ABSTRACT
Hsp40/DnaJ family proteins play important roles in the infection process of various viruses. Porcine DNAJB6 (pDNAJB6) is a major member of this family, but its role in modulating the replication of porcine circovirus type 2 (PCV2) is still unclear. In the present study, pDNAJB6 was found to be significantly upregulated by PCV2 infection, and confirmed to be interacted with PCV2 capsid (Cap) protein and co-localized at both cytoplasm and nucleus in the PCV2-infected cells. Knockout of pDNAJB6 significantly reduced the formation of autophagosomes in PCV2-infected cells or in the cells expressing Cap protein, whereas overexpression of pDNAJB6 showed an opposite effect. In addition, the domain mapping assay showed that the J domain of pDNAJB6 (amino acids (aa) 1-99) and the C terminus of Cap (162-234 aa) were required for the interaction of pDNAJB6 with Cap. Notably, the interaction of pDNAJB6 with Cap was very important to promoting the formation of autophagosomes induced by PCV2 infection or Cap expression and enhancing the replication of PCV2. Taken together, the results presented here show a novel function of pDNAJB6 in regulation of porcine circovirus replication that pDNAJB6 enhances the formation of autophagy to promote viral replication through interacting with viral capsid protein during PCV2 infection.
Subject(s)
Autophagosomes/physiology , Autophagy/genetics , Circoviridae Infections/veterinary , Circovirus/physiology , HSP40 Heat-Shock Proteins/metabolism , Swine Diseases/virology , Virus Replication , Animals , Autophagosomes/genetics , Circoviridae Infections/virology , Gene Knockout Techniques/veterinary , HSP40 Heat-Shock Proteins/deficiency , Mutation , Sus scrofa/metabolism , Swine , Up-Regulation , Virion/physiologyABSTRACT
CRISPR/Cas9-mediated genome editing technology is a simple and highly efficient and specific genome modification approach with wide applications in the animal industry. CRISPR/Cas9-mediated genome editing combined with somatic cell nuclear transfer rapidly constructs gene-edited somatic cell-cloned pigs for the genetic improvement of traits or simulation of human diseases. Chinese Bama pigs are an excellent indigenous minipig breed from Bama County of China. Research on genome editing of Chinese Bama pigs is of great significance in protecting its genetic resource, improving genetic traits and in creating disease models. This study aimed to address the disadvantages of slow growth and low percentage of lean meat in Chinese Bama pigs and to knock out the myostatin gene (MSTN) by genome editing to promote growth and increase lean meat production. We first used CRISPR/Cas9-mediated genome editing to conduct biallelic knockout of the MSTN, followed by somatic cell nuclear transfer to successfully generate MSTN biallelic knockout Chinese Bama pigs, which was confirmed to have significantly faster growth rate and showed myofibre hyperplasia when they reached sexual maturity. This study lays the foundation for the rapid improvement of production traits of Chinese Bama pigs and the generation of gene-edited disease models in this breed.
Subject(s)
CRISPR-Cas Systems , Myostatin/genetics , Swine, Miniature/genetics , Animals , Female , Gene Knockout Techniques/veterinary , Male , Muscle Fibers, Skeletal/physiology , Nuclear Transfer Techniques/veterinary , Pork Meat , Swine , Swine, Miniature/growth & developmentABSTRACT
Vibrio mimicus (V. mimicus) is a significant pathogen in freshwater catfish, though knowledge of virulence determinants and effective vaccine is lacking. Multiplex genome editing by natural transformation (MuGENT) is an easy knockout method, which has successfully used in various bacteria except for V. mimicus. Here, we found V. mimicus strain SCCF01 can uptake exogenous DNA and insert it into genome by natural transformation assay. Subsequently, we exploited this property to make five mutants (â³Hem, â³TS1, â³TS2, â³TS1â³TS2, and â³II), and removed the antibiotic resistance marker by Flp-recombination. Finally, all of the mutants were identified by PCR and RT-PCR. The results showed that combination of natural transformation and FLP-recombination can be applied successfully to generate targeted gene disruptions without the antibiotic resistance marker in V. mimicus. In addition, the five mutants showed mutant could be inherited after several subcultures and a 668-fold decrease in the virulence to yellow catfish (Pelteobagrus fulvidraco). This study provides a convenient method for the genetic manipulation of V. mimicus. It will facilitate the identification and characterization of V. mimicus virulence factors and eventually contribute to a better understanding of V. mimicus pathogenicity and development of attenuated vaccine.
