Porcine germline genome engineering.

Germline editing, the process by which the genome of an individual is edited in such a way that the change is heritable, has been applied to a wide variety of animals [D. A. Sorrell, A. F. Kolb, Biotechnol. Adv. 23, 431-469 (2005); D. Baltimore et al., Science 348, 36-38 (2015)]. Because of its relevancy in agricultural and biomedical research, the pig genome has been extensively modified using a multitude of technologies [K. Lee, K. Farrell, K. Uh, Reprod. Fertil. Dev. 32, 40-49 (2019); C. Proudfoot, S. Lillico, C. Tait-Burkard, Anim. Front. 9, 6-12 (2019)]. In this perspective, we will focus on using pigs as the model system to review the current methodologies, applications, and challenges of mammalian germline genome editing. We will also discuss the broad implications of animal germline editing and its clinical potential.

Single-nucleus transcriptome sequencing reveals hepatic cell atlas in pigs.

BACKGROUND: As the largest substantive organ of animals, the liver plays an essential role in the physiological processes of digestive metabolism and immune defense. However, the cellular composition of the pig liver remains poorly understood. This investigation used single-nucleus RNA sequencing technology to identify cell types from liver tissues of pigs, providing a theoretical basis for further investigating liver cell types in pigs. RESULTS: The analysis revealed 13 cells clusters which were further identified 7 cell types including endothelial cells, T cells, hepatocytes, Kupffer cells, stellate cells, B cells, and cholangiocytes. The dominant cell types were endothelial cells, T cells and hepatocytes in the liver tissue of Dahe pigs and Dahe black pigs, which accounts for about 85.76% and 82.74%, respectively. The number of endothelial cells was higher in the liver tissue of Dahe pigs compared to Dahe black pigs, while the opposite tendency was observed for T cells. Moreover, functional enrichment analysis demonstrated that the differentially expressed genes in pig hepatic endothelial cells were significantly enriched in the protein processing in endoplasmic reticulum, MAPK signaling pathway, and FoxO signaling pathway. Functional enrichment analysis demonstrated that the differentially expressed genes in pig hepatic T cells were significantly enriched in the thyroid hormone signaling pathway, B cell receptor signaling pathway, and focal adhesion. Functional enrichment analysis demonstrated that the differentially expressed genes in pig hepatic hepatocytes were significantly enriched in the metabolic pathways. CONCLUSIONS: In summary, this study provides a comprehensive cell atlas of porcine hepatic tissue. The number, gene expression level and functional characteristics of each cell type in pig liver tissue varied between breeds.

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.

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.

Symptoms of systemic lupus erythematosus are diagnosed in leptin transgenic pigs.

Leptin is a well-known adipokine that plays a critical role in immune responses. To further explore the immunological roles of leptin, we developed a transgenic leptin pig controlled by the pig leptin (pleptin) promoter to overexpress leptin. Symptoms typically associated with systemic lupus erythematosus (SLE) were evident in this transgenic pig strain, including anemia, leukopenia, and thrombocytopenia as well as kidney and liver impairment. Histologically, there were increased immunoglobulin G (IgG) levels, elevated antiplatelet antibody (APA) levels, and deposition of immune complexes in the kidney and liver. In addition, anti-double-stranded DNA antibodies (dsDNAs), antinuclear antibodies (ANAs), and antinucleosome antibodies (ANuAs) were all significantly increased in serum immunological examinations. These findings were also accompanied by repression of the regulatory T cell (Treg) ratio. Significantly, glucocorticoid experimental therapies partially relieved the autoimmune responses and bleeding symptoms observed in these transgenic leptin pigs. Together, these results indicate that leptin plays a critical role in the development of autoimmune disorders and demonstrate that our transgenic leptin pigs can act as a valuable model of SLE.

Improved production of GTKO/hCD55/hCD59 triple-gene-modified Diannan miniature pigs for xenotransplantation by recloning.

