Generation of RAG2 Knockout Immune-Deficient Miniature Pigs.

Recombination-activating genes (RAGs) play a crucial role in the V(D)J recombination process and the development of immune cells. The development of the immune system and its mechanisms in pigs exhibit greater similarity to those of humans compared to other animals, thus rendering pigs a valuable tool for biomedical research. In this study, we utilized CRISPR/Cas9 gene editing and somatic cell nuclear transfer technology to generate RAG2 knockout (KO) pigs. Furthermore, we evaluated the impact of RAG2 KO on the immune organs and immune cell development through morphological observations, blood analysis and flow cytometry technology. RAG2 KO cell lines were used as donors for cloning. The reconstructed embryos were transplanted into 4 surrogate sows, and after 116 days of gestation, 2 sows gave birth to 12 live piglets, all of which were confirmed to be RAG2 KO. The thymus and spleen sizes of RAG2 KO pigs were significantly smaller than those of wild-type (WT) pigs. Hematoxylin-eosin staining results revealed that the thymus and spleen tissue structures of RAG2 KO pigs were disorganized and lacked the characteristic structures, indicating that RAG2 KO leads to dysplasia of the thymus and spleen. Hematological analysis demonstrated that the total number of white blood cells and lymphocytes in the circulation of RAG2 KO pigs was significantly lower, while the number of eosinophils was higher. Flow cytometry results indicated that the proportions of mature T and B lymphocytes were significantly reduced compared to WT pigs. These findings successfully verified the immunodeficiency phenotype of RAG2 KO pigs. This study may provide experimental animals for the development of tumor models and humanized animals.

Unraveling the impact of hyperleptinemia on female reproduction: insights from transgenic pig model.

BACKGROUND: Infertility is a growing global health concern affecting millions of couples worldwide. Among several factors, an extreme body weight adversely affects reproductive functions. Leptin is a well-known adipokine that serves as an endocrine signal between adiposity and fertility. However, the exact mechanisms underlying the effects of high leptin level on female reproduction remain unclear. METHODS: Transgenic pigs overexpressing leptin (♀) were produced by backcrossing and screened for leptin overexpression. The growth curve, fat deposition, reproductive performance, apoptosis, serum hormones and cholesterol production, RNA sequencing, and single-nucleus RNA sequencing (snRNA-seq) of the leptin-overexpressing pigs and wild-type group were evaluated. RESULTS: Transgenic pigs overexpressing leptin (♀) were obtained, which exhibited significantly reduced body weight, body size, and back fat thickness. These pigs manifested a late onset of puberty (330 ± 54.3 vs. 155 ± 14.7 days), irregular estrous behavior characterized by increased inter-estrous interval (29.2 ± 0 vs. 21.3 ± 0.7 days), and more number of matings until pregnancy (at least 3 times). This reproductive impairment in leptin pigs was related to hormonal imbalances characterized by increased levels of FSH, LH, prolactin, E2, P4, and TSH, altered steroidogenesis such as increased levels of serum cholesterol esters along with steroidogenic markers (StAR, CYP19A), and ovarian dysfunctions manifested by neutrophilic infiltration and low expression of caspase-3 positive cells in the ovaries. Moreover, bulk RNA sequencing of the ovaries also revealed neutrophilic infiltration followed by upregulation of inflammation-related genes. Furthermore, snRNA-seq reflected that leptin overexpression triggered immune response, suppressed follicle development and luteinization, resulting in metabolic dysfunction and hormone imbalance in the ovary. CONCLUSIONS: Low body weight in leptin overexpressing pigs adversely affects the reproductive performance, causing delayed puberty, irregular estrous cycles, and reduced breeding efficiency. This is linked to metabolic imbalances, an increased immune response, and altered ovarian functions. This study provides a theoretical basis for the complex mechanisms underlying leptin, and infertility by employing leptin-overexpressing female pigs.

Production of Four-Gene (GTKO/hCD55/hTBM/hCD39)-Edited Donor Pigs and Kidney Xenotransplantation.