Subject(s)
Bacterial Vaccines/immunology , Catfishes , Fish Diseases/immunology , Gene Editing/veterinary , Gene Knockout Techniques/veterinary , Vibrio mimicus/immunology , Animals , Gene Knockout Techniques/methods , Vaccines, Attenuated/immunology , Vibrio Infections/immunology , Vibrio Infections/veterinaryABSTRACT
Our previous studies demonstrated that the deletion of D2 fragment in tilapia Streptococcus agalactiae(GBS) attenuated strain YM001 is the main reason for the loss of virulence to tilapia. In this study, a Δ2 mutant that deletion of D2 fragment in parental virulent strain HN016 was constructed, and the safety, stability, immunogenicity, and growth characteristics, as well as the virulence mechanism of Δ2 mutant were evaluated. The results showed that Δ2 mutant was not pathogenic to tilapia, and the virulent revertants were not observed after 50 generations of passage. The RPS reached 96.11% at 15 days and 93.05% at 30 days, respectively, after intraperitoneal injection, while RPS reached 74.80% at 15 days and 53.16% at 30 days, respectively, after oral immunization. The growth of Δ2 mutant was significantly faster than YM001, and genes that were enriched in the nitrogen metabolism and arginine biosynthesis signaling pathway (arc, glnA, and gdhA) were identified as important candidate genes responsible for growth rate of S. agalactiae. The absence of D2 fragment affected the expression of Sip, therefore influencing the bacterial virulence. Altogether, this study demonstrated that deletion of D2 fragment in HN016 causes the loss of virulence to tilapia, and Δ2 mutant is a promising, better attenuated oral vaccine strain of S. agalactiae compared to YM001.
Subject(s)
Bacterial Vaccines/immunology , Base Sequence , Cichlids/immunology , Fish Diseases/prevention & control , Sequence Deletion , Streptococcal Infections/veterinary , Streptococcus agalactiae/immunology , Animals , Fish Diseases/immunology , Gene Knockout Techniques/veterinary , Serogroup , Streptococcal Infections/immunology , Streptococcal Infections/prevention & control , Streptococcus agalactiae/pathogenicity , Vaccines, Attenuated/immunology , VirulenceABSTRACT
Although the type I interferon-mediated increase of Mx1 and ISG15 gene expression in Epithelioma papulosum cyprini (EPC) cells has been reported, the antiviral role of Mx1 and ISG15 in EPC cells has not been investigated. In this study, to know the anti-viral hemorrhagic septicemia virus (VHSV) role of Mx1 and ISG15 of EPC cells, either Mx1 or ISG15 gene was knocked-out using a CRISPR/Cas9 system, and the progression of cytopathic effects (CPE) and viral growth were analyzed. Mx1 gene and ISG15 gene knockout EPC cells were successfully produced via CRISPR/Cas9 coupled with a single-cell cloning. Through the sequence analysis, one clone showing two heterozygous indel patterns in Mx1 gene and a clone showing three heterozygous indel patterns in ISG15 gene were selected for further analyses. Mx1 knockout EPC cells did not show any differences in VHSV-mediated CPE progression, even when pre-treated with polyinosinic:polycytidylic acid (poly I:C), compared to control EPC cells. These results suggest that Mx1 in EPC cells may be unfunctional to cytoplasmic RNA viruses. In contrast to Mx1, ISG15 knockout cells showed clearly hampered anti-VHSV activity even when pre-treated with poly I:C, indicating that ISG15 plays an important role in type I interferon-mediated anti-viral activity in EPC cells, which allowed VHSV to replicate more efficiently in ISG15 knockout cells than Mx1 knockout and control cells.
Subject(s)
CRISPR-Cas Systems/immunology , Cyprinidae/immunology , Disease Resistance/genetics , Fish Diseases/immunology , Fish Proteins/genetics , Fish Proteins/immunology , Gene Expression , Amino Acid Sequence , Animals , Base Sequence , Cell Line, Tumor , Cyprinidae/genetics , Disease Resistance/immunology , Fish Diseases/genetics , Gene Knockout Techniques/veterinary , Hemorrhagic Septicemia, Viral/genetics , Hemorrhagic Septicemia, Viral/immunology , Interferon Type I/genetics , Novirhabdovirus/physiology , Poly I-C/pharmacology , Sequence Analysis, DNA/veterinary , Sequence Analysis, Protein/veterinaryABSTRACT
PhoP plays an important role as a transcriptional regulator in the two-component phoP/phoQ regulatory system, which is widely present in Gram-negative bacteria. In this study, we used DNA microarray-based technology to evaluate the role of phoP in biofilm formation in avian pathogenic Escherichia coli (APEC). Differences in gene transcription between APEC wild-type and phoP mutant strains were determined. Mutation of the phoP transcriptional regulator affects the expression profile of genes involved in processes such as flagellar assembly, ABC transporters, quorum sensing, and bacterial chemotaxis. Deletion of phoP in APEC reduced biofilm formation, as indicated by crystal violet staining and scanning electron microscopy (SEM). In addition, the phoP gene was found to be associated with changes in bacterial drug resistance and cell-membrane-related properties. This study shows that phoP plays an important regulatory role in APEC biofilm formation, and provides new insights into strategies for preventing and controlling APEC infection.
