Cre-dependent Cas9-expressing pigs enable efficient in vivo genome editing.

Despite being time-consuming and costly, generating genome-edited pigs holds great promise for agricultural, biomedical, and pharmaceutical applications. To further facilitate genome editing in pigs, we report here establishment of a pig line with Cre-inducible Cas9 expression that allows a variety of ex vivo genome editing in fibroblast cells including single- and multigene modifications, chromosome rearrangements, and efficient in vivo genetic modifications. As a proof of principle, we were able to simultaneously inactivate five tumor suppressor genes (TP53, PTEN, APC, BRCA1, and BRCA2) and activate one oncogene (KRAS), achieved by delivering Cre recombinase and sgRNAs, which caused rapid lung tumor development. The efficient genome editing shown here demonstrates that these pigs can serve as a powerful tool for dissecting in vivo gene functions and biological processes in a temporal manner and for streamlining the production of genome-edited pigs for disease modeling.

Generation of Hoxc13 knockout pigs recapitulates human ectodermal dysplasia-9.

Atrichia and sparse hair phenotype cause distress to many patients. Ectodermal dysplasia-9 (ED-9) is a congenital condition characterized by hypotrichosis and nail dystrophy without other disorders, and Hoxc13 is a pathogenic gene for ED-9. However, mice carrying Hoxc13 mutation present several other serious disorders, such as skeletal defects, progressive weight loss and low viability. Mouse models cannot faithfully mimic human ED-9. In this study, we generated an ED-9 pig model via Hoxc13 gene knockout through single-stranded oligonucleotides (c.396C > A) combined with CRISPR/Cas9 and somatic cell nuclear transfer. Eight cloned piglets with three types of biallelic mutations (five piglets with Hoxc13c.396C > A/c.396C > A, two piglets with Hoxc13c.396C > A/c.396C > A + 1 and one piglet with Hoxc13Δ40/Δ40) were obtained. Hoxc13 was not expressed in pigs with all three mutation types, and the expression levels of Hoxc13-regulated genes, namely, Foxn1, Krt85 and Krt35, were decreased. The hair follicles displayed various abnormal phenotypes, such as reduced number of follicles and disarrayed hair follicle cable without normal hair all over the body. By contrast, the skin structure, skeleton phenotype, body weight gain and growth of Hoxc13 knockout pigs were apparently normal. The phenotypes of Hoxc13 mutation in pigs were similar to those in ED-9 patients. Therefore, Hoxc13 knockout pigs could be utilized as a model for ED-9 pathogenesis and as a hairless model for hair regeneration research. Moreover, the hairless pigs without other major abnormal phenotypes generated in this study could be effective models for other dermatological research because of the similarity between pig and human skins.

TRIB1 rs17321515 gene polymorphism increases the risk of coronary heart disease in general population and non-alcoholic fatty liver disease patients in Chinese Han population.

BACKGROUND: Present evidences suggested that TRIB1 rs17321515 polymorphism was tightly associated with the increased risk of NAFLD and CHD. CHD is one of the main complications of NAFLD, whether TRIB1 rs17321515 polymorphism could affect the risk of CHD in general population and NAFLD patients in Chinese Han population was remain unknown. The present study was designed to investigate the association between TRIB1 rs17321515 polymorphism and the risk of CHD in general population and NAFLD patients in Chinese Han population, and investigate the effect of TRIB1 rs17321515 polymorphism on serum lipid levels. PATIENTS AND METHODS: TRIB1 rs17321515 gene polymorphism was genotyped using the polymerase chain reaction (PCR) in healthy controls (n = 175), CHD patients (n = 155), NAFLD patients (n = 146), and NAFLD+CHD patients (n = 156). Serum lipid profiles were determined using biochemical methods. Statistical analyses were performed using SPSS 24.0 statistical software. RESULTS: The TRIB1 rs17321515 AA+GA genotypes were the significant risk factors for the CHD in general population (OR = 1.788; 95% CI: 1.104-2.897; P = 0.018) and in the NAFLD patients (OR = 1.760; 95% CI: 1.071-2.891; P = 0.026). After adjusted for age, gender, and body mass index, the risk for CHD in general population (OR = 1.857; 95% CI: 1.116-3.089; P = 0.017) and NAFLD patients was still significant (OR = 1.723; 95% CI: 1.033-2.873; P = 0.037). In addition, TRIB1 rs17321515 A carriers possess the higher lipid profiles in the included subjects. CONCLUSIONS: TRIB1 rs17321515 AA+GA genotypes were significant associated with the risk of CHD in general population and in NAFLD patients in Chinese Han population. The rs17321515 A allele increases the serum lipid profiles in included subjects.

