous (n = 35) groups and challenged with the JA142 PRRSV strain. Another group of nineteen pigs was kept separately as a negative control group. Serum and peripheral blood mononuclear cells (PBMCs) were collected at 0, 3, 7, 14, 21 and 28 days post-challenge (dpc). Viremia and weight gain were measured in all pigs at each time point, and a flow cytometry analysis of PBMCs was performed to evaluate T cell activation. In addition, 15 pigs (5 pigs per homozygous, heterozygous and negative groups) were sacrificed at 3, 14 and 28 dpc, and the local T cell responses were evaluated in the lungs, bronchoalveolar lavage cells (BALc), lymph nodes and tonsils. The heterozygous pigs showed lower viral loads in the serum and lungs and higher weight gains than the homozygous pigs based on the area under the curve calculation. Consistently, compared with the homozygous pigs, the heterozygous pigs exhibited significantly higher levels of IFN-α in the serum, proliferation of various T cells (γδT, Th1, and Th17) in PBMCs and tissues, and cytotoxic T cells in the lungs and BALc. These results indicate that the higher resistance in the pigs heterozygous for the GBP1E2, WUR and GBP5 markers could be mediated by increased antiviral cytokine (IFN-α) production and T cell activation.

Influence of swine leukocyte antigen haplotype on serum antibody titers against swine erysipelas vaccine and reproductive and meat production traits of SLA-defined selectively bred Duroc pigs.

We investigated possible associations of SLA class II haplotypes with serum antibody titers against a swine erysipelas vaccine, reproductive and meat production traits using a population of selective breeding Duroc pigs. In the selective breeding Duroc pigs, four SLA class II-DRB1 and -DQB1 alleles were assigned by using PCR-sequence specific primer technique. Low-resolution haplotype (Lr)-0.30 and/or Lr-0.13 were deduced from the SLA class II alleles in the population of SLA-defined Duroc pigs. SLA-homozygous piglets with the Lr-0.30 haplotype had relatively lower serum antibody titers against the vaccine compared to those with Lr-0.13. In contrast, there were no statistically significant differences in reproductive performance between the SLA-defined pigs with two SLA class II haplotypes. Weaning and rearing rates until the body weight of 105 kg was reached in homozygous piglets with Lr-0.30 were significantly lower than those in homozygous piglets with Lr-0.13. The SLA-defined pigs had lower birth and weaning weights, body weights at 60 days of age, and daily weight gains than non-selective breeding Duroc pigs. Furthermore, the SLA-defined pigs had slightly lower back fat thickness compared to the non-selective breeding pigs. The rib eye areas of homozygous or heterozygous pigs with Lr-0.13 were larger than those of homozygous pigs with Lr-0.30 and non-selective breeding pigs. These data suggested that SLA haplotypes had the potential as useful genetic markers for selective breeding in the population of SLA-defined Duroc pigs.

Apancreatic pigs cloned using Pdx1-disrupted fibroblasts created via TALEN-mediated mutagenesis.

Pancreatic and duodenal homeobox 1 (PDX1) plays a crucial role in pancreas development, β-cell differentiation, and maintenance of mature β-cell function. In this study, we designed a strategy to produce PDX1-knockout (KO) pigs. A transcription activator-like effector nuclease (TALEN) pair targeting exon 1 of the swine PDX1 gene was constructed. Porcine fetal fibroblasts (PFFs) were transfected with the TALEN plasmids plus a surrogate reporter plasmid. PDX1-mutated PFFs were enriched by magnetic separation and used to produce homozygous PDX1-KO pigs via a two-step somatic cell nuclear transfer (SCNT) cloning process. In the first SCNT step, we obtained eight fetuses, established PFF cell lines, and analyzed PDX1 gene mutations by T7 endonuclease 1 assays and Sanger sequencing. Five fetuses showed mutations at the PDX1 loci with two biallelic mutations and three monoallelic mutations (mutation rate of 62.5%). In the second step, a PDX1 biallelic mutant PFF cell line with a 2 bp deletion in one allele and a 4 bp insertion in the other allele was used as a donor to generate cloned pigs via SCNT. From 462 cloned embryos transferred into two surrogates, nine live piglets were delivered. These piglets at birth were not clearly distinguishable phenotypically from wild-type piglets, but soon developed severe diarrhea and vomiting and all died within 2 days after birth. Dissection of PDX1-KO piglets revealed that the liver, gallbladder, spleen, stomach, common bile duct, and other viscera were present and normal, but the pancreas was absent in all cases.

