Taking the PERVs out of pigs: With the severe shortage of organs needed for transplants, xenotransplantation (transplantation of nonhuman organs to humans) offers an alternative source. Some pig organs have similar size and function to those of humans. The challenge is that the pig genome harbors porcine endogenous retroviruses (PERVs) that can potentially pass to humans with possibly damaging consequences. Niu et al. generated pigs in which all copies of PERVs were inactivated by CRISPR-Cas9 genome engineering (see the Perspective by Denner). Not only does this work provide insights into PERV activity, but it also opens the door to a safer source of organs and tissues for pig-to-human xenotransplantation. Science , this issue p. 1303 ; see also p. 1238

Pig epiblast-derived pluripotent stem cells are considered to have great potential and broad prospects for human therapeutic model development and livestock breeding. Despite ongoing attempts since the 1990s, no stably defined pig epiblast-derived stem cell line has been established. Here, guided by insights from a large-scale single-cell transcriptome analysis of pig embryos from embryonic day (E) 0 to E14, specifically, the tracing of pluripotency changes during epiblast development, we developed an in vitro culture medium for establishing and maintaining stable pluripotent stem cell lines from pig E10 pregastrulation epiblasts (pgEpiSCs). Enabled by chemical inhibition of WNT-related signaling in combination with growth factors in the FGF/ERK, JAK/STAT3, and Activin/Nodal pathways, pgEpiSCs maintain their pluripotency transcriptome features, similar to those of E10 epiblast cells, and normal karyotypes after more than 240 passages and have the potential to differentiate into three germ layers. Strikingly, ultradeep in situ Hi-C analysis revealed functional impacts of chromatin 3D-spatial associations on the transcriptional regulation of pluripotency marker genes in pgEpiSCs. In practice, we confirmed that pgEpiSCs readily tolerate at least three rounds of successive gene editing and generated cloned gene-edited live piglets. Our findings deliver on the long-anticipated promise of pig pluripotent stem cells and open new avenues for biological research, animal husbandry, and regenerative biomedicine.

Dystrophinopathy, including Duchenne muscle dystrophy (DMD) and Becker muscle dystrophy (BMD) is an incurable X-linked hereditary muscle dystrophy caused by a mutation in the DMD gene in coding dystrophin. Advances in further understanding DMD/BMD for therapy are expected. Studies on mdx mice and dogs with muscle dystrophy provide limited insight into DMD disease mechanisms and therapeutic testing because of the different pathological manifestations. Miniature pigs share similar physiology and anatomy with humans and are thus an excellent animal model of human disease. Here, we successfully achieved precise DMD targeting in Chinese Diannan miniature pigs by co-injecting zygotes with Cas9 mRNA and sgRNA targeting DMD. Two piglets were obtained after embryo transfer, one of piglets was identified as DMD-modified individual via traditional cloning, sequencing and T7EN1 cleavage assay. An examination of targeting rates in the DMD-modified piglet revealed that sgRNA:Cas9-mediated on-target mosaic mutations were 70% and 60% of dystrophin alleles in skeletal and smooth muscle, respectively. Meanwhile, no detectable off-target mutations were found, highlighting the high specificity of genetic modification using CRISPR/Cas9. The DMD-modified piglet exhibited degenerative and disordered phenotypes in skeletal and cardiac muscle, and declining thickness of smooth muscle in the stomach and intestine. In conclusion, we successfully generated myopathy animal model by modifying the DMD via CRISPR/Cas9 system in a miniature pig.