BACKGROUND: The number of multigene-modified donor pigs for xenotransplantation is increasing with the advent of gene-editing technologies. However, it remains unclear which gene combination is suitable for specific organ transplantation. METHODS: In this study, we utilized CRISPR/Cas9 gene editing technology, piggyBac transposon system, and somatic cell cloning to construct GTKO/hCD55/hTBM/hCD39 four-gene-edited cloned (GEC) pigs and performed kidney transplantation from pig to rhesus monkey to evaluate the effectiveness of these GEC pigs. RESULTS: First, 107 cell colonies were obtained through drug selection, of which seven were 4-GE colonies. Two colonies were selected for somatic cell nuclear transfer (SCNT), resulting in seven fetuses, of which four were GGTA1 biallelic knockout. Out of these four, two fetuses had higher expression of hCD55, hTBM, and hCD39. Therefore, these two fetuses were selected for two consecutive rounds of cloning, resulting in 97 live piglets. After phenotype identification, the GGTA1 gene of these pigs was inactivated, and hCD55, hTBM, and hCD39 were expressed in cells and multiple tissues. Furthermore, the numbers of monkey IgM and IgG binding to the peripheral blood mononuclear cells (PBMCs) of the 4-GEC pigs were markedly reduced. Moreover, 4-GEC porcine PBMCs had greater survival rates than those from wild-type pigs through complement-mediated cytolysis assays. In pig-to-monkey kidney xenotransplantation, the kidney xenograft successfully survived for 11 days. All physiological and biochemical indicators were normal, and no hyperacute rejection or coagulation abnormalities were found after transplantation. CONCLUSION: These results indicate that the GTKO/hCD55/hTBM/hCD39 four-gene modification effectively alleviates immune rejection, and the pig kidney can functionally support the recipient monkey's life.

Generation and transcriptomic characterization of MIR137 knockout miniature pig model for neurodevelopmental disorders.

BACKGROUND: Neurodevelopmental disorders (NDD), such as autism spectrum disorders (ASD) and intellectual disorders (ID), are highly debilitating childhood psychiatric conditions. Genetic factors are recognized as playing a major role in NDD, with a multitude of genes and genomic regions implicated. While the functional validation of NDD-associated genes has predominantly been carried out using mouse models, the significant differences in brain structure and gene function between mice and humans have limited the effectiveness of mouse models in exploring the underlying mechanisms of NDD. Therefore, it is important to establish alternative animal models that are more evolutionarily aligned with humans. RESULTS: In this study, we employed CRISPR/Cas9 and somatic cell nuclear transplantation technologies to successfully generate a knockout miniature pig model of the MIR137 gene, which encodes the neuropsychiatric disorder-associated microRNA miR-137. The homozygous knockout of MIR137 (MIR137-/-) effectively suppressed the expression of mature miR-137 and led to the birth of stillborn or short-lived piglets. Transcriptomic analysis revealed significant changes in genes associated with neurodevelopment and synaptic signaling in the brains of MIR137-/- miniature pig, mirroring findings from human ASD transcriptomic data. In comparison to miR-137-deficient mouse and human induced pluripotent stem cell (hiPSC)-derived neuron models, the miniature pig model exhibited more consistent changes in critical neuronal genes relevant to humans following the loss of miR-137. Furthermore, a comparative analysis identified differentially expressed genes associated with ASD and ID risk genes in both miniature pig and hiPSC-derived neurons. Notably, human-specific miR-137 targets, such as CAMK2A, known to be linked to cognitive impairments and NDD, exhibited dysregulation in MIR137-/- miniature pigs. These findings suggest that the loss of miR-137 in miniature pigs affects genes crucial for neurodevelopment, potentially contributing to the development of NDD. CONCLUSIONS: Our study highlights the impact of miR-137 loss on critical genes involved in neurodevelopment and related disorders in MIR137-/- miniature pigs. It establishes the miniature pig model as a valuable tool for investigating neurodevelopmental disorders, providing valuable insights for potential applications in human research.

