Heterozygous Pigs Research Articles

Generation of heterozygous PKD1 mutant pigs exhibiting early-onset renal cyst formation

AbstractAutosomal dominant polycystic kidney disease (ADPKD) is the most common inherited kidney disease, manifesting as the progressive development of fluid-filled renal cysts. In approximately half of all patients with ADPKD, end-stage renal disease results in decreased renal function. In this study, we used CRISPR-Cas9 and somatic cell cloning to produce pigs with the unique mutation c.152_153insG (PKD1insG/+). Pathological analysis of founder cloned animals and progeny revealed that PKD1insG/+ pigs developed many pathological conditions similar to those of patients with heterozygous mutations in PKD1. Pathological similarities included the formation of macroscopic renal cysts at the neonatal stage, number and cystogenic dynamics of the renal cysts formed, interstitial fibrosis of the renal tissue, and presence of a premature asymptomatic stage. Our findings demonstrate that PKD1insG/+ pigs recapitulate the characteristic symptoms of ADPKD.Autosomal dominant polycystic kidney disease (ADPKD) is a genetic disorder characterized by the formation of cysts within the kidneys. The authors generated PKD1 heterozygous knockout (PKD1insG/+) pigs by CRISPR-Cas9 and somatic cell cloning techniques. The founder cloned animals and progeny showed the renal cyst formation from the neonatal stage, interstitial fibrosis of the renal tissue, and the presence of a premature asymptomatic stage. Their findings demonstrate that PKD1insG/+ pigs recapitulate the characteristic symptoms of ADPKD.

Viscera Characteristics of MSTN-Edited Heterozygous Pigs.

Myostatin (MSTN) is a protein that negatively regulates growth of skeletal muscle, and inactivation of MSTN improves the mass of skeletal muscle. Our previous work found that MSTN +/− pigs have higher muscle depth and lower fat depth compared to wild type without any developmental problems. Therefore, MSTN-edited pigs are most likely to appear as heterozygotes in the potential future market, but the characteristics of organs in digestive and reproductive system of pigs with MSTN gene editing remains unclear. Here, we investigated the histological of the organs in the digestive system and reproductive system in MSTN gene heterozygotes at adult stages. The length of intestine was further compared between adult heterozygous and wild type pigs. We found no significant differences in histomorphology of organs, including heart, duodenum, jejunum, ileum, cecum, colon, testis, epididymis, ovaries, oviducts and uterus, between individuals from two genotypes. Moreover, there was no significant difference in the average length of intestine in adult pigs. Our data provide a reference for further clarifying the applications of MSTN gene edited pigs.

Immunohistochemical Analyses and Quantitative RT-PCR of Angiogenic Factors in a Porcine Von Willebrand Disease Model