Multiple genetic modification is necessary for successful xenotransplantation from pigs. However, multiple-genetically modified cells usually suffer from various drug selections and long-term in vitro culture, which have a poor performance for somatic cell nuclear transfer (SCNT) to produce genetically modified pigs. We used to generate GTKO/hCD55/hCD59 triple-gene modified pigs by using drug-selective cell lines for SCNT, but the majority of cloned pigs were transgenic-negative individuals. In this study, to improve the production efficiency of multiple genetically modified pigs, we performed the recloning process by using transgenic porcine fetal fibroblast cells. As a result, two fetuses expressing hCD55 and hCD59 were obtained from 12 live-cloned fetuses, and one carrying high transgene expression was selected as a source of donor cells for recloning. Then we obtained 12 cloned piglets, all GTKO and carrying hCD55 and hCD59. Both hCD55 and hCD59 were expressed in fibroblast cells, but the expression levels of hCD55 and hCD59 were different among these piglets. Furthermore, piglet P5# had the highest expression of hCD55 and hCD59 in fibroblast cells than other piglets. Correspondingly, fibroblast cells of piglet P5# had significantly higher resistance against human serum-mediated cytolysis than those of piglet P11#. In conclusion, our results firstly provide support for improving efficiency of generating multiple genetically modified pig by recloning.

Generation of GHR-modified pigs as Laron syndrome models via a dual-sgRNAs/Cas9 system and somatic cell nuclear transfer.

BACKGROUND: Laron syndrome is an autosomal disease resulting from mutations in the growth hormone receptor (GHR) gene. The only therapeutic treatment for Laron syndrome is recombinant insulin-like growth factor I (IGF-I), which has been shown to have various side effects. The improved Laron syndrome models are important for better understanding the pathogenesis of the disease and developing corresponding therapeutics. Pigs have become attractive biomedical models for human condition due to similarities in anatomy, physiology, and metabolism relative to humans, which could serve as an appropriate model for Laron syndrome. METHODS: To further improve the GHR knockout (GHRKO) efficiency and explore the feasibility of precise DNA deletion at targeted sites, the dual-sgRNAs/Cas9 system was designed to target GHR exon 3 in pig fetal fibroblasts (PFFs). The vectors encoding sgRNAs and Cas9 were co-transfected into PFFs by electroporation and GHRKO cell lines were established by single cell cloning culture. Two biallelic knockout cell lines were selected as the donor cell line for somatic cell nuclear transfer for the generation of GHRKO pigs. The genotype of colonies, cloned fetuses and piglets were identified by T7 endonuclease I (T7ENI) assay and sequencing. The GHR expression in the fibroblasts and piglets was analyzed by confocal microscopy, quantitative polymerase chain reaction (q-PCR), western blotting (WB) and immunohistochemical (IHC) staining. The phenotype of GHRKO pigs was recapitulated through level detection of IGF-I and glucose, and measurement of body weight and body size. GHRKO F1 generation were generated by crossing with wild-type pigs, and their genotype was detected by T7ENI assay and sequencing. GHRKO F2 generation was obtained via self-cross of GHRKO F1 pigs. Their genotypes of GHRKO F2 generation was also detected by Sanger sequencing. RESULTS: In total, 19 of 20 single-cell colonies exhibited biallelic modified GHR (95%), and the efficiency of DNA deletion mediated by dual-sgRNAs/Cas9 was as high as 90% in 40 GHR alleles of 20 single-cell colonies. Two types of GHR allelic single-cell colonies (GHR-47/-1, GHR-47/-46) were selected as donor cells for the generation of GHRKO pigs. The reconstructed embryos were transferred into 15 recipient gilts, resulting in 15 GHRKO newborn piglets and 2 fetuses. The GHRKO pigs exhibited slow growth rates and small body sizes. From birth to 13 months old, the average body weight of wild-type pigs varied from 0.6 to 89.5 kg, but that of GHRKO pigs varied from only 0.9 to 37.0 kg. Biochemically, the knockout pigs exhibited decreased serum levels of IGF-I and glucose. Furthermore, the GHRKO pigs had normal reproduction ability, as eighteen GHRKO F1 piglets were obtained via mating a GHRKO pig with wild-type pigs and five GHRKO F2 piglets were obtained by self-cross of F1 generation, indicating that modified GHR alleles can pass to the next generation via germline transmission. CONCLUSION: The dual-sgRNAs/Cas9 is a reliable system for DNA deletion and that GHRKO pigs conform to typical phenotypes of those observed in Laron patients, suggesting that these pigs could serve as an appropriate model for Laron syndrome.