Dysfunction of Complementarity Determining Region 1 Encoded by T Cell Receptor Beta Variable Gene Is Potentially Associated with African Swine Fever Virus Infection in Pigs.

The beta T-cell receptor (TRB) expressed by beta T cells is essential for foreign antigen recognition. The TRB locus contains a TRBV family that encodes three complementarity determining regions (CDRs). CDR1 is associated with antigen recognition and interactions with MHC molecules. In contrast to domestic pigs, African suids lack a 284-bp segment spanning exons 1 and 2 of the TRBV27 gene that contains a sequence encoding CDR1. In this study, we used the African swine fever virus (ASFV) as an example to investigate the effect of deleting the TRBV27-encoded CDR1 on the resistance of domestic pigs to exotic pathogens. We first successfully generated TRBV27-edited fibroblasts with disruption of the CDR1 sequence using CRISPR/Cas9 technology and used them as donor cells to generate gene-edited pigs via somatic cell nuclear transfer. The TRBV-edited and wild-type pigs were selected for synchronous ASFV infection. White blood cells were significantly reduced in the genetically modified pigs before ASFV infection. The genetically modified and wild-type pigs were susceptible to ASFV and exhibited typical fevers (>40 °C). However, the TRBV27-edited pigs had a higher viral load than the wild-type pigs. Consistent with this, the gene-edited pigs showed more clinical signs than the wild-type pigs. In addition, both groups of pigs died within 10 days and showed similar severe lesions in organs and tissues. Future studies using lower virulence ASFV isolates are needed to determine the relationship between the TRBV27 gene and ASFV infection in pigs over a relatively long period.

Generation of stable integration-free pig induced pluripotent stem cells under chemically defined culture condition.

Genome integration-free pig induced pluripotent stem cells (iPSCs) bring tremendous value in pre-clinical testing of regenerative medicine, as well as conservation and exploitation of endangered or rare local pig idioplasmatic resources. However, due to a lack of appropriate culture medium, efficient induction and stable maintenance of pig iPSCs with practical value remains challenging. Here, we established an efficient induction system for exogenous gene-independent iPSCs under chemically defined culture condition previously used for generation of stable pig pre-gastrulation epiblast stem cells (pgEpiSCs). WNT suppression was found to play an essential role in establishment of exogenous gene-independent iPSCs. Strikingly, stable integration-free pig iPSCs could be established from pig somatic cells using episomal vectors in this culture condition. The iPSCs had pluripotency features and transcriptome characteristics approximating pgEpiSCs. More importantly, this induction system may be used to generate integration-free iPSCs from elderly disabled rare local pig somatic cells and the iPSCs could be gene-edited and used as donor cells for nuclear transfer. Our results provide novel insights into potential applications for genetic breeding of livestock species and pre-clinical evaluation of regenerative medicine.

In vitro and in vivo development of interspecies Asian elephant embryos reconstructed with pig enucleated oocytes.

Interspecies somatic cell nuclear transfer (iSCNT) has an immense potential to rescue endangered animals and extinct species like mammoths. In this study, we successfully established an Asian elephant's fibroblast cell lines from ear tissues, performed iSCNT with porcine oocytes and evaluated the in vitro and in vivo development of reconstructed embryos. A total of 7780 elephant-pig iSCNT embryos were successfully reconstructed and showed in vitro development with cleavage rate, 4-cell, 8-cell and blastocyst rate of 73.01, 30.48, 5.64, and 4.73%, respectively. The total number of elephant-pig blastocyte cells and diameter of hatched blastocyte was 38.67 and 252.75 µm, respectively. Next, we designed species-specific markers targeting EDNRB, AGRP and TYR genes to verify the genome of reconstructed embryos with donor nucleus/species. The results indicated that 53.2, 60.8, and 60.8% of reconstructed embryos (n = 235) contained elephant genome at 1-cell, 2-cell and 4-cell stages, respectively. However, the percentages decreased to 32.3 and 32.7% at 8-cell and blastocyst stages, respectively. Furthermore, we also evaluated the in vivo development of elephant-pig iSCNT cloned embryos and transferred 2260 reconstructed embryos into two surrogate gilts that successfully became pregnant and a total of 11 (1 and 10) fetuses were surgically recovered after 17 and 19 days of gestation, respectively. The crown-rump length and width of elephant-pig cloned fetuses were smaller than the control group. Unfortunately, none of these fetuses contained elephant genomes, which suggested that elephant embryos failed to develop in vivo. In conclusion, we successfully obtained elephant-pig reconstructed embryos for the first time and these embryos are able to develop to blastocyst, but the in vivo developmental failure needs further investigated.