Objectives: Von Willebrand Factor (VWF) is a main factor in coagulation. In addition, it was shown to affect angiogenesis and the distribution of angiogenic factors. A severe deficit of this factor, as seen in patients of Von Willebrand Disease (VWD) type 3, can be expected to have impact on all these issues. As women with VWD may develop miscarriage during pregnancy, we analysed VWF as well as the angiogenic factors Angiopoietin-2 (ANG2) and Integrin αVβ3 (ITG) using immunohistochemistry (IHC) and quantitative PCR of uterus and caecum tissues in a female pig model comprising all VWD type 3 genotypes.Methods: Tissue samples of uterus and caecum were harvested from a total of 5 sexually mature female pigs. One of these was affected by VWD type 3, two were heterozygous carriers, and two were wildtype individuals, which served as controls. The expression of VWF, ITG, and ANG2 proteins were compared semiquantitatively by IHC in a double-blind manner. In addition, qRT-PCR was performed for the genes VWF, ANGPT2 (coding for ANG2 protein), and ITGAV (coding for ITG protein subunit)and analysed by relative quantification against the endothel-specific gene PROCR using the ΔΔCT method.Results: The expression of the angiogenic factors analysed showed remarkable differences amongst the genotypes. Using IHC, the pig affected by VWD showed almost no VWF expression within the examined tissues, while in wildtype and heterozygous pigs, expression was obvious in the endothelium. ITG showed strong granular intracellular staining in the uterine epithelium and glands of the VWD pig, but the staining was diffuse and moderate in heterozygous individuals. In wildtype pigs, the strongest staining of the epithelium was detected at the apical cell membrane. On the contrary, the apical cell membrane of the uterine epithelium in the VWD pig showed distinct ANG2 staining. qRT-PCR showed a higher expression of ITGAV in uterus and caecum (p-values: 0.1 and 0.05, respectively) and a significantly higher expression of ANG2 in caecum tissue (p-value: 0.03).Discussion and Conclusion: We were able to show that angiogenic factors in different tissues show alternative expression in pigs affected by VWD compared with heterozygous and wildtype pigs. In pigs affected by VWD, altered expression of ITG as well as ANG2 was seen, next to an extremely reduced expression of VWF. VWF seems to possess a stabilizing effect on ITG on apical membranes. Therefore, ITG in VWD affected pigs was detected to a higher degree in the cytoplasm, probably due to increased internalization. The generally increased expression of ITGAV in VWD affected pigs might be caused by negative feedback due to the reduced level of this protein at its physiological location. The increased expression and the staining pattern of ANG2 in pigs affected by VWD might be caused by the missing of Weibel-Palade-Bodies. The production of these storage organelles depends on VWF. Therefore, no storage of ANG2 can occur in VWD affected pigs and ANG2 may be released steadily. DisclosuresNo relevant conflicts of interest to declare.

Molecular analysis of GPI-anchor biosynthesis pathway genes in rat strains used for the Pig-a gene mutation assay

Recent studies support the assumption that mutation of the X-linked Pig-a gene is most likely responsible for the mutant phenotype of the cells deficient in glycosylphosphatidylinositol (GPI)-anchored proteins quantified in the rodent Pig-a gene mutation assay. In humans, however, mutations in both alleles of one of the 30 other genes involved in GPI-anchor synthesis, e.g., PIG-L and PIG-O, cause reduced expression of surface GPI-anchored proteins. Here, we investigated the possibility that the loss of the GPI-anchor detected by the rat Pig-a assay also could be caused by mutation in other GPI-biosynthesis genes. 31 samples were obtained from 8 inbred and outbred rat strains commonly used for genetic toxicology assays. In order to investigate possible sources of variation in the Pig-a assay, variant DNA sequences were evaluated in Cd59 and 24 GPI-biosynthesis genes. In some genes, such as Pig-n and Pig-u, homozygous variations occurred in all animals, suggesting that these variations are due to deviations in the reference genome. Heterozygous Pig-s, Pig-w, Pig-o, Pig-c, Pgap1, Pgap2, Pig-k and Pig-t variations were found, however, indicating that these genes could serve as targets for mutation in the assay. Protein alignment for these altered genes was conducted with possible human, mouse and rat phenotypic mutants from the literature; this analysis demonstrated that many of the variations that we detected were in non-conserved sequences and that no phenotypes for any of these variants could be inferred from known mutants from the literature. All heterozygous variants were in outbred rats. Overall, the findings of this study cannot totally rule out the possibility that mutations in GPI-biosynthesis genes other than Pig-a are detected in the Pig-a assay, but suggest that if it occurs, it must occur only rarely and therefore mutations in genes other than Pig-a have little impact on rat-based experiments.

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.

Effective generation of maternal genome point mutated porcine embryos by injection of cytosine base editor into germinal vesicle oocytes.