Efficient generation of P53 biallelic knockout Diannan miniature pigs via TALENs and somatic cell nuclear transfer.

BACKGROUND: Pigs have many features that make them attractive as biomedical models for various diseases, including cancer. P53 is an important tumor suppressor gene that exerts a central role in protecting cells from oncogenic transformation and is mutated in a large number of human cancers. P53 mutations occur in almost every type of tumor and in over 50% of all tumors. In a recent publication, pigs with a mutated P53 gene were generated that resulted in lymphoma and renal and osteogenic tumors. However, approximately 80% of human tumors have dysfunctional P53. A P53-deficient pig model is still required to elucidate. METHODS: Transcription activator-like effector nucleases (TALENs) were designed to target porcine P53 exon 4. The targeting activity was evaluated using a luciferase SSA recombination assay. P53 biallelic knockout (KO) cell lines were established from single-cell colonies of fetal fibroblasts derived from Diannan miniature pigs followed by electroporation with TALENs plasmids. One cell line was selected as the donor cell line for somatic cell nuclear transfer (SCNT) for the generation of P53 KO pigs. P53 KO stillborn fetuses and living piglets were obtained. Gene typing of the collected cloned individuals was performed by T7EI assay and sequencing. Fibroblast cells from Diannan miniature piglets with a P53 biallelic knockout or wild type were analyzed for the P53 response to doxorubicin treatment by confocal microscopy and western blotting. RESULTS: The luciferase SSA recombination assay revealed that the targeting activities of the designed TALENs were 55.35-fold higher than those of the control. Eight cell lines (8/19) were mutated for P53, and five of them were biallelic knockouts. One of the biallelic knockout cell lines was selected as nuclear donor cells for SCNT. The cloned embryos were transferred into five recipient gilts, three of them becoming pregnant. Five live fetuses were obtained from one surrogate by caesarean section after 38 days of gestation for genotyping. Finally, six live piglets and one stillborn piglet were collected from two recipients by caesarean section. Sequencing analyses of the target site confirmed the P53 biallelic knockout in all fetuses and piglets, consistent with the genotype of the donor cells. The qPCR analysis showed that the expression of the P53 mRNA had significant reduction in various tissues of the knockout piglets. Furthermore, confocal microscopy and western blotting analyses demonstrated that the fibroblast cells of Diannan miniature piglets with a P53 biallelic knockout were defective in mediating DNA damage when incubated with doxorubicin. CONCLUSION: TALENs combined with SCNT was successfully used to generate P53 KO Diannan miniature pigs. Although these genetically engineered Diannan miniature pigs had no tumorigenic signs, the P53 gene was dysfunctional. We believe that these pigs will provide powerful new resources for preclinical oncology and basic cancer research.

Efficient generation of GGTA1-null Diannan miniature pigs using TALENs combined with somatic cell nuclear transfer.