Development of RAG2 -/- IL2Rγ -/Y immune deficient FAH-knockout miniature pig.

Human hepatocyte transplantation for liver disease treatment have been hampered by the lack of quality human hepatocytes. Pigs with their large body size, longevity and physiological similarities with human are appropriate animal models for the in vivo expansion of human hepatocytes. Here we report on the generation of RAG2-/-IL2Rγ-/YFAH-/- (RGFKO) pigs via CRISPR/Cas9 system and somatic cell nuclear transfer. We showed that thymic and splenic development in RGFKO pigs was impaired. V(D)J recombination processes were also inactivated. Consequently, RGFKO pigs had significantly reduced numbers of porcine T, B and NK cells. Moreover, due to the loss of FAH, porcine hepatocytes continuously undergo apoptosis and consequently suffer hepatic damage. Thus, RGFKO pigs are both immune deficient and constantly suffer liver injury in the absence of NTBC supplementation. These results suggest that RGFKO pigs have the potential to be engrafted with human hepatocytes without immune rejection, thereby allowing for large scale expansion of human hepatocytes.

Small Ruminant Farming in Tribal Areas of Dera Ghazi Khan, Punjab, Pakistan.

Provincially Administered Tribal Areas (PATA) of Punjab-Pakistan are comprised of hilly mountains with small ruminants as a sole source of income. In this study, farming practices, productivity, health and the economic value of sheep were evaluated in PATA through a survey of farmers (n = 138) holding 11,558 heads of sheep. Out of a total population, 87% were non-descriptive flocks, and 9% and 4% were purebred flocks belonging to the Kajli and Thali populations, respectively. Sheep flocks were mainly (86%) reared under the traditional production system and had a delayed onset of puberty. There was low influence of season on the reproduction, and the majority of flocks (78%) were bred throughout the year. The lack of proper vaccination and poor management exposed the flocks to bacterial, viral and parasitic infections, which lead to high mortality in lambs (~22%) and adults (~32%). The share of sheep in farmers livelihood was 42%, and only 20% of producers' living standard was improved with sheep farming, but the rise in rearing more sheep was quite low (20%). Although the livestock department arranged farmers' training, the majority of farmers (83%) never participated in training and had no knowledge of modern technologies. Collectively, the traditional sheep production systems, poor management, lack of vaccination, marketing channels and farmers training hampered the sheep rearing and producers' livelihood in the PATA of Punjab-Pakistan. However, developing model livestock farms, conducting farmer training, establishing a viable market for dairy products, and introducing subsidy policy interventions can improve the sheep farming in these areas.

Production of Triple-Gene (GGTA1, B2M and CIITA)-Modified Donor Pigs for Xenotransplantation.