Cytosine and adenine base editors are promising new tools for introducing precise genetic modifications that are required to generate disease models and to improve traits in pigs. Base editors can catalyze the conversion of C→T (C>T) or A→G (A>G) in the target site through a single guide RNA. Injection of base editors into the zygote cytoplasm can result in the production of offspring with precise point mutations, but most F0 are mosaic, and breeding of F1 heterozygous pigs is time-intensive. Here, we developed a method called germinal vesicle oocyte base editing (GVBE) to produce point mutant F0 porcine embryos by editing the maternal alleles during the GV to MII transition. Injection of cytosine base editor 3 (BE3) mRNA and X-linked Dmd-specific guide RNAs into GVoocytes efficiently edited maternal Dmd during in vitro maturation and did not affect the maturation potential of the oocytes. The edited MII oocytes developed into blastocysts after parthenogenetic activation (PA) or in vitro fertilization (IVF). However, BE3 may reduce the developmental potential of IVF blastocysts from 31.5%±0.8% to 20.4% ±2.1%. There 40%-78.3% diploid PA blastocysts had no more than two different alleles, including up to 10% embryos that had only C>T mutation alleles. Genotyping of IVF blastocysts indicated that over 70% of the edited embryos had one allele or two different alleles of Dmd. Since the male embryos had only a copy of Dmd allele, all five (5/19) F0 male embryos are homozygous and three of them were Dmd precise C>T mutation. Nine (9/19) female IVF embryos had two different alleles including a WT and a C>T mutation. DNA sequencing showed that some of them might be heterozygous embryos. In conclusion, the GVBE method is a valuable method for generating F0 embryos with maternal point mutated alleles in a single step.

A 12.3-kb Duplication Within the VWF Gene in Pigs Affected by Von Willebrand Disease Type 3.

Von Willebrand Disease (VWD) type 3 is a serious and sometimes fatal hereditary bleeding disorder. In pigs, the disease has been known for decades, and affected animals are used as models for the human disease. Due to the recessive mode of inheritance of VWD type 3, severe bleeding is typically seen in homozygous individuals. We sequenced the complete porcine VWF (Von Willebrand Factor) complementary DNA (cDNA) and detected a tandem duplication of exons 17 and 18, causing a frameshift and a premature termination codon (p.Val814LeufsTer3) in the affected pig. Subsequent next generation sequencing on genomic DNA proved the existence of a 12.3-kb tandem duplication associated with VWD. This duplication putatively originates from porcine Short Interspersed Nuclear Elements (SINEs) located within VWF introns 16 and 18 with high identity. The premature termination truncates the VWF open reading frame by a large part, resulting in an almost entire loss of the mature peptide. It is therefore supposed to account for the severe VWD type 3. Our results further indicate the presence of strong, nonsense-mediated decay in VWF messenger RNA (mRNA) containing the duplication, which was supported by the almost complete absence of the complete VWF protein in immunohistochemistry analysis of the VWD-affected pig. In the past, differentiation of wild-type and heterozygous pigs in this VWD colony had to rely on clinical examinations and additional laboratory methods. The present study provides the basis to distinguish both genotypes by performing a rapid and simple genetic analysis.

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.

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.

Creation of miniature pig model of human Waardenburg syndrome type 2A by ENU mutagenesis.

Human Waardenburg syndrome 2A (WS2A) is a dominant hearing loss (HL) syndrome caused by mutations in the microphthalmia-associated transcription factor (MITF) gene. In mouse models with MITF mutations, WS2A is transmitted in a recessive pattern, which limits the study of hearing loss (HL) pathology. In the current study, we performed ENU (ethylnitrosourea) mutagenesis that resulted in substituting a conserved lysine with a serine (p. L247S) in the DNA-binding domain of the MITF gene to generate a novel miniature pig model of WS2A. The heterozygous mutant pig (MITF +/L247S) exhibits a dominant form of profound HL and hypopigmentation in skin, hair, and iris, accompanied by degeneration of stria vascularis (SV), fused hair cells, and the absence of endocochlear potential, which indicate the pathology of human WS2A. Besides hypopigmentation and bilateral HL, the homozygous mutant pig (MITF L247S/L247S) and CRISPR/Cas9-mediated MITF bi-allelic knockout pigs both exhibited anophthalmia. Three WS2 patients carrying MITF mutations adjacent to the corresponding region were also identified. The pig models resemble the clinical symptom and molecular pathology of human WS2A patients perfectly, which will provide new clues for better understanding the etiology and development of novel treatment strategies for human HL.

Fiber-type distribution and expression of myosin heavy chain isoforms in newborn heterozygous myostatin-knockout pigs.