RAG1/2 knockout pigs with severe combined immunodeficiency.

Pigs share many physiological, biochemical, and anatomical similarities with humans and have emerged as valuable large animal models for biomedical research. Considering the advantages in immune system resemblance, suitable size, and longevity for clinical practical and monitoring purpose, SCID pigs bearing dysfunctional RAG could serve as important experimental tools for regenerative medicine, allograft and xenograft transplantation, and reconstitution experiments related to the immune system. In this study, we report the generation and phenotypic characterization of RAG1 and RAG2 knockout pigs using transcription activator-like effector nucleases. Porcine fetal fibroblasts were genetically engineered using transcription activator-like effector nucleases and then used to provide donor nuclei for somatic cell nuclear transfer. We obtained 27 live cloned piglets; among these piglets, 9 were targeted with biallelic mutations in RAG1, 3 were targeted with biallelic mutations in RAG2, and 10 were targeted with a monoallelic mutation in RAG2. Piglets with biallelic mutations in either RAG1 or RAG2 exhibited hypoplasia of immune organs, failed to perform V(D)J rearrangement, and lost mature B and T cells. These immunodeficient RAG1/2 knockout pigs are promising tools for biomedical and translational research.

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.

Transgenic expression of human cytoxic T-lymphocyte associated antigen4-immunoglobulin (hCTLA4Ig) by porcine skin for xenogeneic skin grafting.

Porcine skin is frequently used as a substitute of human skin to cover large wounds in clinic practice of wound care. In our previous work, we found that transgenic expression of human cytoxicT-lymphocyte associated antigen4-immunoglobulin (hCTLA4Ig) in murine skin graft remarkably prolonged its survival in xenogeneic wounds without extensive immunosuppression in recipients, suggesting that transgenic hCTLA4Ig expression in skin graft may be an effective and safe method to prolong xenogeneic skin graft survival. In this work, using a transgene construct containing hCTLA4Ig coding sequence under the drive of human Keratine 14 (k14) promoter, hCTLA4Ig transgenic pigs were generated by somatic nuclear transfer. The derived transgenic pigs were healthy and exhibited no signs of susceptibility to infection. The hCTLA4Ig transgene was stably transmitted through germline over generations, and thereby a transgenic pig colony was established. In the derived transgenic pigs, hCTLA4Ig expression in skin was shown to be genetically stable over generations, and detected in heart, kidney and corneal as well as in skin. Transgenic hCTLA4Ig protein in pigs exhibited expected biological activity as it suppressed human lymphocyte proliferation in human mixed lymphocyte culture to extents comparable to those of commercially purchased purified hCTLA4Ig protein. In skin grafting from pigs to rats, transgenic porcine skin grafts exhibited remarkably prolonged survival compared to the wild-type skin grafts derived from the same pig strain (13.33 ± 3.64 vs. 6.25 ± 2.49 days, P < 0.01), further indicating that the transgenic hCTLA4Ig protein was biologically active and capable of extending porcine skin graft survival in xenogeneic wounds. The transgenic pigs generated in this work can be used as a reproducible resource to provide porcine skin grafts with extended survival for wound coverage, and also as donors to investigate the impacts of hCTLA4Ig on xenotransplantation of other organs (heart, kidney and corneal) due to the ectopic transgenic hCTLA4Ig expression.