Effect of Single Nucleotide Polymorphisms in IGFBP2 and IGFBP3 Genes on Litter Size Traits in Berkshire Pigs

ABSTRACTLitter size is among the most important traits in swine breeding. However, information on the genetics of litter size in pigs is lacking. In this study, we identified single nucleotide polymorphisms (SNPs) in the insulin-like growth factor binding protein 2 and 3 (IGFBP2 and IGFBP3) genes in Berkshire pigs and analyzed their association with litter size traits. The IGFBP2 SNP was located on chromosome 15 intron 2 (455, A > T) and the IGFBP3 SNP was on chromosome 18 intron 2 (53, A > G). The AT type of IGFBP2 and the GG type of IGFBP3 had the highest values for all litter size traits including total number born (TNB), number of pigs born alive, and breeding value according to TNB. Homozygous GG pigs expressed higher levels of IGFBP3 mRNA in the endometrium than pigs of other genotypes, and a positive correlation was observed between litter size traits and IGFBP3 but not IGFBP2 expression level. These results suggest that SNPs in the IGFBP2 and the IGFBP3 gene are useful biomarkers for increasing the reproductive productivity of Berkshire pigs.

Squalene epoxidase plays a critical role in determining pig meat quality by regulating adipogenesis, myogenesis, and ROS scavengers

In mammals, Squalene epoxidase (SQLE) is an enzyme that converts squalene to 2,3-oxidosqualene, in the early stage of cholesterol generation. Here, we identified single nucleotide polymorphisms (SNPs) in the SQLE gene (c.2565 G > T) by RNA Sequencing from the liver tissue of Berkshire pigs. Furthermore, we found that homozygous GG pigs expressed more SQLE mRNA than GT heterozygous and TT homozygous pigs in longissimus dorsi tissue. Next, we showed that the SNP in the SQLE gene was associated with several meat quality traits including backfat thickness, carcass weight, meat colour (yellowness), fat composition, and water-holding capacity. Rates of myogenesis and adipogenesis induced in C2C12 cells and 3T3-L1 cells, respectively, were decreased by Sqle knockdown. Additionally, the expression of myogenic marker genes (Myog, Myod, and Myh4) and adipogenic marker genes (Pparg, Cebpa, and Adipoq) was substantially downregulated in cells transfected with Sqle siRNA. Moreover, mRNA expression levels of ROS scavengers, which affect meat quality by altering protein oxidation processes, were significantly downregulated by Sqle knockdown. Taken together, our results suggest the molecular mechanism by which SNPs in the SQLE gene can affect meat quality.

Development of α 1,3-galactosyltransferase Inactivated and Human Membrane Cofactor Protein Expressing Homozygous Transgenic Pigs for Xenotransplantation

Development of α 1,3-galactosyltransferase Inactivated and Human Membrane Cofactor Protein Expressing Homozygous Transgenic Pigs for Xenotransplantation

The AMPK V199i Mutation in Berkshire Pigs Decreases the Rate of Postmortem pH Decline in Skeletal Muscle