To explore the effects of heterozygous myostatin-knockout (MSNT+/-) on muscle characteristics, specifically fiber-type distribution and expression of myosin heavy chain isoforms in pigs. The fiber cross-sectional area of the semitendinosus and semimembranosus muscles were much larger in MSTN+/- pigs at birth than in wild-type (WT) pigs. MSTN+/- pigs had a higher proportion of fast-type fibers and lower succinate dehydrogenase activity in muscles than WT pigs. The myosin heavy chain IIB mRNA level in both two muscles was~threefold higher in MSTN+/- pigs compared with WT pigs. MSTN+/- pigs exhibit a disproportionate increase in muscle mass and can have a higher body weight due to fiber hypertrophy, a change in the fiber-type distribution, and alteration of myosin heavy chain isoforms levels, leading to more fast glycolytic fibers.

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.

Pigs with Severe Combined Immunodeficiency Are Impaired in Controlling Influenza A Virus Infection

Influenza A viruses (IAV) infect many host species, including humans and pigs. Severe combined immunodeficiency (SCID) is a condition characterized by a deficiency of T, B, and/or natural killer (NK) cells. Animal models of SCID have great value for biomedical research. Here, we evaluated the pathogenesis and the innate immune response to the 2009 H1N1 pandemic IAV (H1N1pdm09) using a recently identified line of naturally occurring SCID pigs deficient in T and B lymphocytes that still have functional NK cells. SCID pigs challenged with H1N1pdm09 showed milder lung pathology compared to the non-SCID heterozygous carrier pigs. Viral titers in the lungs and nasal swabs of challenged SCID pigs were significantly higher than in carrier pigs 7 days postinfection, despite higher levels of IL-1β and IFN-α in the lungs of SCID pigs. The lower levels of pulmonary pathology were associated with the T and B cell absence in response to infection. The higher viral titers, prolonged shedding, and delayed viral clearance indicated that innate immunity was insufficient for controlling IAV in pigs. This recently identified line of SCID pigs provides a valuable model to understand the immune mechanisms associated with influenza protection and recovery in a natural host.

Immunohistochemical Analyses on Angiogenesis in a Porcine Von Willebrand Disease Model

Objectives: Women with von Willebrand Disease (VWD) type 3 may develop miscarriage during pregnancy. There is evidence that besides its role in coagulation von Willebrand Factor (VWF) is also involved in angiogenesis and defects of this pathway could play a role in miscarriage. For this reason, VWF as well as proteins with known functions in angiogenesis were analysed by immunohistochemistry of several tissues in a pig model comprising all VWD type 3 genotypes (VWD, heterozygous carriers, wildtype).Methods: Uterus tissue samples including the endometrium were collected from a total of 6 pigs. Of these, two were affected by VWD type 3, two were heterozygous carriers, and the remaining two were wildtype individuals, which served as controls. Hematoxylin-eosin-staining was implemented for morphological evaluation of the tissues, especially with regard to possible differences in structure and pattern of blood vessels. In a second step immunohistochemical analyses comparing the expression of angiogenic factors including VWF were performed in VWD-animals, genetic carriers and wildtype pigs.Results: Hematoxylin-eosin-staining of histological sections of the porcine uteri comprising the different genotypes revealed differences in the vascular architecture within the lamina propria. Compared to non pregnant wild type pigs where blood vessels in the lamina propria of the endometrium usually are of smaller caliber (mostly categorized as arterioles, venules and capillaries), the lamina propria of VWD type 3 animals contained multiple large arteries and veins (of the type usually found in the uterine wall). The immunohistochemical analysis for VWF showed almost no VWF expression in the pigs affected by VWD. In wildtype and heterozygous pigs, expression was obvious in the endothelium. A narrow band of VWF expression apically on the epithel cells as well as partial expression in glands was seen only in wildtype pigs, but not in heterozygous pigs. For some, but not all further angiogenic factors analysed, expression differences were present among the porcine VWD genotypes.Conclusion: The results of our study confirm effects of VWF on the uterine vascular architecture and on the expression of specific angiogenic factors. Pigs affected by VWD showed almost no expression of VWF and the blood vessels within the uterine lamina propria had a surprisingly high percentage of large arteries and veins indicating a crucial role of vWF for uterine angiogenesis. Expression differences among the genotypes were obvious for specific angiogenic factors. DisclosuresVon Depka:Octapharma: Other: Study Investigator, Speakers Bureau.