Activation of human immune T-cells by swine leukocyte antigens class I (SLA-I) and class II (SLA-II) leads to xenograft destruction. Here, we generated the GGTA1, B2M, and CIITA (GBC) triple-gene-modified Diannan miniature pigs, analyzed the transcriptome of GBC-modified peripheral blood mononuclear cells (PBMCs) in the pig's spleen, and investigated their effectiveness in anti-immunological rejection. A total of six cloned piglets were successfully generated using somatic cell nuclear transfer, one of them carrying the heterozygous mutations in triple genes and the other five piglets carrying the homozygous mutations in GGTA1 and CIITA genes, but have the heterozygous mutation in the B2M gene. The autopsy of GBC-modified pigs revealed that a lot of spot bleeding in the kidney, severe suppuration and necrosis in the lungs, enlarged peripulmonary lymph nodes, and adhesion between the lungs and chest wall were found. Phenotyping data showed that the mRNA expressions of triple genes and protein expressions of B2M and CIITA genes were still detectable and comparable with wild-type (WT) pigs in multiple tissues, but α1,3-galactosyltransferase was eliminated, SLA-I was significantly decreased, and four subtypes of SLA-II were absent in GBC-modified pigs. In addition, even in swine umbilical vein endothelial cells (SUVEC) induced by recombinant porcine interferon gamma (IFN-γ), the expression of SLA-I in GBC-modified pig was lower than that in WT pigs. Similarly, the expression of SLA-II DR and DQ also cannot be induced by recombinant porcine IFN-γ. Through RNA sequencing (RNA-seq), 150 differentially expressed genes were identified in the PBMCs of the pig's spleen, and most of them were involved in immune- and infection-relevant pathways that include antigen processing and presentation and viral myocarditis, resulting in the pigs with GBC modification being susceptible to pathogenic microorganism. Furthermore, the numbers of human IgM binding to the fibroblast cells of GBC-modified pigs were obviously reduced. The GBC-modified porcine PBMCs triggered the weaker proliferation of human PBMCs than WT PBMCs. These findings indicated that the absence of the expression of α1,3-galactosyltransferase and SLA-II and the downregulation of SLA-I enhanced the ability of immunological tolerance in pig-to-human xenotransplantation.

Construction of PIK3C3 Transgenic Pig and Its Pathogenesis of Liver Damage.

As a member of the PIKs family, PIK3C3 participates in autophagy and plays a central role in liver function. Several studies demonstrated that the complete suppression of PIK3C3 in mammals can cause hepatomegaly and hepatosteatosis. However, the function of PIK3C3 overexpression on the liver and other organs is still unknown. In this study, we successfully generated PIK3C3 transgenic pigs through somatic cell nuclear transfer (SCNT) by designing a specific vector for the overexpression of PIK3C3. Plasmid identification was performed through enzyme digestion and transfected into the fetal fibroblasts derived from Diannan miniature pigs. After 2 weeks of culturing, six positive colonies obtained from a total of 14 cell colonies were identified through PCR. One positive cell line was selected as the donor cell line for SCNT for the construction of PIK3C3transgenic pigs. Thirty single blastocysts were collected and identified as PIK3C3 transgenic-positive blastocysts. Two surrogates became pregnant after transferring the reconstructed embryos into four surrogates. Fetal fibroblasts of PIK3C3-positive fetuses identified through PCR were used as donor cells for SCNT to generate PIK3C3 transgenic pigs. To further explore the function of PIK3C3 overexpression, genotyping and phenotyping of the fetuses and piglets obtained were performed by PCR, immunohistochemical, HE, and apoptosis staining. The results showed that inflammatory infiltration and vacuolar formation in hepatocytes and apoptotic cells, and the mRNA expression of NF-κB, TGF-ß1, TLR4, TNF-α, and IL-6 significantly increased in the livers of PIK3C3 transgenic pigs when compared with wild-type (WT) pigs. Immunofluorescence staining showed that LC3B and LAMP-1-positive cells increased in the livers of PIK3C3 transgenic pigs. In the EBSS-induced autophagy of the porcine fibroblast cells (PFCs), the accumulated LC3II protein was cleared faster in PIK3C3 transgenic (PFCs) thanWT (PFCs). In conclusion, PIK3C3 overexpression promoted autophagy in the liver and associated molecular mechanisms related to the activation of ULK1, AMBR1, DRAM1, and MTOR, causing liver damage in pigs. Therefore, the construction of PIK3C3 transgenic pigs may provide a new experimental animal resource for liver diseases.

Inhibition of FOXO1­mediated autophagy promotes paclitaxel­induced apoptosis of MDA­MB­231 cells.