ObjectivesAMP-activated protein kinase (AMPK) is a heterotrimeric enzyme that participates in the regulation of cellular energy homeostasis and is comprised of an ɑ, β, and γ subunit. In pigs, two known genetic mutations in the skeletal muscle specific γ3 regulatory subunit alter fresh pork quality. The first mutation, AMPKγ3R200Q (Rendement Napole mutation), arises from an arginine to glutamine substitution and makes AMPK constitutively active. Animals with this mutation produce meat with an abnormally low ultimate pH known as acid meat. The second mutation, AMPKγ3V199I, arises from a valine to isoleucine substitution making AMPK unresponsive to AMP. While the AMPKγ3V199I mutation has been associated with improved pork quality, little information is available elucidating the biochemical mechanism responsible. Therefore, our objective was to investigate the role of the AMPKγ3V199I mutation in determining fresh meat quality during the conversion of muscle to meat.Materials and MethodsWe investigated this mutation using Berkshire pigs, a breed known to carry the AMPKg3V199I mutation with a high frequency (80 to 90% homozygous mutants). Homozygous mutant (n = 12) and heterozygous Berkshire pigs (n = 9) were harvested at The Ohio State University’s Meat Laboratory. Muscle samples were collected from the longissimus lumborum et thoracis at 0, 30, 60, 120, 240, and 1440 min postmortem and frozen in liquid nitrogen to measure pH decline. To determine the rate of pH decline, a single exponential decay curve was fit to the data to derive a rate constant for each pig. After 1440 min, carcass data were measured for dressing percentage, backfat thickness, loin eye area, and Minolta color (L*, a*, and b*). Data were analyzed with a mixed model in JMP. The LSMeans were compared using a Student’s t test and considered significant at P ≤ 0.05.ResultsNo differences were detected in any carcass measurements. The homozygous mutant pigs exhibited a decreased rate of postmortem pH decline (P = 0.0325). This difference primarily arose from the elevated muscle pH of homozygous mutant muscle at 30 min (P = 0.0036) and 60 min (P = 0.0391) postmortem compared to the heterozygous pigs. The slower postmortem pH decline may explain the superior meat quality associated with Berkshire pigs and may be due to a decreased glycolytic capacity in the homozygous pigs.ConclusionThe AMPKγ3V199I mutation results in a slower pH decline in homozygous mutants compared to heterozygotes. The data also suggest that the AMPKγ3V199I mutation may be a recessive mutation compared to the dominant AMPKγ3R200Q mutation.

Production of a Locus- and Allele-Specific Monoclonal Antibody for the Characterization of SLA-1*0401 mRNA and Protein Expression Levels in MHC-Defined Microminipigs.

The class I major histocompatibility complex (MHC) presents self-developed peptides to specific T cells to induce cytotoxity against infection. The MHC proteins are encoded by multiple loci that express numerous alleles to preserve the variability of the antigen-presenting ability in each species. The mechanism regulating MHC mRNA and protein expression at each locus is difficult to analyze because of the structural and sequence similarities between alleles. In this study, we examined the correlation between the mRNA and surface protein expression of swine leukocyte antigen (SLA)-1*0401 after the stimulation of peripheral blood mononuclear cells (PBMCs) by Staphylococcus aureus superantigen toxic shock syndrome toxin-1 (TSST-1). We prepared a monoclonal antibody (mAb) against a domain composed of Y102, L103 and L109 in the α2 domain. The Hp-16.0 haplotype swine possess only SLA-1*0401, which has the mAb epitope, while other haplotypes possess 0 to 3 SLA classical class I loci with the mAb epitopes. When PBMCs from SLA-1*0401 homozygous pigs were stimulated, the SLA-1*0401 mRNA expression level increased until 24 hrs and decreased at 48 hrs. The kinetics of the interferon regulatory transcription factor-1 (IRF-1) mRNA level were similar to those of the SLA-1*0401 mRNA. However, the surface protein expression level continued to increase until 72 hrs. Similar results were observed in the Hp-10.0 pigs with three mAb epitopes. These results suggest that TSST-1 stimulation induced both mRNA and surface protein expression of class I SLA in the swine PBMCs differentially and that the surface protein level was sustained independently of mRNA regulation.

