Animal cloning is of great importance to the production of transgenic and genome-edited livestock. Especially for multiple gene-editing operations, recloning is one of the most feasible methods for ...livestock. In addition, a multiple-round cloning method is practically necessary for animal molecular breeding. However, cloning efficiency remains extremely low, especially for serial cloning, which seriously impedes the development of livestock breeding based on genome editing technology. The incomplete reprogramming and failure in oocyte activation of some pluripotent factors were deemed to be the main reason for the low efficiency of animal recloning. Here, to overcome this issue, which occurred frequently in the process of animal recloning, we established a reporter system in which fluorescent proteins were driven by pig
or
promoter to monitor the reprogramming process in cloned and recloned pig embryos. We studied the effect of different histone deacetylase (HDAC) inhibitors on incomplete reprogramming. Our results showed that Trichostatin A (TSA) could activate pluripotent factors and significantly enhance the development competence of recloned pig embryos, while the other two inhibitors, valproic acid (VPA) and Scriptaid, had little effect on that. Furthermore, we found no difference in
mRNA abundance between TSA-treated and untreated embryos. These findings suggest that TSA remarkably improves the reprogramming state of pig recloned embryos by restoring the expression of incompletely activated pluripotent genes
and
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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.
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.
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.
The IRF-1 expressed in the ICM of pigs' early blastocyst enhances the pluripotency of piPSCs, in part through promoting the JAK-STAT pathway.
NANOG functions as the gateway for the generation of pluripotent stem cells (PSCs) in mice and humans. NANOG is a transcription factor highly expressed in pig pre-implantation embryos, indicating ...that it is a conserved pluripotency-associated factor. However, pig NANOG reporter PSCs have yet to be established, and the regulation of pluripotency by NANOG is not fully understood in this animal.
In this study, pig NANOG tdTomato knock-in reporter positive PC-iPS cells were established using CRISPR/Cas9. The resulting cell line was treated with several cytokines and their corresponding inhibitors to identify pathways that regulate NANOG expression. The pathways examined were LIF (leukemia inhibitory factor)/IL6 (interleukin 6)-STAT3, FGF (fibroblast growth factor)/ERK, IGF1 (insulin-like growth factor 1)/PIP3 (phosphoinositide 3-kinase)-AKT, Activin A/SMAD, and BMP4 (bone morphogenetic proteins)/SMAD.
Our experiments showed that the Activin A/SMAD pathway is directly associated with activation of NANOG expression in the pig, as is also the case in mice and humans. Activin A directly regulates the expression of pig NANOG via SMAD2/3; inhibition of this pathway by SB431542 resulted in inhibition of NANOG expression.
Our results show that Activin A plays an important regulatory role in NANOG-mediated pluripotency in pig iPS cells. Activin A treatment may be therefore an effective method for de novo derivation of authentic embryonic stem cells (ESCs) from pig pre-implantation embryos.
Pluripotency maintenance and exit in embryonic stem cells is a focal topic in stem cell biology. However, the effects of screening under very stringent culture conditions (e.g., differentiation ...medium, no leukemia inhibitory factor, no chemical inhibitors such as PD0325901 and CHIR99021, and no feeder cells) and of prolonging culture for key factors that regulate pluripotency exit, have not yet been reported. Here, we used a genome-wide CRISPR library to perform such a screen in mouse embryonic stem cells. Naïve NANOG-GFP mESCs were first transfected with a mouse genome-wide CRISPR knockout library to obtain a mutant mESCs library, followed by screening for two months in a strict N2B27 differentiation medium. The clones that survived our stringent screening were analyzed to identify the inserted sgRNAs. In addition to identifying the enriched genes that were reported in previous studies (Socs3, Tsc1, Trp53, Nf2, Tcf7l1, Csnk1a1, and Dhx30), we found 17 unreported genes, among which Zfp771 and Olfr769 appeared to be involved in pluripotency exit. Furthermore, Zfp771 knockout ESCs showed a differentiation delay in embryonic chimera experiments, indicating Zfp771 played an important role in pluripotency exit. Our results show that stringent screening with the CRISPR library can reveal key regulators of pluripotency exit.
