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.

[Activation of renal outer medullary potassium channel in the renal distal convoluted tubule by high potassium diet].

Renal outer medullary potassium (ROMK) channel is an important K+ excretion channel in the body, and K+ secreted by the ROMK channels is most or all source of urinary potassium. Previous studies focused on the ROMK channels of thick ascending limb (TAL) and collecting duct (CD), while there were few studies on the involvement of ROMK channels of the late distal convoluted tubule (DCT2) in K+ excretion. The purpose of the present study was mainly to record the ROMK channels current in renal DCT2 and observe the effect of high potassium diet on the ROMK channels by using single channel and whole-cell patch-clamp techniques. The results showed that a small conductance channel current with a conductance of 39 pS could be recorded in the apical membrane of renal DCT2, and it could be blocked by Tertiapin-Q (TPNQ), a ROMK channel inhibitor. The high potassium diet significantly increased the probability of ROMK channel current occurrence in the apical membrane of renal DCT2, and enhanced the activity of ROMK channel, compared to normal potassium diet (P < 0.01). Western blot results also demonstrated that the high potassium diet significantly up-regulated the protein expression levels of ROMK channels and epithelial sodium channel (ENaC), and down-regulated the protein expression level of Na+-Cl- cotransporter (NCC). Moreover, the high potassium diet significantly increased urinary potassium excretion. These results suggest that the high potassium diet may activate the ROMK channels in the apical membrane of renal DCT2 and increase the urinary potassium excretion by up-regulating the expression of renal ROMK channels.

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.

A Novel Regulatory Mechanism of Smooth Muscle α-Actin Expression by NRG-1/circACTA2/miR-548f-5p Axis.

RATIONALE: Neuregulin-1 (NRG-1) includes an extracellular epidermal growth factor-like domain and an intracellular domain (NRG-1-ICD). In response to transforming growth factor-ß1, its cleavage by proteolytic enzymes releases a bioactive fragment, which suppresses the vascular smooth muscle cell (VSMC) proliferation by activating ErbB (erythroblastic leukemia viral oncogene homolog) receptor. However, NRG-1-ICD function in VSMCs remains unknown. OBJECTIVE: Here, we characterize the function of NRG-1-ICD and underlying mechanisms in VSMCs. METHODS AND RESULTS: Immunofluorescence staining, Western blotting, and quantitative real-time polymerase chain reaction showed that NRG-1 was expressed in rat, mouse, and human VSMCs and was upregulated and cleaved in response to transforming growth factor-ß1. In the cytoplasm of HASMCs (human aortic smooth muscle cells), the NRG-1-ICD participated in filamentous actin formation by interacting with α-SMA (smooth muscle α-actin). In the nucleus, the Nrg-1-ICD induced circular ACTA2 (alpha-actin-2; circACTA2) formation by recruitment of the zinc-finger transcription factor IKZF1 (IKAROS family zinc finger 1) to the first intron of α-SMA gene. We further confirmed that circACTA2, acting as a sponge binding microRNA (miR)-548f-5p, interacted with miR-548f-5p targeting 3' untranslated region of α-SMA mRNA, which in turn relieves miR-548f-5p repression of the α-SMA expression and thus upregulates α-SMA expression, thereby facilitating stress fiber formation and cell contraction in HASMCs. Accordingly, in vivo studies demonstrated that the localization of the interaction of circACTA2 with miR-548f-5p is significantly decreased in human intimal hyperplastic arteries compared with normal arteries, implicating that dysregulation of circACTA2 and miR-548f-5p expression is involved in intimal hyperplasia. CONCLUSIONS: These results suggest that circACTA2 mediates NRG-1-ICD regulation of α-SMA expression in HASMCs via the NRG-1-ICD/circACTA2/miR-548f-5p axis. Our data provide a molecular basis for fine-tuning α-SMA expression and VSMC contraction by transcription factor, circular RNA, and microRNA.

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.

Low toxic and high soluble camptothecin derivative 2-47 effectively induces apoptosis of tumor cells in vitro.

