pregnancy-nursing-management-for-embryo-transferred-genetically

Pregnancy and Nursing Management for Embryo-Transferred and Genetically Modified Rabbits

With the advancement of scientific research, the demand for gene-edited rabbit models is increasing. However, there are limited pregnancy and feeding ...

Wuyi University

Hairless mice have been widely applied in skin-related researches, while hairless pigs will be an ideal model for skin-related study and other biomedical researches because of the similarity of skin structure with humans. The previous study revealed that hairlessness phenotype in nude mice is caused by insufficient expression of phospholipase C-delta 1 (PLCD1), an essential molecule downstream of Foxn1, which encouraged us to generate PLCD1-deficient pigs. In this study, we plan to firstly produce PLCD1 knockout (KO) mice by CRISPR/Cas9 technology, which will lay a solid foundation for the generation of hairless PLCD1 KO pigs.

Abnormalities of hair structure and skin histology derived from CRISPR/Cas9-based knockout of phospholipase C-delta 1 in mice.

Clinical evidence suggests that there exists a strong correlation between Zika virus (ZIKV) infection and abnormal development of the nervous system. However, the underlying mechanisms remain to be elusive. In this study, recombinant lentiviral vectors coding for ZIKV structural proteins and truncations (prM-Env, M-Env and Env) were transduced into PC12 cells. Envelope (Env) overexpression induced significant inhibition of proliferation and triggered G2/M cell cycle arrest and apoptosis in PC12 cells. Flow cytometry and western blot analysis showed that the apoptosis was associated with up-regulation of both p53 and p21 and down-regulation of cyclin B1. Presence of aberrant nuclei clusters were confirmed by immunofluorescence staining analysis. The data indicate that overexpression of prM-Env, M-Env or Env led to apoptosis via an intrinsic cell death signaling pathway that is dependent on the activation of caspase-9 and caspase-3 and accompanied by an increased ratio of Bax to Bcl-2 in transduced PC12 cells. In summary, our results suggest that ZIKV Env protein causes apoptosis in PC12 cells via an intrinsic cell death signaling pathway, which may contribute to ZIKV-induced abnormal development of the nervous system.

Zika Virus Envelope Protein induces G2/M Cell Cycle Arrest and Apoptosis via an Intrinsic Cell Death Signaling Pathway in Neuroendocrine PC12 Cells.

Mesenchymal stromal cells (MSCs) were originally characterized by the ability to differentiate into different mesenchymal lineages in vitro, and their immunomodulatory and trophic functions have recently aroused significant interest in the application of MSCs in cell-based regenerative medicine. However, a major problem in clinical practice is the replicative senescence of MSCs, which limits the cell proliferation potential of MSCs after large-scale expansion. Telomeric zinc finger-associated protein (TZAP), a novel specific telomere-binding protein, was recently found to stimulate telomere trimming and prevent excessive telomere elongation. The aim of this study was to elucidate the role of TZAP in regulating MSCs senescence, differentiation and proliferation.

TZAP plays an inhibitory role in the self-renewal of porcine mesenchymal stromal cells and is implicated the regulation of premature senescence via the p53 pathway.

The Tibet minipig is a rare highland pig breed worldwide and has many applications in biomedical and agricultural research. However, Tibet minipigs are not like domesticated pigs in that their ovulation number is low, which is unfavourable for the collection of zygotes. Partly for this reason, few studies have reported the successful generation of genetically modified Tibet minipigs by zygote injection. To address this issue, we described an efficient way to generate gene-edited Tibet minipigs, the major elements of which include the utilization of synchronized oestrus instead of superovulation to obtain zygotes, optimization of the preparation strategy, and co-injection of clustered regularly interspaced short palindromic repeat sequences associated protein 9 (Cas9) mRNA and single-guide RNAs (sgRNAs) into the cytoplasm of zygotes. We successfully obtained allelic  gene knockout ( ) Tibet minipigs with a typical albino phenotype (i.e., red-coloured eyes with light pink-tinted irises and no pigmentation in the skin and hair) as well as     and     Tibet minipigs with typical phenotypes of albinism and immunodeficiency, which was characterized by thymic atrophy and abnormal immunocyte proportions. The overall gene editing efficiency was 75% for the  single gene knockout, while for  and  dual gene editing, the values were 25% and 75%, respectively. No detectable off-target mutations were observed. By intercrossing F generation minipigs, targeted genetic mutations can also be transmitted to gene-edited minipigs' offspring through germ line transmission. This study is a valuable exploration for the efficient generation of gene-edited Tibet minipigs with medical research value in the future.

Optimization Strategy for Generating Gene-edited Tibet Minipigs by Synchronized Oestrus and Cytoplasmic Microinjection.