Expression of Huntington's disease protein results in apoptotic neurons in the brains of cloned transgenic pigs.

Neurodegeneration is a hallmark of many neurological diseases, including Alzheimer's, Parkinson's and the polyglutamine diseases, which are all caused by misfolded proteins that accumulate in neuronal cells of the brain. Although apoptosis is believed to contribute to neurodegeneration in these cases, genetic mouse models of these diseases often fail to replicate apoptosis and overt neurodegeneration in the brain. Using nuclear transfer, we generated transgenic Huntington's disease (HD) pigs that express N-terminal (208 amino acids) mutant huntingtin with an expanded polyglutamine tract (105Q). Postnatal death, dyskinesia and chorea-like movement were observed in some transgenic pigs that express mutant huntingtin. Importantly, the transgenic HD pigs, unlike mice expressing the same transgene, displayed typical apoptotic neurons with DNA fragmentation in their brains. Also, expression of mutant huntingtin resulted in more neurons with activated caspase-3 in transgenic pig brains than that in transgenic mouse brains. Our findings suggest that species differences determine neuropathology and underscore the importance of large mammalian animals for modeling neurological disorders.

Interactive effects of low temperature and roscovitine (ROS) on meiotic resumption and developmental potential of goat oocytes.

The objective of this article was to study the effects of low temperature and roscovitine (ROS) on meiotic resumption and developmental potential of goat oocytes. Goat oocytes were cultured at different temperatures in medium containing different concentrations of ROS, and at the end of culture, oocytes were either matured or processed for light/confocal microscopy. The matured oocytes were activated chemically or fertilized in vitro for embryo development. Meiotic arrest was successfully maintained for 24 hr with 0, 50, and 200 microM ROS at 5, 20, and 38.5 degrees C, respectively. Following chemical activation, morulae/blastocysts (M/B) rates similar to untreated oocytes were obtained in oocytes that had been inhibited for 24 hr at 5 degrees C without ROS (Protocol 5C) or at 20 degrees C with 50 microM ROS (Protocol 20C) or for 8 hr at 38.5 degrees C with 200 microM ROS (Protocol 8 hr), but no blastulation was observed after oocytes were inhibited at 38.5 degrees C with 200 microM ROS for 24 hr. Following fertilization, however, while M/B rates similar to controls were achieved in oocytes treated with protocols 5C and 20C, few oocytes inhibited with Protocol 8 hr developed into morulae, due to a high incidence of polyspermy. Changes in GV chromatin configuration were not observed after inhibition with Protocol 5C, but were apparent after inhibition with protocols 20C and 8 hr, leading to a precocious germinal vesicle breakdown (GVBD) during subsequent maturation. Cortical granule (CG) migration and the formation of microtubule organizing centers occurred during inhibition and were more obvious in the absence of ROS. Significantly more oocytes inhibited by protocols 5C and 20C than by Protocol 8 hr completed CG migration after maturation. In conclusion, goat oocytes were tolerant to chilling and culture at lower temperatures with less ROS was better than culture at higher temperatures with more ROS for oocyte GVBD inhibition.

Chromatin Accessibility Dynamics during Chemical Induction of Pluripotency.

Despite its exciting potential, chemical induction of pluripotency (CIP) efficiency remains low and the mechanisms are poorly understood. We report the development of an efficient two-step serum- and replating-free CIP protocol and the associated chromatin accessibility dynamics (CAD) by assay for transposase-accessible chromatin (ATAC)-seq. CIP reorganizes the somatic genome to an intermediate state that is resolved under 2iL condition by re-closing previously opened loci prior to pluripotency acquisition with gradual opening of loci enriched with motifs for the OCT/SOX/KLF families. Bromodeoxyuridine, a critical ingredient of CIP, is responsible for both closing and opening critical loci, at least in part by preventing the opening of loci enriched with motifs for the AP1 family and facilitating the opening of loci enriched with SOX/KLF/GATA motifs. These changes differ markedly from CAD observed during Yamanaka-factor-driven reprogramming. Our study provides insights into small-molecule-based reprogramming mechanisms and reorganization of nuclear architecture associated with cell-fate decisions.