Alpha-1,3-galactosyltransferase-deficient miniature pigs produced by serial cloning using neonatal skin fibroblasts with loss of heterozygosity.

ObjectiveProduction of alpha-1,3-galactosyltransferase (αGT)-deficient pigs is essential to overcome xenograft rejection in pig-to-human xenotransplantation. However, the production of such pigs requires a great deal of cost, time, and labor. Heterozygous αGT knockout pigs should be bred at least for two generations to ultimately obtain homozygote progenies. The present study was conducted to produce αGT-deficient miniature pigs in much reduced time using mitotic recombination in neonatal ear skin fibroblasts.MethodsMiniature pig fibroblasts were transfected with αGT gene-targeting vector. Resulting gene-targeted fibroblasts were used for nuclear transfer (NT) to produce heterozygous αGT gene-targeted piglets. Fibroblasts isolated from ear skin biopsies of these piglets were cultured for 6 to 8 passages to induce loss of heterozygosity (LOH) and treated with biotin-conjugated IB4 that binds to galactose-α-1,3-galactose, an epitope produced by αGT. Using magnetic activated cell sorting, cells with monoallelic disruption of αGT were removed. Remaining cells with LOH carrying biallelic disruption of αGT were used for the second round NT to produce homozygous αGT gene-targeted piglets.ResultsMonoallelic mutation of αGT gene was confirmed by polymerase chain reaction in fibroblasts. Using these cells as nuclear donors, three heterozygous αGT gene-targeted piglets were produced by NT. Fibroblasts were collected from ear skin biopsies of these piglets, and homozygosity was induced by LOH. The second round NT using these fibroblasts resulted in production of three homozygous αGT knockout piglets.ConclusionThe present study demonstrates that the time required for the production of αGT-deficient miniature pigs could be reduced significantly by postnatal skin biopsies and subsequent selection of mitotic recombinants. Such procedure may be beneficial for the production of homozygote knockout animals, especially in species, such as pigs, that require a substantial length of time for breeding.

A Translational Model for Diet-related Atherosclerosis: Effect of Statins on Hypercholesterolemia and Atherosclerosis in a Minipig.

Models of atherosclerosis are used in preclinical studies but often fail to translate to humans. A model that better reflects human atherosclerosis is necessary. We recently engineered the ExeGen™ low-density lipoprotein receptor (LDLR) miniswine, in which the LDL receptor gene is modified to drive hypercholesterolemia and atherosclerosis, and showed diet-related exacerbation of these phenotypes. Five groups of animals, either wild type (+/+) or heterozygous (+/-), were fed either a normal or high-fat diet for 6 months. One group of heterozygous pigs fed a high-fat diet was also administered atorvastatin at 3 mg/kg/day. Clinical chemistry and anatomic pathology parameters were measured biweekly and at termination. The high-fat diet resulted in increased adiposity and interspersion of adipocytes within the salivary glands. The heterozygous pigs on the high-fat diet gained more weight and had significant increases in total cholesterol, high-density lipoprotein, and LDL compared to wild-type animals or heterozygous animals fed a normal diet. Atorvastatin attenuated these parameters, indicating the statin had a beneficial effect, even in a high-fat diet scenario. Atorvastatin treatment also reduced the intensity of Oil Red O staining in pigs on high-fat diet. Atorvastatin-related amelioration of several indices of cardiovascular pathophysiology in this model underscores its utility for drug discovery.

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 a miniature pig disease model for human Laron syndrome.