Triple­negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, and it often becomes resistant to paclitaxel (PTX) therapy. Autophagy plays an important cytoprotective role in PTX­induced tumor cell death, and targeting autophagy has been promising for improving the efficacy of tumor chemotherapy in recent years. The aim of the present study was to clarify the mechanism of PTX inducing autophagy in TNBC cells to provide a potential clinical chemotherapy strategy of PTX for TNBC. The present study reported that PTX induced both apoptosis and autophagy in MDA­MB­231 cells and that inhibition of autophagy promoted apoptotic cell death. Furthermore, it was found that forkhead box transcription factor O1 (FOXO1) enhanced PTX­induced autophagy through a transcriptional activation pattern in MDA­MB­231 cells, which was associated with the downstream target genes autophagy related 5, class III phosphoinositide 3­kinase vacuolar protein sorting 34, autophagy related 4B cysteine peptidase, beclin 1 and microtubule associated protein 1 light chain 3ß. Knocking down FOXO1 attenuated the survival of MDA­MB­231 cells in response to PTX treatment. These findings may be beneficial for improving the treatment efficacy of PTX and to develop autophagic targeted therapy for TNBC.

Delayed body development with reduced triglycerides levels in leptin transgenic pigs.

Leptin is a well-known adipokine that plays critical role in adiposity. To further investigate the role of leptin in adiposity, we utilized leptin overexpressing transgenic pigs and evaluated the effect of leptin on growth and development, fat deposition, and lipid metabolism at tissue and cell level. Leptin transgenic pigs were produced and divided into two groups: elevated leptin expression (leptin ( +)) and normal leptin expression group (control). Results indicated that leptin ( +) pigs had elevated leptin protein and mRNA expression levels and exhibited sluggish growth and development followed by decreased subcutaneous fat thickness, low serum triglycerides, saturated, unsaturated fatty acids and high cholesterol esters (p < 0.05). There were differences in the lipid metabolism related genes at different fat depots, including upregulation of PPARγ, AGPAT6, PLIN2, HSL and ATGL in subcutaneous, PPARγ in perirenal, and FAT/CD36 and PLIN2 in mesenteric adipose tissues and downregulation of AGPAT6 and ATGL in perirenal and AGPAT6 in mesenteric adipose tissues (p < 0.05). Additionally, in-vitro cultured leptin ( +) preadipocytes exhibited upregulation of PPARγ, FAT/CD36, ACACA, AGPAT, PLIN2, ATGL and HSL as compared to control (p < 0.05). These findings suggested that homeostasis imbalance in lipolysis and lipogenesis at adipose tissue and adipocytes levels led to low subcutaneous fat depots in leptin overexpression pigs. These pigs can act as model for obesity and related metabolic disorder.

Generation and characterization of stable pig pregastrulation epiblast stem cell lines.

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.

Efficient Generation of P53 Biallelic Mutations in Diannan Miniature Pigs Using RNA-Guided Base Editing.

The base editing 3 (BE3) system, a single-base gene editing technology developed using CRISPR/Cas9n, has a broad range of applications for human disease model construction and gene therapy, as it is highly efficient, accurate, and non-destructive. P53 mutations are present in more than 50% of human malignancies. Due to the similarities between humans and pigs at the molecular level, pig models carrying P53 mutations can be used to research the mechanism of tumorigenesis and improve tumor diagnosis and treatment. According to pathogenic mutations of the human P53 gene at W146* and Q100*, sgRNAs were designed to target exon 4 and exon 5 of the porcine P53 gene. The target editing efficiencies of the two sgRNAs were 61.9% and 50.0%, respectively. The editing efficiency of the BE3 system was highest (about 60%) when C (or G) was at the 5th base. Puromycin screening revealed that 75.0% (21/28) and 68.7% (22/32) of cell colonies contained a P53 mutation at sgRNA-Exon5 and sgRNA-Exon4, respectively. The reconstructed embryos from sgRNA-Exon5-5# were transferred into six recipient gilts, all of which aborted. The reconstructed embryos from sgRNA-Exon4-7# were transferred into 6 recipient gilts, 3 of which became pregnant, resulting in 14 live and 3 dead piglets. Sequencing analyses of the target site confirmed 1 P53 monoallelic mutation and 16 biallelic mutations. The qPCR analysis showed that the P53 mRNA expression level was significantly decreased in different tissues of the P53 mutant piglets (p < 0.05). Additionally, confocal microscopy and western blot analysis revealed an absence of P53 expression in the P53 mutant fibroblasts, livers, and lung tissues. In conclusion, a porcine cancer model with a P53 point mutation can be obtained via the BE3 system and somatic cell nuclear transfer (SCNT).