BMI1 enables interspecies chimerism with human pluripotent stem cells.

Human pluripotent stem cells (hPSCs) exhibit very limited contribution to interspecies chimeras. One explanation is that the conventional hPSCs are in a primed state and so unable  to form chimeras in pre-implantation embryos. Here, we show that the conventional hPSCs undergo rapid apoptosis when injected into mouse pre-implantation embryos. While, forced-expression of BMI1, a polycomb factor in hPSCs overcomes the apoptosis and enables hPSCs to integrate into mouse pre-implantation embryos and subsequently contribute to chimeras with both embryonic and extra-embryonic tissues. In addition, BMI1 also enables hPSCs to integrate into pre-implantation embryos of other species, such as rabbit and pig. Notably, BMI1 high expression and anti-apoptosis are also indicators for naïve hPSCs to form chimera in mouse embryos. Together, our findings reveal that the apoptosis is an initial barrier in interspecies chimerism using hPSCs and provide a rational to improve it.

XIST Derepression in Active X Chromosome Hinders Pig Somatic Cell Nuclear Transfer.

Pig cloning by somatic cell nuclear transfer (SCNT) remains extremely inefficient, and many cloned embryos undergo abnormal development. Here, by profiling transcriptome expression, we observed dysregulated chromosome-wide gene expression in every chromosome and identified a considerable number of genes that are aberrantly expressed in the abnormal cloned embryos. In particular, XIST, a long non-coding RNA gene, showed high ectopic expression in abnormal embryos. We also proved that nullification of the XIST gene in donor cells can normalize aberrant gene expression in cloned embryos and enhance long-term development capacity of the embryos. Furthermore, the increased quality of XIST-deficient embryos was associated with the global H3K9me3 reduction. Injection of H3K9me demethylase Kdm4A into NT embryos could improve the development of pre-implantation stage embryos. However, Kdm4A addition also induced XIST derepression in the active X chromosome and thus was not able to enhance the in vivo long-term developmental capacity of porcine NT embryos.

Generation of Knock-In Pigs Carrying Oct4-tdTomato Reporter through CRISPR/Cas9-Mediated Genome Engineering.

The porcine pluripotent cells that can generate germline chimeras have not been developed. The Oct4 promoter-based fluorescent reporter system, which can be used to monitor pluripotency, is an important tool to generate authentic porcine pluripotent cells. In this study, we established a porcine Oct4 reporter system, wherein the endogenous Oct4 promoter directly controls red fluorescent protein (RFP). 2A-tdTomato sequence was inserted to replace the stop codon of the porcine Oct4 gene by homogenous recombination (HR). Thus, the fluorescence can accurately show the activation of endogenous Oct4. Porcine fetal fibroblast (PFF) lines with knock-in (KI) of the tdTomato gene in the downstream of endogenous Oct4 promoter were achieved using the CRISPR/CAS9 system. Transgenic PFFs were used as donor cells for somatic cell nuclear transfer (SCNT). Strong RFP expression was detected in the blastocysts and genital ridges of SCNT fetuses but not in other tissues. Two viable transgenic piglets were also produced by SCNT. Reprogramming of fibroblasts from the fetuses and piglets by another round of SCNT resulted in tdTomato reactivation in reconstructed blastocysts. Result indicated that a KI porcine reporter system to monitor the pluripotent status of cells was successfully developed.

Cytoplasmic mislocalization of RNA splicing factors and aberrant neuronal gene splicing in TDP-43 transgenic pig brain.

