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Autosomal 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.

There have been several recent attempts to generate, de novo, a functional whole kidney from stem cells using the organogenic niche or blastocyst complementation methods. However, none of these attempts succeeded in constructing a urinary excretion pathway for the stem cell-generated embryonic kidney. First, we transplanted metanephroi from cloned pig fetuses into gilts; the metanephroi grew to about 3 cm and produced urine, although hydronephrosis eventually was observed because of the lack of an excretion pathway. Second, we demonstrated the construction of urine excretion pathways in rats. Rat metanephroi or metanephroi with bladders (developed from cloacas) were transplanted into host rats. Histopathologic analysis showed that tubular lumina dilation and interstitial fibrosis were reduced in kidneys developed from cloacal transplants compared with metanephroi transplantation. Then we connected the host animal’s ureter to the cloacal-developed bladder, a technique we called the “stepwise peristaltic ureter” (SWPU) system. The application of the SWPU system avoided hydronephrosis and permitted the cloacas to differentiate well, with cloacal urine being excreted persistently through the recipient ureter. Finally, we demonstrated a viable preclinical application of the SWPU system in cloned pigs. The SWPU system also inhibited hydronephrosis in the pig study. To our knowledge, this is the first report showing that the SWPU system may resolve two important problems in the generation of kidneys from stem cells: construction of a urine excretion pathway and continued growth of the newly generated kidney.

The imprinted gene ZDBF2 is regulated through a unique mechanism involving a transient paternal transcript in early embryos, rather than persistent gametic DNA methylation. In humans and mice, this transcript— CMKLR2-AS (also known as GPR1-AS ) or the long isoform of Zdbf2 ( Liz/Zdbf2linc/Platr12 )—arises from the unmethylated paternal allele and initiates secondary epigenetic marks that maintain ZDBF2 expression. Here, we investigate the evolutionary origin of this mechanism, and show that the first exon of human GPR1-AS overlaps with a MER21C long terminal repeat (LTR), a retrotransposon subfamily specific to Boreoeutherian mammals. Comparative analyses revealed that this MER21C insertion occurred in the common ancestor of Euarchontoglires, including primates, rodents, and rabbits. Although not annotated, the first exon of mouse Liz displays conserved features with the MER21C-overlapping exon in humans. In rabbit and nonhuman primate placentas, GPR1-AS orthologs with LTR-embedded first exons were also identified. In contrast, in non-Euarchontoglire mammals such as cow and tammar wallaby, ZDBF2 is biallelically expressed, suggesting absence of imprinting. These findings suggest that ZDBF2 imprinting emerged in Euarchontoglires via MER21C insertion. Together with our prior work on LTR-driven imprinting in oocytes, our findings demonstrate that post-fertilization activation of retrotransposons can also drive lineage-specific acquisition of imprinting.

To combat organ shortage in transplantation medicine, a novel strategy has been proposed to generate human organs from exogenous pluripotent stem cells utilizing the developmental mechanisms of pig embryos/foetuses. Genetically modified pigs missing specific organs are key elements in this strategy. In this study, we demonstrate the feasibility of using a genome-editing approach to generate anephrogenic foetuses in a genetically engineered pig model. SALL1 knockout (KO) was successfully induced by injecting genome-editing molecules into the cytoplasm of pig zygotes, which generated the anephrogenic phenotype. Extinguished SALL1 expression and marked dysgenesis of nephron structures were observed in the rudimentary kidney tissue of SALL1 -KO foetuses. Biallelic KO mutations of the target gene induced nephrogenic defects; however, biallelic mutations involving small in-frame deletions did not induce the anephrogenic phenotype. Through production of F1 progeny from mutant founder pigs, we identified mutations that could reliably induce the anephrogenic phenotype and hence established a line of fertile SALL1 -mutant pigs. Our study lays important technical groundwork for the realization of human kidney regeneration through the use of an empty developmental niche in pig foetuses.

Genetic cardiomyopathy is a group of intractable cardiovascular disorders involving heterogeneous genetic contribution. This heterogeneity has hindered the development of life-saving therapies for this serious disease. Genetic mutations in dystrophin and its associated glycoproteins cause cardiomuscular dysfunction. Large animal models incorporating these genetic defects are crucial for developing effective medical treatments, such as tissue regeneration and gene therapy. In the present study, we knocked out the δ-sarcoglycan (δ-SG) gene ( SGCD ) in domestic pig by using a combination of efficient de novo gene editing and somatic cell nuclear transfer. Loss of δ-SG expression in the SGCD knockout pigs caused a concomitant reduction in the levels of α-, β-, and γ-SG in the cardiac and skeletal sarcolemma, resulting in systolic dysfunction, myocardial tissue degeneration, and sudden death. These animals exhibited symptoms resembling human genetic cardiomyopathy and are thus promising for use in preclinical studies of next-generation therapies. Gene δ-sarcoglycan was knocked out in pigs via gene editing and somatic cell cloning. Loss of expression led to α-, β-, and γ-sarcoglycan depletion in the cardiac and skeletal sarcolemma. Pigs exhibiting systolic dysfunction, myocardial tissue degeneration, and sudden death are promising for studying next-generation therapies for human genetic cardiomyopathy.

