Abstract Background Despite years of research, porcine-induced pluripotent stem cells (piPSCs) with germline chimeric capacity have not been established. Furthermore, the key transcription factors (TFs) defining the naïve state in piPSCs also remain elusive, even though TFs in the inner cell mass (ICM) are believed to be key molecular determinants of naïve pluripotency. In this study, interferon regulatory factor 1 (IRF-1) was screened to express higher in ICM than trophectoderm (TE). But the impact of IRF-1 on maintenance of pluripotency in piPSCs was not determined. Methods Transcriptome profiles of the early ICM were analyzed to determine highly interconnected TFs. Cells carrying these TFs’ reporter were used to as donor cells for somatic cell nuclear transfer to detect expression patterns in blastocysts. Next, IRF1-Flag was overexpressed in DOX-hLIF-2i piPSCs and AP staining, qRT-PCR, and RNA-seq were conducted to examine the effect of IRF-1 on pluripotency. Then, the expression of IRF-1 in DOX-hLIF-2i piPSCs was labeled by GFP and qRT-PCR was conducted to determine the difference between GFP-positive and GFP-negative cells. Next, ChIP-Seq was conducted to identify genes target by IRF-1. Treatment with IL7 in wild-type piPSCs and STAT3 phosphorylation inhibitor in IRF-1 overexpressing piPSCs was conducted to confirm the roles of JAK-STAT3 signaling pathway in IRF-1’s regulation of pluripotency. Moreover, during reprogramming, IRF-1 was overexpressed and knocked down to determine the change of reprogramming efficiency. Results IRF-1 was screened to be expressed higher in porcine ICM than TE of d6~7 SCNT blastocysts. First, overexpression of IRF-1 in the piPSCs was observed to promote the morphology, AP staining, and expression profiles of pluripotency genes as would be expected when cells approach the naïve state. Genes, KEGG pathways, and GO terms related to the process of differentiation were also downregulated. Next, in the wild-type piPSCs, high-level fluorescence activated by the IRF-1 promoter was associated with higher expression of naïve related genes in piPSCs. Analysis by ChIP-Seq indicated that genes related to the JAK-STAT pathway, and expression of IL7 and STAT3 were activated by IRF-1. The inhibitor of STAT3 phosphorylation was observed could revert the expression of primed genes in IRF-1 overexpressing cells, but the addition of IL7 in culture medium had no apparent change in the cell morphology, AP staining results, or expression of pluripotency related genes. In addition, knockdown of IRF-1 during reprogramming appeared to reduce reprogramming efficiency, whereas overexpression exerted the converse effect. Conclusion The IRF-1 expressed in the ICM of pigs’ early blastocyst enhances the pluripotency of piPSCs, in part through promoting the JAK-STAT pathway.
Compared with rodents, pigs are closer to humans in terms of anatomy, metabolism and physiology, so they are ideal animal models of human diseases and xenotransplantation donors. In addition, as one of the most important livestock in China, pigs are closely related to our lives in terms of breeding improvement, disease prevention and animal welfare. In this review, we mainly summarize the research progress and future application of genetically modified pig models in the fields of xenotransplantation, molecular breeding and human disease models. We wish to take this opportunity to raise the awareness of researchers in related fields on cutting-edge technologies such as gene editing and understand the significance of genetically modified pig models in life science research.猪在解剖结构、代谢、生理生化等特征方面比啮齿类动物更接近人类，因此在模拟某些人类疾病以及提供异种移植器官等方面具有其他动物不可替代的优势，是理想的人类疾病动物模型和异种器官的供体。另外，猪作为我国畜牧业最重要的物种之一，猪的品种改良、疫病防控以及动物福利等问题都与人民生活息息相关。本文主要介绍了遗传修饰猪模型在分子育种、人类疾病模型以及异种器官移植领域的研究进展及未来应用前景，希望增进相关领域研究人员对基因编辑等前沿技术的了解，理解遗传修饰猪模型在生命科学研究中的重要意义。.