BACKGROUND: TAR DNA-binding protein 43 (TDP-43) is a nuclear protein, but it is redistributed in the neuronal cytoplasm in both amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Because small transgenic animal models often lack cytoplasmic TDP-43, how the cytoplasmic accumulation of TDP-43 contributes to these diseases remains unclear. The current study is aimed at studying the mechanism of cytoplasmic pathology of TDP-43. RESULTS: We established transgenic pigs expressing mutant TDP-43 (M337V). This pig model shows severe phenotypes and early death. We found that transgenic TDP-43 is also distributed in the cytoplasm of neuronal cells in the spinal cord and brain. Transgenic TDP-43 interacts with PSF, an RNA splicing factor that associates with NeuN to regulate neuronal RNA splicing. The interaction of TDP-43, PSF and NeuN causes PSF and NeuN mislocalize into the neuronal cytoplasm in transgenic pigs. Consistently, abnormal PSF-related neuronal RNA splicing is seen in TDP-43 transgenic pigs. The cytoplasmic localization of PSF and NeuN as well as abnormal PSF-related neuronal RNA splicing was also found in ALS patient brains. CONCLUSION: Our findings from a large mammalian model suggest that cytoplasmic mutant TDP-43 could reduce the nuclear function of RNA splicing factors, contributing to neuropathology.

Production of transgenic pigs over-expressing the antiviral gene Mx1.

The myxovirus resistance gene (Mx1) has a broad spectrum of antiviral activities. It is therefore an interesting candidate gene to improve disease resistance in farm animals. In this study, we report the use of somatic cell nuclear transfer (SCNT) to produce transgenic pigs over-expressing the Mx1 gene. These transgenic pigs express approximately 15-25 times more Mx1 mRNA than non-transgenic pigs, and the protein level of Mx1 was also markedly enhanced. We challenged fibroblast cells isolated from the ear skin of transgenic and control pigs with influenza A virus and classical swine fever virus (CFSV). Indirect immunofluorescence assay (IFA) revealed a profound decrease of influenza A proliferation in Mx1 transgenic cells. Growth kinetics showed an approximately 10-fold reduction of viral copies in the transgenic cells compared to non-transgenic controls. Additionally, we found that the Mx1 transgenic cells were more resistant to CSFV infection in comparison to non-transgenic cells. These results demonstrate that the Mx1 transgene can protect against viral infection in cells of transgenic pigs and indicate that the Mx1 transgene can be harnessed to develop disease-resistant pigs.

Species-dependent neuropathology in transgenic SOD1 pigs.

Mutations in the human copper/zinc superoxide dismutase 1 (hSOD1) gene cause familial amyotrophic lateral sclerosis (ALS). It remains unknown whether large animal models of ALS mimic more pathological events seen in ALS patients via novel mechanisms. Here, we report the generation of transgenic pigs expressing mutant G93A hSOD1 and showing hind limb motor defects, which are germline transmissible, and motor neuron degeneration in dose- and age-dependent manners. Importantly, in the early disease stage, mutant hSOD1 did not form cytoplasmic inclusions, but showed nuclear accumulation and ubiquitinated nuclear aggregates, as seen in some ALS patient brains, but not in transgenic ALS mouse models. Our findings revealed that SOD1 binds PCBP1, a nuclear poly(rC) binding protein, in pig brain, but not in mouse brain, suggesting that the SOD1-PCBP1 interaction accounts for nuclear SOD1 accumulation and that species-specific targets are key to ALS pathology in large mammals and in humans.

Highly efficient generation of GGTA1 biallelic knockout inbred mini-pigs with TALENs.