Hereditary tyrosinemia type I (HT1) results from the loss of fumarylacetoacetate hydrolase (FAH) activity and can lead to lethal liver injury (LLI). Therapeutic options for HT1 remain limited. The   pig, a well-characterized animal model of HT1, represents a promising candidate for testing novel therapeutic approaches to treat this condition. Here, we report an improved single-step method to establish a biallelic (  ) mutant porcine model using CRISPR-Cas9 and cytoplasmic microinjection. We also tested the feasibility of rescuing HT1 pigs through inactivating the 4-hydroxyphenylpyruvic acid dioxygenase () gene, which functions upstream of the pathogenic pathway, rather than by directly correcting the disease-causing gene as occurs with traditional gene therapy. Direct intracytoplasmic delivery of CRISPR-Cas9 targeting  before intrauterine death reprogrammed the tyrosine metabolism pathway and protected pigs against  deficiency-induced LLI. Characterization of the F1 generation revealed consistent liver-protective features that were germline transmissible. Furthermore,  ablation ameliorated oxidative stress and inflammatory responses and restored the gene profile relating to liver metabolism homeostasis. Collectively, this study not only provided a novel large animal model for exploring the pathogenesis of HT1, but also demonstrated that CRISPR-Cas9-mediated  ablation alleviated LLI in HT1 pigs and represents a potential therapeutic option for the treatment of HT1.

Genetically blocking  CRISPR-Cas9 protects against lethal liver injury in a pig model of tyrosinemia type I.

Adenosine-to-inosine (A-to-I) RNA editing, catalyzed by ADAR enzymes, is an essential post-transcriptional modification. Although hundreds of thousands of RNA editing sites have been reported in mammals, brain-wide analysis of the RNA editing in the mammalian brain remains rare. Here, a genome-wide RNA-editing investigation is performed in 119 samples, representing 30 anatomically defined subregions in the pig brain. We identify a total of 682,037 A-to-I RNA editing sites of which 97% are not identified before. Within the pig brain, cerebellum and olfactory bulb are regions with most edited transcripts. The editing level of sites residing in protein-coding regions are similar across brain regions, whereas region-distinct editing is observed in repetitive sequences. Highly edited conserved recoding events in pig and human brain are found in neurotransmitter receptors, demonstrating the evolutionary importance of RNA editing in neurotransmission functions. Although potential data biases caused by age, sex or health status are not considered, this study provides a rich resource to better understand the evolutionary importance of post-transcriptional RNA editing.

A porcine brain-wide RNA editing landscape.

Gene therapy's entrance into clinical settings has made it an ever more attractive field of study for various diseases. However, relatively little progress has been made in targeting kidney diseases due to poor gene delivery efficiency in renal cells. The development of novel gene therapy vectors for medical intervention to treat kidney diseases is needed. In this study, we designed and produced a pseudotyped lentiviral vector with envelope glycoproteins of Zika virus (ZIKV), and evaluated its potential use in viral vector entry, neutralization assay, and gene delivery especially in the renal context. The lentiviral vector, simplified as ZIKV-E, is pseudotyped with Env/G-TC representing the transmembrane (TM) and cytoplasmic (Y) domains of Env replaced with the TM and CY domains of the glycoprotein (G) of the vesicular stomatitis virus.  results show that ZIKV-E induced efficient transduction in tubular epithelial cells in mouse kidneys, demonstrating >100-fold higher expression of exogenous green fluorescent protein gene compared with that achieved by vesicular stomatitis virus G (VSV-G) protein pseudotyped lentiviral vector. The results also showed that the vector ZIKV-E transduced cells in a pH-independent manner and the transduction was inhibited by anti-ZIKV Env domain III antibodies. Results also show that ZIKV-E can be used as a surrogate for studies of ZIKV entry mechanisms and neutralization antibody assay. In all, this study successfully demonstrated a novel pseudotyped lentiviral vector ZIKV-E for inducing high transduction efficiency in renal tubular epithelial cells that could serve as a foundation for gene therapy for the treatment of inherited renal diseases in humans.

Efficient Gene Transfer to Kidney Using a Lentiviral Vector Pseudotyped with Zika Virus Envelope Glycoprotein.

Acute ischaemic stroke (AIS) is a complex, systemic, pathological, and physiological process. Systemic inflammatory responses and disorders of the gut microbiome contribute to increased mortality and disability following AIS. We conducted 16S high-throughput sequencing and ultra-performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry-based non-targeted metabolomic analyses of the plasma from a Tibetan miniature pig middle cerebral artery occlusion (MCAO) model. A significant decrease in the abundance of Firmicutes and a significant increase in the abundance of Actinobacteria were observed after the onset of AIS. Among the plasma metabolites, the levels of phospholipids and amino acids were considerably altered. Loading values and differential metabolite-bacterial group association analyses of the metabolome and microbiome indicated a correlation between the microbiome and metabolome of Tibetan miniature pigs after MCAO. Furthermore, significant changes were observed in the ABC transporter pathway and purine metabolism in the gut microbiome-plasma metabolome during the early stage of AIS. Kyoto Encyclopaedia of Genes and Genomes enrichment analysis showed that arginine, proline, and cyanoamino acid metabolism was upregulated while ABC transporter metabolism pathway and carbohydrate digestion and absorption were substantially downregulated. The results of this study suggest that AIS affects the gut microbiota and plasma metabolites in Tibetan miniature pigs and that faecal microbiota transplantation could be a potential therapeutic approach for AIS.