Improving porcine SCNT efficiency by selecting donor cells size.

Considerable advancements have recently been achieved in porcine somatic cell nuclear transfer (SCNT), but the efficiency remains low. Donor cell size might play an important role in SCNT, but its effects in pigs remain unclear. This study aimed to evaluate the efficiency of porcine SCNT by selecting donor cells of suitable size. Porcine fetal fibroblasts (PFFs) were divided into three groups, group S (small, d ≤ 13 µm), group M (medium, 13 µm 18 µm), and their biological characteristics were analyzed. Next, SCNT was performed using PFFs of different sizes to evaluate the developmental potential of reconstructed embryos. The data showed that PFFs in groups S, M and L accounted for 17.5%, 47.7% and 34.8% of cells, respectively. Morphologically, cells in group S exhibited clear and regular cell membranes and nuclei, whereas cells in groups M and L displayed varying degrees of cell membrane protuberance, karyo-pyknosis, autophagy and mitochondrial abnormalities. In addition, the growth status and proliferation capabilities of cells in group S were significantly better than those of group M and group L. The percentage of cells at G0/G1 in group S and M were significantly greater than group L. The senescence rate of group S was lower than group M and group L. The apoptosis rate of group S was significantly lower than that of group L but comparable to that of group M . The cleavage rate of group S was also significantly greater than that of group M but comparable to that of group L . The blastocyst rate of group S was significantly greater than that of group M and group L. The blastocyst cell numbers of group S were also significantly greater than those of group M and group L. These findings suggested that small PFFs with a diameter of less than 13 µm are more suitable donor cells for SCNT in pigs.Abbreviations: DMEM: Dulbecco's modified Eagle's medium; FBS: Fetal bovine serum; PBS: Phosphate buffer saline; PFFs: Porcine fetal fibroblast cells; SCNT: Somatic cell nuclear transfer.

Extensive germline genome engineering in pigs.

The clinical applicability of porcine xenotransplantation-a long-investigated alternative to the scarce availability of human organs for patients with organ failure-is limited by molecular incompatibilities between the immune systems of pigs and humans as well as by the risk of transmitting porcine endogenous retroviruses (PERVs). We recently showed the production of pigs with genomically inactivated PERVs. Here, using a combination of CRISPR-Cas9 and transposon technologies, we show that pigs with all PERVs inactivated can also be genetically engineered to eliminate three xenoantigens and to express nine human transgenes that enhance the pigs' immunological compatibility and blood-coagulation compatibility with humans. The engineered pigs exhibit normal physiology, fertility and germline transmission of the 13 genes and 42 alleles edited. Using in vitro assays, we show that cells from the engineered pigs are resistant to human humoral rejection, cell-mediated damage and pathogenesis associated with dysregulated coagulation. The extensive genome engineering of pigs for greater compatibility with the human immune system may eventually enable safe and effective porcine xenotransplantation.

Successful vitrification of early-stage porcine cloned embryos.

The objective of this study was to investigate the survival and development of porcine cloned embryos vitrified by Cryotop carrier at the zygote, 2- and 4-cell stages. The quality of resultant blastocysts was evaluated according to their total cell number, apoptotic cell rate, reactive oxygen species (ROS) production, glutathione (GSH) content and mRNA expression levels of genes related to embryonic development. The survival rates of zygotes, 2- and 4-cell embryos after vitrification did not differ from those of their fresh counterparts. Vitrification still resulted in significantly decreased blastocyst formation rates of these early-stage embryos. Moreover, the total cells, apoptotic rate, ROS and GSH levels in resultant blastocysts were unaffected by vitrification. The mRNA expression levels of PCNA, CPT1, POU5F1 and DNMT3B in the blastocysts derived from vitrified early-stage embryos were significantly higher than those in the fresh blastocysts, but there was no change in expression of CDX2 and DNMT3A genes. In conclusion, our data demonstrate that the early-stage porcine cloned embryos including zygotes, 2- and 4-cells can be successfully vitrified, with respectable blastocyst yield and quality.