This article was originally published under Nature Research’s License to Publish, but has now been made available under a [CC BY 4.0] license. The PDF and HTML versions of the article have been modified accordingly.

Purpose This study aims to demonstrate vitrification methods that provide reliable cryopreservation for embryos with compromised cryotolerance. Methods Two‐cell stage mouse embryos and in vitro produced porcine embryos were vitrified using the hollow fiber vitrification (HFV) and Cryotop (CT) methods. The performance of these two methods was compared by the viability of the vitrified‐rewarmed embryos. Results Regardless of the method used, 100% of the mouse 2‐cell embryos developed successfully after vitrification‐rewarming into the blastocyst stage, whereas vitrification tests using porcine morulae with the HFV method produced significantly better results. The developmental rates of vitrified porcine morula into the blastocyst stage, as well as blastocyst cell number, were 90.3% and 112.3 ± 6.9 in the HFV group compared with 63.4% and 89.5 ± 8.1 in the CT group (P < .05). Vitrification tests using 4‐ to 8‐cell porcine embryos resulted in development into the blastocyst stage (45.5%) in the HFV group alone, demonstrating its better efficacy. The HFV method did not impair embryo viability, even after spontaneous rewarming at room temperature for vitrified embryos, which is generally considered a contraindication. Conclusion Vitrification test using embryos with compromised cryotolerance allows for more precise determining of effective cryopreservation methods and devices.

To develop novel medical technologies, pig disease models are invaluable especially in the final stages of translational research. Recently, we established a genetically engineered ornithine transcarbamylase-deficient (OTCD) pig strain. Here, we report its characterization and treatment responsiveness. OTCD pigs were obtained by mating an OTCD carrier female (OTC-Xc.186_190delXWT) with a wild-type male. Due to the X-linked recessive mode of inheritance, the disease phenotype emerged only in males. Medication with nitrogen-scavenging agents was based on a clinical protocol. OTCD pigs were born smaller than their wild-type and carrier littermates, showing anemia and faltering. Biochemically, high levels of urinary orotic acid and loss of OTC activity were observed. The natural life course of OTCD pigs was characterized by a decrease in arterial percentage saturation of oxygen and body temperature, as well as an increase in blood ammonia levels; the pigs died in 24.0 ± 5.0 h (mean ± SD, n = 6). The established standard medication composed with nitrogen-scavenging agents and transfusion nearly doubled the survival time to 42.4 ± 13.7 h (n = 6). Our OTCD pig model appropriately mimicked the human pathology. Along with established protocols in handling and medication, this is a first step in developing a large animal disease model that is useful for translational research into novel medical technologies, such as cell transplantation and gene therapy, as well as in relation to urea cycle disorder.

IntroductionPancreatic duodenum homeobox 1 (Pdx1) expression is crucial for pancreatic organogenesis and is a key regulator of insulin gene expression. Hairy and enhancer of split 1 (Hes1) controls tissue morphogenesis by maintaining undifferentiated cells. Hes1 encodes a basic helix loop helix (bHLH) transcriptional repressor and functionally antagonizes positive bHLH genes, such as the endocrine determination gene neurogenin-3. Here, we generated a new pig model for diabetes by genetic engineering Pdx1 and Hes1 genes.Research design and methodsA transgenic (Tg) chimera pig with germ cells carrying a construct expressing Hes1 under the control of the Pdx1 promoter was used to mate with wild-type gilts to obtain Tg piglets.ResultsThe Tg pigs showed perinatal death; however, this phenotype could be rescued by insulin treatment. The duodenal and splenic lobes of the Tg pigs were slender and did not fully develop, whereas the connective lobe was absent. β cells were not detected, even in the adult pancreas, although other endocrine cells were detected, and exocrine cells functioned normally. The pigs showed no irregularities in any organs, except diabetes-associated pathological alterations, such as retinopathy and renal damage.ConclusionPdx1-Hes1 Tg pigs were an attractive model for the analysis of pancreatic development and testing of novel treatment strategies for diabetes.

Genetically engineered pigs play an indispensable role in the study of rare monogenic diseases. Pigs harboring a gene responsible for a specific disease can be efficiently generated via somatic cell cloning. The generation of somatic cell-cloned pigs from male cells with mutation(s) in an X chromosomal gene is a reliable and straightforward method for reproducing X-linked genetic diseases (XLGDs) in pigs. However, the severe symptoms of XLGDs are often accompanied by impaired growth and reproductive disorders, which hinder the reproduction of these valuable model animals. Here, we generated unique chimeric boars composed of mutant cells harboring a lethal XLGD and normal cells. The chimeric boars exhibited the cured phenotype with fertility while carrying and transmitting the genotype of the XLGD. This unique reproduction system permits routine production of XLGD model pigs through the male-based breeding, thereby opening an avenue for translational research using disease model pigs.