BACKGROUND: α1,3-Galactosyltransferase (GGTA1) is essential for the biosynthesis of glycoproteins and therefore a simple and effective target for disrupting the expression of galactose α-1,3-galactose epitopes, which mediate hyperacute rejection (HAR) in xenotransplantation. Miniature pigs are considered to have the greatest potential as xenotransplantation donors. A GGTA1-knockout (GTKO) miniature pig might mitigate or prevent HAR in xenotransplantation. METHODS: Transcription activator-like effector nucleases (TALENs) were designed to target exon 6 of porcine GGTA1 gene. The targeting activity was evaluated using a luciferase SSA recombination assay. Biallelic GTKO cell lines were established from single-cell colonies of fetal fibroblasts derived from Diannan miniature pigs following transfection by electroporation with TALEN plasmids. One cell line was selected as donor cell line for somatic cell nuclear transfer (SCNT) for the generation of GTKO pigs. GTKO aborted fetuses, stillborn fetuses and live piglets were obtained. Genotyping of the collected cloned individuals was performed. The Gal expression in the fibroblasts and one piglet was analyzed by fluorescence activated cell sorting (FACS), confocal microscopy, immunohistochemical (IHC) staining and western blotting. RESULTS: The luciferase SSA recombination assay revealed that the targeting activities of the designed TALENs were 17.1-fold higher than those of the control. Three cell lines (3/126) showed GGTA1 biallelic knockout after modification by the TALENs. The GGTA1 biallelic modified C99# cell line enabled high-quality SCNT, as evidenced by the 22.3 % (458/2068) blastocyst developmental rate of the reconstructed embryos. The reconstructed GTKO embryos were subsequently transferred into 18 recipient gilts, of which 12 became pregnant, and six miscarried. Eight aborted fetuses were collected from the gilts that miscarried. One live fetus was obtained from one surrogate by caesarean after 33 d of gestation for genotyping. In total, 12 live and two stillborn piglets were collected from six surrogates by either caesarean or natural birth. Sequencing analyses of the target site confirmed the homozygous GGTA1-null mutation in all fetuses and piglets, consistent with the genotype of the donor cells. Furthermore, FACS, confocal microscopy, IHC and western blotting analyses demonstrated that Gal epitopes were completely absent from the fibroblasts, kidneys and pancreas of one GTKO piglet. CONCLUSIONS: TALENs combined with SCNT were successfully used to generate GTKO Diannan miniature piglets.

Porcine Zygote Injection with Cas9/sgRNA Results in DMD-Modified Pig with Muscle Dystrophy.

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.

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.

What Have We Learned From In Vitro Studies About Pig-to-primate Organ Transplantation?

Natural preformed and de novo antibodies against pig antigens are a major cause of pig xenograft rejection in nonhuman primates (NHPs). In vivo studies in pig-to-NHP models are time consuming. In vitro assays, for example, antibody binding to pig cells, complement-dependent cytotoxicity assays, provide valuable information quickly and inexpensively. Using in vitro assays for several years, it has been documented that (1) during the first year of life, humans and NHPs develop anti-wild-type pig antibodies, but humans develop no or minimal antibody to triple-knockout (TKO) pig cells. (2) Some adult humans have no or minimal antibodies to TKO pig cells and are therefore unlikely to rapidly reject a TKO organ, particularly if the organ also expresses human "protective" proteins. (3) There is good correlation between immunoglobulin (Ig)M (but no t IgG) binding and complement injury. (4) All Old World NHPs develop antibodies to TKO pig cells and are not optimal recipients of TKO organs. (5) galactosyltransferase gene-knockout/ß4GalNT2KO pigs are preferred for Old World NHPs. (6) Humans develop anti-pig IgE and IgA antibodies against pig cells, but their role remains uncertain. (7) In a small percentage of allosensitized humans, antibodies that cross-react with swine leukocyte antigens may be detrimental to a pig organ xenograft. (8) Prior sensitization to pig antigens is unlikely to be detrimental to a subsequent allograft. (9) Deletion of expression of Gal and Neu5Gc is associated with a reduction in the T-cell response to pig cells. All of these valuable observations have largely predicted the results of in vivo studies.

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.

Expression levels of candidate genes for intramuscular fat deposition in two Banna mini-pig inbred lines divergently selected for fatness traits.