A great deal of research has been directed towards the problem of the treatment of waste water contaminated by uranium, and lots of processes available in nuclear facility. However, the key problem is that the existing processes are mainly applied for depleted uranium (DU), but not for enriched uranium (EU). So, it's imperative to develop a process for EU waste water treatment which comes from nuclear facility decommission or others. Consequently, the new process coagulation micro-filtration (CMF) combined with anion exchange (AE) was established to deal with the EU waste water. The experiments was divided into three parts CMF, AE and CMF+AE, respectively. The experiment results revealed that CMF is capable to deal with the waste water(the concentration of uranium in original waste water was 4.29 mg/L) at the pH 6.0-7.0, and its decontamination factor (DF) reached 103;AE is good at treating the original waste water when the concentration of which is not more than 500 g/L, meanwhile, the residence time is not less than 10 minutes, therefore, the treated water by AE meets the discharge standard; The DF number of the combined CMF + AE process might get 104. According to our research, the CMF + AE process provides a new choice for EU waste water treatment.
CRISPR-Cas9-mediated genome editing in sheep is of great use in both agricultural and biomedical applications. While targeted gene knockout by CRISPR-Cas9 through non-homologous end joining (NHEJ) has worked efficiently, the knockin efficiency via homology-directed repair (HDR) remains lower, which severely hampers the application of precise genome editing in sheep. Here, in sheep fetal fibroblasts (SFFs), we optimized several key parameters that affect HDR, including homology arm (HA) length and the amount of double-stranded DNA (dsDNA) repair template; we also observed synchronization of SFFs in G2/M phase could increase HDR efficiency. Besides, we identified three potent small molecules, RITA, Nutlin3, and CTX1, inhibitors of p53-MDM2 interaction, that caused activation of the p53 pathway, resulting in distinct G2/M cell-cycle arrest in response to DNA damage and improved CRISPR-Cas9-mediated HDR efficiency by 1.43- to 4.28-fold in SFFs. Furthermore, we demonstrated that genetic knockout of p53 could inhibit HDR in SFFs by suppressing the expression of several key factors involved in the HDR pathway, such as BRCA1 and RAD51. Overall, this study offers an optimized strategy for the usage of dsDNA repair template, more importantly, the application of MDM2 antagonists provides a simple and efficient strategy to promote CRISPR/Cas9-mediated precise genome editing in sheep primary cells.
Transplantation is an effective approach for treating end-stage organ failure. There has been a long-standing interest in xenotransplantation as a means of increasing the number of available organs. In the past decade, there has been tremendous progress in xenotransplantation accelerated by the development of rapid gene-editing tools and immunosuppressive therapy. Recently, the heart and kidney from pigs were transplanted into the recipients, which suggests that xenotransplantation has entered a new era. The genetic discrepancy and molecular incompatibility between pigs and primates results in barriers to xenotransplantation. An increasing body of evidence suggests that innate immune responses play an important role in all aspects of the xenogeneic rejection. Simultaneously, the role of important cellular components like macrophages, natural killer (NK) cells, and neutrophils, suggests that the innate immune response in the xenogeneic rejection should not be underestimated. Here, we summarize the current knowledge about the innate immune system in xenotransplantation and highlight the key issues for future investigations. A better understanding of the innate immune responses in xenotransplantation may help to control the xenograft rejection and design optimal combination therapies.
: Table S6. KEGG pathway enrichment analysis for differentially expressed genes (DEGs) identified using RNA-Seq data obtained from NANOG tdTomato knock-in positive PC-iPS cells treated with Activin A or SB431542.
Protein drug production encompasses various methods, among which animal bioreactors are emerging as a transgenic system. Animal bioreactors have the potential to reduce production costs and increase efficiency, thereby producing recombinant proteins that are crucial for therapeutic applications. Various species, including goats, cattle, rabbits, and poultry, have been genetically engineered to serve as bioreactors. This review delves into the analysis and comparison of different expression systems for protein drug production, highlighting the advantages and limitations of microbial, yeast, plant cell, and mammalian cell expression systems. Additionally, the emerging significance of genetically modified chickens as a potential bioreactor system for producing protein-based drugs is highlighted. The avian bioreactor enables the expression of target genes in ovarian cells, resulting in the production of corresponding gene expression products in egg whites. This production method boasts advantages such as a short cycle, high production efficiency, low research costs, and the expression products being closer to their natural state and easier to purify. It demonstrates immense potential in production applications, scientific research, and sustainable development. The utilization of advanced gene editing technologies, such as CRISPR/Cas9, has revolutionized the precision and efficiency of generating genetically modified chickens. This has paved the way for enhanced production of recombinant therapeutic proteins with desired glycosylation patterns and reduced immunogenic responses.
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