Integration of 16S rRNA gene sequencing and LC/MS-based metabolomic analysis of early biomarkers of acute ischaemic stroke in Tibetan miniature pigs.

Zika virus can infect the fetus through the placental barrier, causing ZIKV congenital syndrome and even miscarriage, which can cause great harm to pregnant women and infants. Currently, there is no vaccine and drug available to combat the Zika virus. In this study, we designed a fusion protein named EDIII-Fc, including the EDIII region of Zika E protein and human IgG Fc fragment, and obtained 293T cells that stably secreted EDIII-Fc protein using the lentiviral expression system. Mice were immunized with the EDIII-Fc protein, and it was observed that viral replication was significantly inhibited in the immunized mice compared to non-immunized mice. In rhesus macaques, we found that EDIII-Fc effectively induce the secretion of neutralizing antibodies and T cell immunity. These experimental data provide valid data for further use of Zika virus E protein to prepare an effective, safe, affordable Zika vaccine.

EDIII-Fc induces protective immune responses against the Zika virus in mice and rhesus macaque.

Reactive oxygen species (ROS)-induced adaptive/maladaptive mitophagy plays an essential role in the pathophysiology of acute ischemic stroke (AIS). However, most studies have been conducted using rodent models, which limits their clinical application. In this study, we aimed to develop porcine models of permanent stroke and observe the pathophysiological alterations caused by acute ischemic stroke, focusing on ROS-induced mitophagy. Miniature pigs were subjected to lateral frontotemporal electrocoagulation, which resulted in permanent middle cerebral artery occlusion. We investigated global brain damage and mechanisms of adaptive/maladaptive mitophagy caused by ROS and global brain inflammation after AIS. An early neuroinflammatory response was observed in the ipsilateral hemisphere. ROS levels were significantly elevated in the ipsilateral hemisphere and slightly elevated in the contralateral hemisphere. ROS accumulation may be attributed to the increased production and impaired elimination of ROS. In addition, mitophagy and apoptosis were detected in the ischemic core, which may be attributed to ROS accumulation. We propose "distinct-area targeting" interventions aimed at maladaptive mitophagy within the ischemic core of the infarct hemisphere, which may provide new therapeutic targets for the treatment of AIS.

Article	Year
Abnormalities of hair structure and skin histology derived from CRISPR/Cas9-based knockout of phospholipase C-delta 1 in mice. Journal of translational medicine\| PubMed ID: 29793503	2018
Zika Virus Envelope Protein induces G2/M Cell Cycle Arrest and Apoptosis via an Intrinsic Cell Death Signaling Pathway in Neuroendocrine PC12 Cells. International journal of biological sciences\| PubMed ID: 29989100	2018
TZAP plays an inhibitory role in the self-renewal of porcine mesenchymal stromal cells and is implicated the regulation of premature senescence via the p53 pathway. Journal of translational medicine\| PubMed ID: 30845965	2019
Optimization Strategy for Generating Gene-edited Tibet Minipigs by Synchronized Oestrus and Cytoplasmic Microinjection. International journal of biological sciences\| PubMed ID: 31754342	2019
Genetically blocking CRISPR-Cas9 protects against lethal liver injury in a pig model of tyrosinemia type I. Molecular therapy. Methods & clinical development\| PubMed ID: 33997102	2021
A porcine brain-wide RNA editing landscape. Communications biology\| PubMed ID: 34112917	2021
Efficient Gene Transfer to Kidney Using a Lentiviral Vector Pseudotyped with Zika Virus Envelope Glycoprotein. Human gene therapy\| PubMed ID: 35904396	2022
Integration of 16S rRNA gene sequencing and LC/MS-based metabolomic analysis of early biomarkers of acute ischaemic stroke in Tibetan miniature pigs. Journal of microbiological methods\| PubMed ID: 37863204	2023
EDIII-Fc induces protective immune responses against the Zika virus in mice and rhesus macaque. PLoS neglected tropical diseases\| PubMed ID: 37983259	2023
Ischemic Stroke Induces ROS Accumulation, Maladaptive Mitophagy, and Neuronal Apoptosis in Minipigs. Journal of microbiology and biotechnology\| PubMed ID: 39631782	2024

Weiwang Gu

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