Pigs have emerged as one of the most popular large animal models in biomedical research, which in many cases is considered as a superior choice over rodent models. In addition, transplantation studies using pig pluripotent stem (PS) cell derivatives may serve as a testbed for safety and efficacy prior to human trials. Recently, it has been shown that mouse and human PS cells cultured in LCDM (recombinant human LIF, CHIR 99021, (S)-(+)-dimethindene maleate, minocycline hydrochloride) medium exhibited extended developmental potential (designated as extended pluripotent stem cells, or EPS cells), which could generate both embryonic and extraembryonic tissues in chimeric mouse conceptus. Whether stable pig induced pluripotent stem (iPS) cells can be generated in LCDM medium and their chimeric competency remains unknown. iPS cells were generated by infecting pig pericytes (PC) and embryonic fibroblasts (PEFs) with a retroviral vector encoding Oct4, Sox2, Klf4, and cMyc reprogramming factors and subsequently cultured in a modified LCDM medium. The pluripotency of PC-iPS and PEF-iPS cells was characterized by examining the expression of pluripotency-related transcription factors and surface markers, transcriptome analysis, and in vitro and in vivo differentiation capabilities. Chimeric contribution of PC-iPS cells to mouse and pig conceptus was also evaluated with fluorescence microscopy, flow cytometry, and PCR analysis. In this study, using a modified version of the LCDM medium, we successfully generated iPS cells from both PCs and PEFs. Both PC-iPS and PEF-iPS cells maintained the stable “dome-shaped” morphology and genome stability after long-term culture. The immunocytochemistry analyses revealed that both PC-iPS and PEF-iPS cells expressed OCT4, SOX2, and SALL4, but only PC-iPS cells expressed NANOG and TRA-1-81 (faint). PC-iPS and PEF-iPS cells could be differentiated into cell derivatives of all three primary germ layers in vitro. The transcriptome analysis showed that PEF-iPS and PC-iPS cells clustered with pig ICM, Heatmap and volcano plot showed that there were 1475 differentially expressed genes (DEGs) between PC-iPS and PEF-iPS cells (adjusted p value < 0.1), and the numbers of upregulated genes and downregulated genes in PC-iPS cells were 755 and 720, respectively. Upregulated genes were enriched with GO terms including regulation of stem cell differentiation, proliferation, development, and maintenance. And KEGG pathway enrichment in upregulated genes revealed Wnt, Jak-STAT, TGF-β, P53, and MAPK stem cell signaling pathways. Fluorescence microscopy and genomic PCR analyses using pig mtDNA-specific and GFP primers showed that the PC-iPS cell derivatives could be detected in both mouse and pig pre-implantation blastocysts and post-implantation conceptuses. Quantitative analysis via flow cytometry revealed that the chimeric contribution of pig PC-iPS cells in mouse conceptus was up to 0.04%. Our findings demonstrate that stable iPS cells could be generated in LCDM medium, which could give rise to both embryonic and extraembryonic cells in vivo. However, the efficiency and level of chimeric contribution of pig LCDM-iPS cells were found low.

Somatic cell nuclear transfer (SCNT) is an important method of breeding quality varieties, expanding groups, and preserving endangered species. However, the viability of SCNT embryos is poor, and the cloned rate of animal production is low in pig. This study aims to investigate the gene function and establish a disease model of Banna miniature inbred pig. SCNT with donor cells derived from fetal, newborn, and adult fibroblasts was performed, and the cloning efficiencies among the donor cells were compared. The results showed that the cleavage and blastocyst formation rates did not significantly differ between the reconstructed embryos derived from the fetal (74.3% and 27.4%) and newborn (76.4% and 21.8%) fibroblasts of the Banna miniature inbred pig (P>0.05). However, both fetal and newborn fibroblast groups showed significantly higher rates than the adult fibroblast group (61.9% and 13.0%; P<0.05). The pregnancy rates of the recipients in the fetal and newborn fibroblast groups (60% and 80%, respectively) were higher than those in the adult fibroblast group. Eight, three, and one cloned piglet were obtained from reconstructed embryos of the fetal, newborn, and adult fibroblasts, respectively. Microsatellite analyses results indicated that the genotypes of all cloning piglets were identical to their donor cells and that the genetic homozygosity of the Banna miniature inbred pig was higher than those of the recipients. Therefore, the offspring was successfully cloned using the fetal, newborn, and adult fibroblasts of Banna miniature inbred pig as donor cells.