Industrial wastewater discharge leads to serious eutrophication of water bodies, but most of the adsorbents are difficult to selectively remove phosphorus and are difficult to use multiple times, ...therefore, developing an efficient and reusable material for removal phosphate is extremely necessary. In this work, a kind of highly selective phosphate adsorbent, microporous carbon material (MCM), based on glucose was synthesized by hydrothermal and activation method. The MCM were characterized by SEM, XPS, BET, element analysis, et al. The phosphate adsorption mechanism of MCM were investigated by batch adsorption experiment and model calculation. Results showed that MCM had a high adsorption capacity for phosphate in a wide range of pH (1.5–10). Langmuir model and pseudo-second-order kinetic revealed that the process was endothermic and involved both physical and chemical adsorption. The main phosphate adsorption mechanisms of MCM are electrostatic attraction, ion complexation, hydrogen bonding, and physical adsorption. The ions competition simulation experiment indicated that the MCM was highly selective for phosphate removal. Furthermore, the phosphate adsorption tests were carried out on five kinds of water, and the removal rates were all above 99.98%. The 20 regenerative cycles experiment revealed that the MCM had high reusability. Therefore, this kind of novel glucose-based highly selective phosphate adsorbent with multi-cycle phosphorus removal performance can improve the eutrophication of water. This study provides a new idea for phosphate removal and expands the application range of glucose-based carbon materials.
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•A kind of phosphate adsorption microporous carbon material (MCM) was prepared.•MCM has a high effect on phosphate removal over a wide pH range (1.5–10).•The phosphate removal rate of MCM was more than 99% in five kinds of water.•The phosphate adsorption mechanism of MCM was investigated.•MCM maintains >80% adsorption capacity after 20 regeneration cycles.
Abstract This paper proposes a risk prediction algorithm for power operation and maintenance data networks based on the grey theory, aiming at the real-time, healthy, and high stability requirements ...of power operation and maintenance data networks in work. By combining the entropy weight method to assign weight to risk indicators, the risk prediction of data networks in dynamic networks is achieved. By using two indicators of risk value and risk dispersion, the overall system is predicted for risk. Through experimental comparison, the prediction algorithm based on grey theory designed in this paper has a smaller error than existing prediction methods, which more scientifically realizes the risk prediction mechanism, ensures the stability of power operation and maintenance data, and ensures the reliable operation of the power grid.
Animal infectious diseases pose a significant threat to the global agriculture and biomedicine industries, leading to significant economic losses and public health risks. The emergence and spread of ...viral infections such as African swine fever virus (ASFV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine epidemic diarrhea virus (PEDV), and avian influenza virus (AIV) have highlighted the need for innovative approaches to develop resilient and disease-resistant animal populations. Gene editing technologies, such as CRISPR/Cas9, offer a promising avenue for generating animals with enhanced disease resistance. This review summarizes recent advances in molecular breeding strategies for generating disease-resistant animals, focusing on the development of disease-resistant livestock. We also highlight the potential applications of genome-wide CRISPR/Cas9 library screening and base editors in producing precise gene modified livestock for disease resistance in the future. Overall, gene editing technologies have the potential to revolutionize animal breeding and improve animal health and welfare.
•We summarized literature on genetic manipulation, specifically genome editing, for disease resistance in livestock and poultry.•Three gene editing strategies enhance host immunity and target pathogen vulnerabilities.•CRISPR libraries and novel tools offer precise disease-resistant breeding opportunities for livestock and poultry research.
Abstract During early embryonic development, the transition from totipotency to pluripotency is a fundamental and critical process for proper development. However, the regulatory mechanisms governing ...this transition remain elusive. Here, we conducted a comprehensive genome-wide CRISPR/Cas9 screen to investigate the 2-cell-like cells (2CLCs) phenotype in mouse embryonic stem cells (mESCs). This effort led to the identification of ten regulators that play a pivotal role in determining cell fate during this transition. Notably, our study revealed Mdm2 as a significant negative regulator of 2CLCs, as perturbation of Mdm2 resulted in a higher proportion of 2CLCs. Mdm2 appears to influence cell fate through its impact on cell cycle progression and H3K27me3 epigenetic modifications. In summary, the results of our CRISPR/Cas9 screen have uncovered several genes with distinct functions in regulating totipotency and pluripotency at various levels, offering a valuable resource for potential targets in future molecular studies.
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