Laron syndrome is a rare disease caused by mutations of the growth hormone receptor (GHR), inheriting in an autosomal manner. To better understand the pathogenesis and to develop therapeutics, we generated a miniature pig model for this disease by employing ZFNs to knock out GHR gene. Three types of F0 heterozygous pigs (GHR+/4bp, GHR+/2bp, GHR+/3bp) were obtained and in which no significant phenotypes of Laron syndrome were observed. Prior to breed heterozygous pigs to homozygosity (GHR4bp/4bp), pig GHR transcript with the 4 bp insert was evaluated in vitro and was found to localize to the cytoplasm rather than the membrane. Moreover, this mutated transcript lost most of its signal transduction capability, although it could bind bGH. GHR4bp/4bp pigs showed a small body size and reduced body weight. Biochemically, these pigs exhibited significantly elevated levels of GH and decreased levels of IGF-I. These results resemble the phenotype observed in Laron patients, suggesting that these pigs could serve as an ideal model for Laron syndrome to bridge the gaps between mouse model and human.

Gene expression of beta-adrenergic receptors and myosin heavy chain isoforms induced by ractopamine feeding duration in pigs not carrying the ryanodine receptor mutation

To evaluate the effects of ractopamine hydrochloride (RAC) feeding duration on mRNA abundance of beta-adrenergic receptors (β-AR) and myosin heavy chain (MyHC) isoforms in the Longissimus dorsi (LD) muscle of pigs not carrying the ryanodine receptor type I (RyR1) mutation, 80 finishing barrows (initial body weight=69.4±7.87kg) were randomly assigned to 1 of 5 treatments with 8 replicate pens per treatment and 2 pigs per pen. Pigs were fed a corn–soybean meal-based diet with no added RAC (control) or with 10mg/kg RAC fed for 7, 14, 21, or 28d before slaughter. On d 28, hair root samples were collected for RyR1 genotyping and LD muscle samples were collected to measure β-AR (β1- and β2-subtypes) and MyHC isoforms (Iβ, IIa, IIx/d, and IIb) gene expression using the quantitative real-time PCR method. Six heterozygous negative pigs for the RyR1 mutation were detected, and therefore not considered for statistical analysis. Increasing RAC feeding duration did not affect the abundance of β1-AR mRNA, but resulted in a tendency (P=0.073) towards a linear decrease in β2-AR mRNA abundance. Even though MyHC Iβ mRNA abundance was linearly decreased (P=0.002) with increasing RAC feeding duration, both MyHC IIa and IIx/d gene expressions were not affected by the RAC treatment. There was a linear increase (P=0.029) in MyHC IIb mRNA abundance as RAC feeding duration was increased. In conclusion, RAC feeding alters gene expression of β2-AR and MyHC isoforms in the LD muscle of pigs not carrying the RyR1 mutation in a time-dependent manner.

Production of CMAH Knockout Preimplantation Embryos Derived From Immortalized Porcine Cells Via TALE Nucleases

Although noncancerous immortalized cell lines have been developed by introducing genes into human and murine somatic cells, such cell lines have not been available in large domesticated animals like pigs. For immortalizing porcine cells, primary porcine fetal fibroblasts were isolated and cultured using the human telomerase reverse transcriptase (hTERT) gene. After selecting cells with neomycin for 2 weeks, outgrowing colonized cells were picked up and subcultured for expansion. Immortalized cells were cultured for more than 9 months without changing their doubling time (~24 hours) or their diameter (< 20 µm) while control cells became replicatively senescent during the same period. Even a single cell expanded to confluence in 100 mm dishes. Furthermore, to knockout the CMAH gene, designed plasmids encoding a transcription activator-like effector nuclease (TALENs) pairs were transfected into the immortalized cells. Each single colony was analyzed by the mutation-sensitive T7 endonuclease I assay, fluorescent PCR, and dideoxy sequencing to obtain three independent clonal populations of cells that contained biallelic modifications. One CMAH knockout clone was chosen and used for somatic cell nuclear transfer. Cloned embryos developed to the blastocyst stage. In conclusion, we demonstrated that immortalized porcine fibroblasts were successfully established using the human hTERT gene, and the TALENs enabled biallelic gene disruptions in these immortalized cells.