A galactopoiesis accordant yield of functional recombinant human factor IX from homozygous transgenic pigs requires a large amount of vitamin K supplementation

Transgenic pigs failed to accord milk yield curve to lactate rhFIX-a vitamin K (VK) dependent protein even fed with VK enriched to 8 times higher than nutritional requirement. A further higher VK supplementation may be required. Homozygous transgenic sows (n=4, 200kg) at their 3rd nursing were divided into control and treatment groups and respectively received VK enriched and further menadione (soluble VK) supplemented diet (220mg/kg VK enriched diet) for 33days. At next lactation, control sows than received treatment and previous treated were fed on control diet. Results revealed that menadione treatment increased milk bioactivity of rhFIX from the 7th day of 73 to the 21st day of 153IU/mL; it gradually decreased to 96IU/mL on 35th day of lactation. Under control feeding, bioactivity remained relatively unchanged. However, milk rhFIX concentration and ratio of activated rhFIX responded little to the treatment. The menadione-induced bioactivity curve agrees with the known lactation pattern of sow means rhFIX secretion is still galactopoietic but requires high VK intake to show. The ineffectual VK spend on lactational carboxylation might be common in other mammary VK dependent expression system but can be effectively overcome by a high supplementation of menadione with a 5-folds improvement in quality.

Analysis of Swine Leukocyte Antigen Haplotypes in Yucatan Miniature Pigs Used as Biomedical Model Animal.

The porcine major histocompatibility complex (MHC) is called swine leukocyte antigen (SLA), which controls immune responses and transplantation reactions. The SLA is mapped on pig chromosome 7 (SSC7) near the centromere. In this study, 3 class I (SLA-1, SLA-3, and SLA-2) and 3 class II (DRB1, DQB1, and DQA) genes were used for investigation of SLA haplotypes in Yucatan miniature pigs in Korea. This pig breed is a well-known model organism for biomedical research worldwide. The current study indicated that Korean Yucatan pig population had 3 Class I haplotypes (Lr-4.0, Lr-6.0, and Lr-25.0) and 3 class II haplotypes (Lr-0.5, Lr-0.7, and Lr-0.25). The combinations of SLA class I and II haplotype together, 2 homozygous (Lr-4.5/4.5 and Lr-6.7/6.7) and 3 heterozygous (Lr-4.5/6.7, Lr-4.5/25.25, and Lr-6.7/25.25) haplotypes were identified, including previously unidentified new heterozygous haplotypes (Lr-4.5/4.7). In addition, a new SLA allele typing method using Agilent 2100 bioanalyzer was developed that permitted more rapid identification of SLA haplotypes. These results will facilitate the breeding of SLA homozygous Yucatan pigs and will expedite the possible use of these pigs for the biomedical research, especially xenotransplantation research.

FUT1 genetic variants impact protein glycosylation of porcine intestinal mucosa.

A massive use of antibiotics in industrial pig production is a major cause of the rapidly rising bacterial resistance to antibiotics. An enhanced understanding of infectious diseases and of host-microbe interactions has the potential to explore alternative ways to improve pig health and reduce the need for antibiotics. Host-microbe interactions depend on host-expressed glycans and microbe-carrying lectins. In this study, a G > A (nucleotide 307) missense mutation in the porcine α1,2fucosyltransferase 1 gene (FUT1), which has been reported to prevent infections by the common porcine enteric pathogen F18 fimbriated Escherichia coli, provided a unique opportunity to study glycan structures potentially involved in intestinal infections. N- and O-Linked glycans of the intestinal mucosa proteins were characterized in detail using LC-MS/MS. Relative abundances of all glycans were determined and compared between four heterozygous pigs (FUT1-307(A/G)) and four age-matched homozygous pigs from the same 2 litters carrying the missense FUT1 gene constellation (FUT1-307(A/A)). None of the characterized 48 N-linked glycans was found to be regulated by the FUT1 missense mutation, while 11 of the O-linked glycans showed significantly altered abundances between the two genotypes. The overall abundance of H-antigen carrying structures was decreased fivefold, while H-antigen precursors and sialylated structures were relatively more abundant in pigs with the FUT1 missense mutation. These results provide insight into the role of FUT1 on intestinal glycosylation, improve our understanding of how variation in FUT1 can modulate host-microbe interactions, and suggest that the FUT1 genetic variant may help to improve pig gut health.