The cytotoxic activity of camptothecin derivatives is so high that these compounds need to be further modified before their successful application as anti-cancer agents clinically. In this study, we reported the synthesis and biological evaluation of a novel camptothecin derivative called compound 2-47. The changes in structure did not reduce its activity to inhibit DNA topoisomerase I. Compound 2-47 induced apoptosis of many tumor cells including leukemia cells K562, Jurkat, HL-60, breast cancer cell BT-549, colon cancer cell HT-29 and liver cancer cell HepG2 with a half maximal inhibitory concentration (IC50) of 2- to 3-fold lower than HCPT as a control. In particular, 2-47 inhibited the proliferation of Jurkat cells with an IC50 of as low as 40 nM. By making use of Jurkat cell as a model, following treatment of Jurkat cells, compound 2-47 activated caspase-3 and PARP, resulting in a decreased Bcl-2/Bax ratio. These data showed that compound 2-47 induces Jurkat cell death through the mitochondrial apoptotic pathway. In addition, compound 2-47 showed a decreased cytotoxic activity against normal cells and an improved solubility in low-polar solvent. For example, compound 2-47 solutes in CHCl3 130-fold higher than HCPT. Taken together, our data demonstrated that camptothecin derivative 2-47 notably inhibits the tumor cell proliferation through mitochondrial-mediated apoptosis in vitro.

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.

5-(2-carboxyethenyl) isatin derivative induces G2/M cell cycle arrest and apoptosis in human leukemia K562 cells.

Our previous study successfully identified that the novel isatin derivative (E)-methyl 3-(1-(4-methoxybenzyl)-2,3-dioxoindolin-5-yl) acrylate (HKL 2H) acts as an anticancer agent at an inhibitory concentration (IC50) level of 3nM. In this study, the molecular mechanism how HKL 2H induces cytotoxic activity in the human chronic myelogenous leukemia K562 cells was investigated. Flow cytometric analysis showed that the cells were arrested in the G2/M phase and accumulated subsequently in the sub-G1 phase in the presence of HKL 2H. HKL 2H treatment down-regulated the expressions of CDK1 and cyclin B but up-regulated the level of phosphorylated CDK1. Annexin-V staining and the classic DNA ladder studies showed that HKL 2H induced the apoptosis of K562 cells. Our study further showed that HKL 2H treatment caused the dissipation of mitochondrial membrane potential, activated caspase-3 and lowered the Bcl-2/Bax ratio in K562 cells, suggesting that the HKL 2H-causing programmed cell death of K562 cells was caused via the mitochondrial apoptotic pathway. Taken together, our data demonstrated that HKL 2H, a 5-(2-carboxyethenyl) isatin derivative, notably induces G2/M cell cycle arrest and mitochondrial-mediated apoptosis in K562 cells, indicating that this compound could be a promising anticancer candidate for further investigation.

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.

The complete chloroplast genome and phylogenetic analysis of Lemmaphyllum carnosum var. drymoglossoides (baker) X. P. Wei, 2013.

Lemmaphyllum carnosum var. drymoglossoides (Baker) X. P. Wei, 2013 is a valuable medicinal fern in China. Its complete chloroplast genome was determined using Illumina paired-end sequencing. The genome was 157,571 bp in length with 130 genes, including 87 protein-coding genes, eight ribosomal RNA genes, and 35 tRNA genes. It displayed a quadripartite structure consisting of a small single-copy (SSC) of 21,691 bp, a large single-copy (LSC) of 81,106 bp, and two inverted repeats (IRs) of 27,387 bp, respectively. The phylogenetic results indicated that L. carnosum var. drymoglossoides exhibited the closest relationship with L. intermedium, and this study provided new information for the phylogenetic relationship of the Polypodiaceae family.

Exosomes-transferred LINC00668 Contributes to Thrombosis by Promoting NETs Formation in Inflammatory Bowel Disease.

Epidemiological studies show an association between inflammatory bowel disease (IBD) and increased risk of thrombosis. However, how IBD influences thrombosis remains unknown. The current study shows that formation of neutrophil extracellular traps (NETs) significantly increased in the dextran sulfate sodium (DSS)-induced IBD mice, which in turn, contributes to thrombus formation in a NETs-dependent fashion. Furthermore, the exosomes isolated from the plasma of the IBD mice induce arterial and venous thrombosis in vivo. Importantly, proinflammatory factors-exposed intestinal epithelial cells (inflamed IECs) promote neutrophils to release NETs through their secreted exosomes. RNA sequencing revealed that LINC00668 is highly enriched in the inflamed IECs-derived exosomes. Mechanistically, LINC00668 facilitates the translocation of neutrophil elastase (NE) from the cytoplasmic granules to the nucleus via its interaction with NE in a sequence-specific manner, thereby inducing NETs release and thrombus formation. Importantly, berberine (BBR) suppresses the nuclear translocation of NE and subsequent NETs formation by inhibiting the interaction of LINC00668 with NE, thus exerting its antithrombotic effects. This study provides a novel pathobiological mechanism linking IBD and thrombosis by exosome-mediated NETs formation. Targeting LINC00668 can serve as a novel molecular treatment strategy to treat IBD-related thrombosis.

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.

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.

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.