Inbred mini-pigs are ideal organ donors for future human xenotransplantations because of their clear genetic background, high homozygosity, and high inbreeding endurance. In this study, we chose fibroblast cells from a highly inbred pig line called Banna mini-pig inbred line (BMI) as donor nuclei for nuclear transfer, combining with transcription activator-like effector nucleases (TALENs) and successfully generated α-1,3-galactosyltransferase (GGTA1) gene biallelic knockout (KO) pigs. To validate the efficiency of TALEN vectors, in vitro-transcribed TALEN mRNAs were microinjected into one-cell stage parthenogenetically activated porcine embryos. The efficiency of indel mutations at the GGTA1-targeting loci was as high as 73.1% (19/26) among the parthenogenetic blastocysts. TALENs were co-transfected into porcine fetal fibroblasts of BMI with a plasmid containing neomycin gene. The targeting efficiency reached 89.5% (187/209) among the survived cell clones after a 10 d selection. More remarkably 27.8% (58/209) of colonies were biallelic KO. Five fibroblast cell lines with biallelic KO were chosen as nuclear donors for somatic cell nuclear transfer (SCNT). Three miniature piglets with biallelic mutations of the GGTA1 gene were achieved. Gal epitopes on the surface of cells from all the three biallelic KO piglets were completely absent. The fibroblasts from the GGTA1 null piglets were more resistant to lysis by pooled complement-preserved normal human serum than those from wild-type pigs. These results indicate that a combination of TALENs technology with SCNT can generate biallelic KO pigs directly with high efficiency. The GGTA1 null piglets with inbred features created in this study can provide a new organ source for xenotransplantation research.

Establishment of a porcine Oct-4 promoter-driven EGFP reporter system for monitoring pluripotency of porcine stem cells.

Porcine pluripotent cells with the capacity to generate germ line chimeras have not been developed yet. The transcription factor Oct-4 is an important marker of undifferentiating status and a central regulator of pluripotency in cells. Establishment of an Oct-4 promoter-based reporter system, such as that used in mice, will be a useful tool for monitoring the differentiating statuses of porcine cells both in vivo and in vitro. In the present study, we constructed a vector, pOGN2, in which enhanced green fluorescent protein (EGFP) was driven by the porcine Oct-4 promoter. In pigs containing this vector, EGFP was expected to be specifically expressed in pluripotent cells. We delivered the vectors into porcine fetal fibroblasts (PEFs) using liposomes. After transfected PEFs were selected with G418, we established eight cell lines containing the pOGN2 vector. When transgenic cells were used as donor nuclei to make somatic cell nuclear transfer (SCNT) embryos, SCNT embryos derived from four transgenic cell lines expressed green fluorescence. When PEFs with pOGN2 vectors were infected with retroviral vectors encoding the four transcription factors (Oct-4, Sox2, Klf4, and c-Myc), EGFP-expressing iPS cell colonies were observed at day 20. This work lays a foundation that can be used to generate a pig strain with an Oct4-EGFP reporter system, which would be greatly helpful in studying the differentiating and reprogramming mechanisms of pig embryos.

Use of the 2A peptide for generation of multi-transgenic pigs through a single round of nuclear transfer.

Multiple genetic modifications in pigs can essentially benefit research on agriculture, human disease and xenotransplantation. Most multi-transgenic pigs have been produced by complex and time-consuming breeding programs using multiple single-transgenic pigs. This study explored the feasibility of producing multi-transgenic pigs using the viral 2A peptide in the light of previous research indicating that it can be utilized for multi-gene transfer in gene therapy and somatic cell reprogramming. A 2A peptide-based double-promoter expression vector that mediated the expression of four fluorescent proteins was constructed and transfected into primary porcine fetal fibroblasts. Cell colonies (54.3%) formed under G418 selection co-expressed the four fluorescent proteins at uniformly high levels. The reconstructed embryos, which were obtained by somatic cell nuclear transfer and confirmed to express the four fluorescent proteins evenly, were transplanted into seven recipient gilts. Eleven piglets were delivered by two gilts, and seven of them co-expressed the four fluorescent proteins at equivalently high levels in various tissues. The fluorescence intensities were directly observed at the nose, hoof and tongue using goggles. The results suggest that the strategy of combining the 2A peptide and double promoters efficiently mediates the co-expression of the four fluorescent proteins in pigs and is hence a promising methodology to generate multi-transgenic pigs by a single nuclear transfer.