Advances in pig models of human diseases Hou, Naipeng; Du, Xuguang; Wu, Sen
Animal models and experimental medicine,
April 2022, Letnik:
5, Številka:
2
Journal Article
Recenzirano
Odprti dostop
Animal models of human diseases play a critical role in medical research. Pigs are anatomically and physiologically more like humans than are small rodents such as mice, making pigs an attractive ...option for modeling human diseases. Advances in recent years in genetic engineering have facilitated the rapid rise of pig models for use in studies of human disease. In the present review, we summarize the current status of pig models for human cardiovascular, metabolic, neurodegenerative, and various genetic diseases. We also discuss areas that need to be improved. Animal models of human diseases play a critical role in medical research. Advances in recent years in genetic engineering have facilitated the rapid rise of pig models for use in studies of human disease. In the present review, we summarize the current status of pig models for human cardiovascular, metabolic, neurodegenerative, various genetic diseases and xenotransplantation.
Animal models of human diseases play an important role in the research of medical field. In the current review, we attempt to summarize the advances of the pig models for human cardiovascular diseases, metabolic diseases, neurodegenerative diseases, and some other genetic diseases. We also discuss the areas that need to be improved.
Efficient exogenous DNA integration can be mediated by Cas9 through the non-homology end-joining pathway. However, such integrations are often imprecise and contain a variety of mutations at the ...junctions between the external DNA and the genomic loci. Here we describe a microhomology-dependent targeted integration method, designated MITI, for precise site-specific gene insertions. We found that the MITI strategy yielded higher knock-in accuracy than Cas9 HITI for the insertion of external DNA and tagging endogenous genes. Furthermore, in combination with negative selection and four different CrRNAs targeting donor vectors and genome-targeted sites with a CrRNA array, MITI facilitated precise ligation at all junctions. Therefore, our Cas12a-based MITI method increases the repertoire of precision genome engineering approaches and provides a useful tool for various gene editing applications.
The interaction between foot-and-mouth disease virus (FMDV) and the host is extremely important for virus infection, but there are few researches on it, which is not conducive to vaccine development ...and FMD control. In this study, we designed a porcine genome-scale CRISPR/Cas9 knockout library containing 93,859 single guide RNAs targeting 16,886 protein-coding genes, 25 long ncRNAs, and 463 microRNAs. Using this library, several previously unreported genes required for FMDV infection are highly enriched post-FMDV selection in IBRS-2 cells. Follow-up studies confirmed the dependency of FMDV on these genes, and we identified a functional role for one of the FMDV-related host genes: TOB1 (Transducer of ERBB2.1). TOB1-knockout significantly inhibits FMDV infection by positively regulating the expression of RIG-I and MDA5. We further found that TOB1-knockout led to more accumulation of mRNA transcripts of transcription factor CEBPA, and thus its protein, which further enhanced transcription of RIG-I and MDA5 genes. In addition, TOB1-knockout was shown to inhibit FMDV adsorption and internalization mediated by EGFR/ERBB2 pathway. Finally, the FMDV lethal challenge on TOB1-knockout mice confirmed that the deletion of TOB1 inhibited FMDV infection in vivo. These results identify TOB1 as a key host factor involved in FMDV infection in pigs.
Objective
Soil organic carbon (SOC) in deeper soils may be more protected by mineral particles than SOC in shallower soils; thus, the vertical SOC distribution is closely related to the overall SOC ...stability. This study aimed to verify the relationship between the solubility temperature dependence of water extractable SOC fractions and their vertical distribution characteristics.
Methods
Soil samples collected from each soil horizon in natural dragon spruce (
Picea asperata
) and red birch (
Betula albosinensis
) forests were analyzed. Cold water-extracted organic carbon (CWEOC) and hot water-extracted organic carbon (HWEOC) were extracted at 20 °C and 80 °C, respectively. The sum of CWEOC and HWEOC was considered the water-extracted organic carbon (WEOC) content. The carbohydrate-C (Car-C) and phenolic-C (Phe-C) contents extracted by hot water were also determined.
Results
The CWEOC, HWEOC, Car-C, and Phe-C contents varied significantly (
P
< 0.001) among soil horizons. The WEOC/SOC ratio increased significantly from the decomposed organic (H-) horizon to the surface mineral (A-) horizon (
P
< 0.05) but was not significantly different among the surface (A-), the subsurface (B-), and the bottom (C-) mineral horizons (Brunisols, according to the Canadian Soil Classification System). In both forests, CWEOC/HWEOC increased from the H- horizon to the B- horizon but was not significantly different between the B- and C- horizons. The Car-C/Phe-C ratios in the mineral soil horizons were significantly higher than those in the H-horizon and litter.