Generation of α1,3-galactosyltransferase and cytidine monophospho-&lt;i&gt;N&lt;/i&gt;-acetylneuraminic acid hydroxylase gene double-knockout pigs

Zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) are new tools for producing gene knockout (KO) animals. The current study reports produced genetically modified pigs, in which two endogenous genes were knocked out. Porcine fibroblast cell lines were derived from homozygous α1,3-galactosyltransferase (GalT) KO pigs. These cells were subjected to an additional KO for the cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH) gene. A pair of ZFN-encoding mRNAs targeting exon 8 of the CMAH gene was used to generate the heterozygous CMAH KO cells, from which cloned pigs were produced by somatic cell nuclear transfer (SCNT). One of the cloned pigs obtained was re-cloned after additional KO of the remaining CMAH allele using the same ZFN-encoding mRNAs to generate GalT/CMAH-double homozygous KO pigs. On the other hand, the use of TALEN-encoding mRNAs targeting exon 7 of the CMAH gene resulted in efficient generation of homozygous CMAH KO cells. These cells were used for SCNT to produce cloned pigs homozygous for a double GalT/CMAH KO. These results demonstrate that the combination of TALEN-encoding mRNA, in vitro selection of the nuclear donor cells and SCNT provides a robust method for generating KO pigs.

Generation of CRISPR/Cas9-mediated gene-targeted pigs via somatic cell nuclear transfer.

The domestic pig has been widely used as an important large animal model. Precise and efficient genetic modification in pig provides a great promise in biomedical research. Recently, clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system has been successfully used to produce many gene-targeted animals. However, these animals have been generated by co-injection of Cas9 mRNA and single-guide RNA (sgRNA) into one-cell stage embryos, which mostly resulted in mosaicism of the modification. One or two rounds of further breeding should be performed to obtain homozygotes with identical genotype and phenotype. To address this issue, gene-targeted somatic cells can be used as donor for somatic cell nuclear transfer (SCNT) to produce gene-targeted animals with single and identical mutations. In this study, we applied Cas9/sgRNAs to effectively direct gene editing in porcine fetal fibroblasts and then mutant cell colonies were used as donor to generate homozygous gene-targeted pigs through single round of SCNT. As a result, we successfully obtained 15 tyrosinase (TYR) biallelic mutant pigs and 20 PARK2 and PINK1 double-gene knockout (KO) pigs. They were all homozygous and no off-target mutagenesis was detected by comprehensive analysis. TYR (-/-) pigs showed typical albinism and the expression of parkin and PINK1 were depleted in PARK2 (-/-)/PINK1 (-/-) pigs. The results demonstrated that single- or double-gene targeted pigs can be effectively achieved by using the CRISPR/Cas9 system combined with SCNT without mosaic mutation and detectable off-target effects. This gene-editing system provides an efficient, rapid, and less costly manner to generate genetically modified pigs or other large animals.

The effects of ryanodine receptor 1 (RYR1) mutation on plasma cytokines and catecholamines during prolonged restraint in pigs

In the current study, plasma cytokines, including interleukin (IL) 1, IL-2, IL-6, IL-10, IL-12, interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) and catecholamines (adrenaline and noradrenaline) were evaluated during 4h restraint and recovery phase in 60 male pigs. Blood samples were collected from three groups of pigs representing different RYR1 genotypes, namely NN homozygotes (wild-type), Nn heterozygous and nn homozygous (mutant). The 4h restraint evoked an increase in plasma cytokine concentrations in each of the RYR1 genotype groups. During the restraint, the greatest concentrations of plasma IL-6, IL-10, IL-12 and TNF-α in nn homozygous pigs and IFN-γ in NN homozygous were observed. Interleukin 1, IL-2, IL-10, and TNF-α measures were positively intercorrelated in each of the three RYR1 genotype group. A positive correlation was seen between all measured cytokines (with the exception of IL-6) and plasma catecholamine concentrations in Nn heterozygous and nn homozygous pigs. The results suggest that of the cytokine parameters evaluated, IL-6, IL-10, IL-12 and TNF-α of the nn homozygous group seem to show a stronger stress-related response as compared with those of the other two (NN and Nn) groups.