Intramuscular fat (IMF) content plays an important role in meat quality. Many genes involved in lipid and energy metabolism were identified as candidate genes for IMF deposition, since genetic polymorphisms within these genes were associated with IMF content. However, there is less information on the expression levels of these genes in the muscle tissue. This study aimed at investigating the expression levels of sterol regulating element binding protein-1c (SREBP-1c), diacylglycerol acyltransferase (DGAT-1), heart-fatty acids binding protein (H-FABP), leptin receptor (LEPR) and melanocortin 4 receptor (MC4R) genes and proteins in two divergent Banna mini-pig inbred lines (BMIL). A similar growth performance was found in both the fat and the lean BMIL. The fat meat and IMF content in the fat BMIL were significantly higher than in the lean BMIL, but the lean meat content was lower. The serum triacylglycerol (TAG) and free fatty acid (FFA) contents were significantly higher in the fat than in the lean BMIL. The expression levels of SREBP-1c, DGAT-1 and H-FABP genes and proteins in fat BMIL were increased compared to the lean BMIL. However, the expression levels of LEPR and MC4R genes and proteins were lower.

Towards Improving the Outcomes of Multiple Ovulation and Embryo Transfer in Sheep, with Particular Focus on Donor Superovulation.

Considerable improvements in sheep multiple ovulation and embryo transfer (MOET)protocols have been made; however, unlike for cattle, MOET is poorly developed in sheep, and thus has not been broadly applicable as a routine procedure. The tightly folded nature of the ewe cervix, the inconsistent ovarian response to various superovulatory treatments, and the requirement of labor to handle animals, particularly during large-scale production, has limited the implementation of successful MOET in sheep. Moreover, several extrinsic factors (e.g., sources, the purity of gonadotrophins and their administration) and intrinsic factors (e.g., breed, age, nutrition, reproductive status) severely limit the practicability of MOET in sheep and other domestic animals. In this review, we summarize the effects of different superovulatory protocols, and their respective ovarian responses, in terms of ovulation rate, and embryo recovery and transfer. Furthermore, various strategies, such as inhibin immunization, conventional superovulation protocols, and melatonin implants for improving the ovarian response, are discussed in detail. Other reproductive techniques and their relative advantages and disadvantages, such as artificial insemination (AI), and donor embryo recovery and transfer to the recipient through different procedures, which must be taken into consideration for achieving satisfactory results during any MOET program in sheep, are also summarized in this article.

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.

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.

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.

A multiple-tissue-specific magnetic resonance imaging model for diagnosing Parkinson's disease: a brain radiomics study.

Brain radiomics can reflect the characteristics of brain pathophysiology. However, the value of T1-weighted images, quantitative susceptibility mapping, and R2* mapping in the diagnosis of Parkinson's disease (PD) was underestimated in previous studies. In this prospective study to establish a model for PD diagnosis based on brain imaging information, we collected high-resolution T1-weighted images, R2* mapping, and quantitative susceptibility imaging data from 171 patients with PD and 179 healthy controls recruited from August 2014 to August 2019. According to the inclusion time, 123 PD patients and 121 healthy controls were assigned to train the diagnostic model, while the remaining 106 subjects were assigned to the external validation dataset. We extracted 1408 radiomics features, and then used data-driven feature selection to identify informative features that were significant for discriminating patients with PD from normal controls on the training dataset. The informative features so identified were then used to construct a diagnostic model for PD. The constructed model contained 36 informative radiomics features, mainly representing abnormal subcortical iron distribution (especially in the substantia nigra), structural disorganization (e.g., in the inferior temporal, paracentral, precuneus, insula, and precentral gyri), and texture misalignment in the subcortical nuclei (e.g., caudate, globus pallidus, and thalamus). The predictive accuracy of the established model was 81.1 ± 8.0% in the training dataset. On the external validation dataset, the established model showed predictive accuracy of 78.5 ± 2.1%. In the tests of identifying early and drug-naïve PD patients from healthy controls, the accuracies of the model constructed on the same 36 informative features were 80.3 ± 7.1% and 79.1 ± 6.5%, respectively, while the accuracies were 80.4 ± 6.3% and 82.9 ± 5.8% for diagnosing middle-to-late PD and those receiving drug management, respectively. The accuracies for predicting tremor-dominant and non-tremor-dominant PD were 79.8 ± 6.9% and 79.1 ± 6.5%, respectively. In conclusion, the multiple-tissue-specific brain radiomics model constructed from magnetic resonance imaging has the ability to discriminate PD and exhibits the advantages for improving PD diagnosis.