Conclusions
The more water-soluble SOC fractions tended to be more distributed in the deeper horizons. Therefore, WEOC in mineral soils may be more stable than is generally believed.
Various methods for ex utero culture systems have been explored. However, limitations remain regarding the in vitro culture platforms used before implanting mouse embryos and the normal development ...of mouse blastocysts in vitro. Furthermore, vascular niche support during mouse embryo development from embryonic day (E) 3.5 to E7.5 is unknown in vitro.
This study established a three-dimensional (3D) "sandwich" vascular niche culture system with in vitro culture medium (IVCM) using human placenta perivascular stem cells (hPPSCs) and human umbilical vein endothelial cells (hUVECs) as supportive cells (which were seeded into the bottom layer of Matrigel) to test mouse embryos from E3.5 to E7.5 in vitro. The development rates and greatest diameters of mouse embryos from E3.5 to E7.5 were quantitatively determined using SPSS software statistics. Pluripotent markers and embryo transplantation were used to monitor mouse embryo quality and function in vivo.
Embryos in the IVCM + Cells (hPPSCs + hUVECs) group showed higher development rates and greater diameters at each stage than those in the IVCM group. Embryos in the IVCM + Cells group cultured to E5.5 morphologically resembled natural egg cylinders and expressed specific embryonic cell markers, including Oct4 and Nanog. These features were similar to those of embryos developed in vivo. After transplantation, the embryos were re-implanted in the internal uterus and continued to develop to a particular stage.
The 3D in vitro culture system enabled embryo development from E3.5 to E7.5, and the vascularization microenvironment constructed by Matrigel, hPPSCs, and hUVECs significantly promoted the development of implanted embryos. This system allowed us to further study the physical and molecular mechanisms of embryo implantation in vitro.
N6-methyladenosine (m6A) methyltransferase Mettl3 is involved in conventional T cell immunity; however, its role in innate immune cells remains largely unknown. Here, we show that Mettl3 ...intrinsically regulates invariant natural killer T (iNKT) cell development and function in an m6A-dependent manner. Conditional ablation of Mettl3 in CD4+CD8+ double-positive (DP) thymocytes impairs iNKT cell proliferation, differentiation, and cytokine secretion, which synergistically causes defects in B16F10 melanoma resistance. Transcriptomic and epi-transcriptomic analyses reveal that Mettl3 deficiency disturbs the expression of iNKT cell-related genes with altered m6A modification. Strikingly, Mettl3 modulates the stability of the Creb1 transcript, which in turn controls the protein and phosphorylation levels of Creb1. Furthermore, conditional targeting of Creb1 in DP thymocytes results in similar phenotypes of iNKT cells lacking Mettl3. Importantly, ectopic expression of Creb1 largely rectifies such developmental defects in Mettl3-deficient iNKT cells. These findings reveal that the Mettl3-m6A-Creb1 axis plays critical roles in regulating iNKT cells at the post-transcriptional layer.
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•Mettl3-dependent m6A methylation plays a key role in iNKT cell development•A comprehensive m6A landscape of low-input iNKT cells is established by m6A-LACE-seq•Mettl3 deficiency disrupts iNKT cell-related gene network in an m6A-dependent manner•The Mettl3-m6A-Creb1 axis is critical for sustaining iNKT cell differentiation and function
You et al. demonstrate that Mettl3 is essential for regulating iNKT cell proliferation, differentiation, and melanoma resistance. The study highlights the critical role of Mettl3-dependent m6A modification in controlling iNKT cell-related gene expression, particularly in maintaining the stability of Creb1 and c-Myc transcripts.
Substantial rates of fetal loss plague all in vitro procedures involving embryo manipulations, including human-assisted reproduction, and are especially problematic for mammalian cloning where over ...90% of reconstructed nuclear transfer embryos are typically lost during pregnancy. However, the epigenetic mechanism of these pregnancy failures has not been well described. Here we performed methylome and transcriptome analyses of pig induced pluripotent stem cells and associated cloned embryos, and revealed that aberrant silencing of imprinted genes, in particular the retrotransposon-derived RTL1 gene, is the principal epigenetic cause of pregnancy failure. Remarkably, restoration of RTL1 expression in pig induced pluripotent stem cells rescued fetal loss. Furthermore, in other mammals, including humans, low RTL1 levels appear to be the main epigenetic cause of pregnancy failure.
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.
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Li et al. show that RITA, Nutlin3, and CTX1, inhibitors of p53-MDM2 interaction, could increase the homology-directed repair efficiency by 1.43- to 4.28-fold, which provides a simple and efficient strategy to promote CRISPR-Cas9-mediated precise genome editing in sheep primary cells.
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