Effect of CRISPR/Cas9-mediated PD-1 gene knockout on proliferation, phenotype,IFN-γ and IL-2 secretion of T cells in Cynomolgus monkeys / 中国肿瘤生物治疗杂志

@#[Abstract] Objective: To investigate the effects of CRISPR/Cas9 gene editing mediated PD-1 knockdown on the proliferation, phenotype, IFN-γ and IL-2 secretion of T cells in Cynomolgus monkeys. Methods: gRNA targeting PD-1 gene of Cynomolgus monkey was designed, and the corresponding plasmid was constructed and extracted. peripheral blood mononuclear cells (PBMCs) of Cynomolgus monkeys were isolated, and plasmid DNAs were added for transfection by using Lonza 4D electrorotometer. FACS analysis and fluorescence microscopy were used to detect transfection efficiency at 48 h after transfection. Genomic DNA of T cells was extracted for PCR amplification and T7E1 digestion identification. The proliferation of T cells was induced under the stimulation of human CD3 antibody and IL-2, and the cell growth curve was drawn. PI staining flow cytometry was used to detect cell cycle and the expression levels of CD4 and CD8, and ELISA was used to detect the secretion of IFN- γ and IL-2. Results: At 48 h after transfection, the cells with green fluorescent protein expression in experimental group were observed under fluorescence microscopy with a transfection efficiency of (21.6±3.2)%. T7E1 enzyme digestion results showed that the PCR product of genomic DNA of cells in experimental group showed 3 bands after digestion, including the target cleavage bands(243，197 bp). Compared with non-transfected cells, the cells in experimental group exhibited slow proliferation, delayed colony formation, with small volume and weak refraction; the number of T cells at G0/G1 phase of the experimental group was significantly increased (P<0.05), while the number of cells at G2/M phase was significantly reduced (P<0.05); and the secretion levels of IFN-γ and IL-2 in the cells of the experimental group increased significantly (both P<0.05). However, the difference in the expression levels of CD4 and CD8 was not statistically significant between the two groups (both P>0.05). Conclusion: PD-1 gene knockout can arrest T cells in Cynomolgus monkey at G0/G1 phase, thereby inhibiting its proliferation and increasing the secretion of IFN-γ and IL-2 in the meanwhile.

Endogenous leptin promotes autophagy in EBSS-induced PFCs.

Leptin is an important adipokine and plays a vital role in animals. However, the role of leptin in the autophagic response of pig fibroblast cells (PFCs) has not been fully elucidated. In this study, we investigated the relationship between leptin and autophagy as well as underlying molecular basis. We found that PFCs treated with EBSS could secrete leptin, and the leptin concentration in the supernatant of leptin transgenic PFCs was higher than that of WT PFCs. We found an increase in LC3-II protein level and a decrease in p62 protein level in treated leptin transgenic PFCs compared with treated WT PFCs. Meanwhile, we observed an increase of autophagosomes by transmission electron microscopy and an enhancement of the accumulation of LC3 puncta in the cytoplasm of treated leptin transgenic PFCs, and these effects were further augmented by Baf A1 treatment. Furthermore, we detected the expression levels of 7 autophagy signaling pathway genes and 17 autophagy-related (ATG) genes by q-PCR. We found that between the two types of EBSS-treated cells 3 genes expression pattern were significantly different among the 7 autophagy signaling pathway genes and 8 genes expression pattern were significantly differernt among the ATG genes. These results indicated that leptin may promote autophagy and involving the downregulation of FOXO1 and LMNA genes via an unknown pathway which causes the upregulation of the 4 genes and the downregulation of 4 genes.