Subject(s)
Biofilms/growth & development , Chickens/microbiology , Escherichia coli Infections/veterinary , Escherichia coli Proteins/physiology , Escherichia coli/physiology , Poultry Diseases/microbiology , Agglutination , Animals , Drug Resistance, Multiple , Escherichia coli/genetics , Escherichia coli/pathogenicity , Escherichia coli Infections/microbiology , Gene Knockout Techniques/veterinary , Hydrophobic and Hydrophilic Interactions , Mutation , Oligonucleotide Array Sequence Analysis/veterinary , RNA, Bacterial/isolation & purification , Real-Time Polymerase Chain Reaction/veterinaryABSTRACT
X-linked hypophosphatemia (XLH) is the most common cause of inheritable rickets, with an incidence of 1/20 000 in humans. Inactivation or mutation of the gene PHEX, a phosphate-regulating endopeptidase, leads to hypophosphatemia and defective bone mineralization in XLH patients. Presently, there is no adequate animal model for safety assessments of physiotherapies and drug screening for XLH rickets. In this study, an XLH model was generated via PHEX gene knockout (KO) through coinjection of clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9)/sgRNA mRNA into rabbit zygotes. The typical phenotypes of growth retardation, hypophosphatemia, elevated serum FGF23 and bone mineralization were observed in the PHEX KO rabbits but not in normal controls. In summary, for the first time, we have successfully obtained PHEX KO rabbits and recapitulated human XLH using the CRISPR/Cas9 system. This novel XLH rabbit model could be utilized as a drug screening model for XLH prevention and preclinical therapy.
Subject(s)
Disease Models, Animal , Familial Hypophosphatemic Rickets/genetics , Animals , Calcification, Physiologic/genetics , Familial Hypophosphatemic Rickets/metabolism , Female , Fibroblast Growth Factor-23 , Gene Knockout Techniques/methods , Gene Knockout Techniques/veterinary , Genetic Diseases, X-Linked/genetics , Humans , Hypophosphatemia/etiology , Hypophosphatemia/genetics , Mutation , PHEX Phosphate Regulating Neutral Endopeptidase/genetics , PHEX Phosphate Regulating Neutral Endopeptidase/metabolism , Phosphates , Rabbits , Rickets/etiologyABSTRACT
The artificially synthesized polyinosinic-polycytidylic acid (poly IC) has been widely used to induce type I IFN responses in various vertebrates including fish. However, as poly IC is too expensive to use in aquaculture, the development of another economical long dsRNA producing method is needed to practically use long dsRNAs in aquaculture farms for the control of infectious diseases. In the present study, to produce long dsRNAs economically, we developed a novel long dsRNA production system based on the RNase III gene deleted auxotrophic mutant E. tarda (ΔalrΔrncΔasd E. tarda) and a long dsRNA-producing vector that was equipped with two modified λ phage PR promoters arranged in a head-to-head fashion. As the present genetically engineered E. tarda cannot live without supplementation of d-alanine and DAP, environmental and medicinal risks are minimized. Olive flounder (Paralichthys olivaceus) fingerlings administered the long dsRNA-producing auxotrophic E. tarda mutant (Δalr ΔrncΔasd E. tarda) showed significantly higher expressions of TLR22, Mx1, and ISG15 genes, indicating a potential to increase type I interferon responses.
Subject(s)
Aquaculture/methods , Bacterial Proteins/metabolism , Edwardsiella tarda/genetics , Flatfishes/immunology , RNA, Bacterial/metabolism , RNA, Double-Stranded/administration & dosage , Ribonuclease III/genetics , Adjuvants, Immunologic/administration & dosage , Adjuvants, Immunologic/metabolism , Animals , Gene Knockout Techniques/veterinary , Immunization/veterinary , RNA, Double-Stranded/genetics , RNA, Double-Stranded/metabolismABSTRACT
The broad diversity of neurons is vital to neuronal functions. During vertebrate development, the spinal cord is a site of sensory and motor tasks coordinated by interneurons and the ongoing neurogenesis. In the spinal cord, V2-interneuron (V2-IN) progenitors (p2) develop into excitatory V2a-INs and inhibitory V2b-INs. The balance of these two types of interneurons requires precise control in the number and timing of their production. Here, using zebrafish embryos with altered Notch signaling, we show that different combinations of Notch ligands and receptors regulate two functions: the maintenance of p2 progenitor cells and the V2a/V2b cell fate decision in V2-IN development. Two ligands, DeltaA and DeltaD, and three receptors, Notch1a, Notch1b, and Notch3 redundantly contribute to p2 progenitor maintenance. On the other hand, DeltaA, DeltaC, and Notch1a mainly contribute to the V2a/V2b cell fate determination. A ubiquitin ligase Mib, which activates Notch ligands, acts in both functions through its activation of DeltaA, DeltaC, and DeltaD. Moreover, p2 progenitor maintenance and V2a/V2b fate determination are not distinct temporal processes, but occur within the same time frame during development. In conclusion, V2-IN cell progenitor proliferation and V2a/V2b cell fate determination involve signaling through different sets of Notch ligand-receptor combinations that occur concurrently during development in zebrafish.
Subject(s)
Interneurons/cytology , Neural Stem Cells/cytology , Neurogenesis/genetics , Receptors, Notch/genetics , Spinal Cord/embryology , Zebrafish/embryology , Animals , Cell Proliferation , Gene Expression Regulation, Developmental , Gene Knockout Techniques/veterinary , Homeodomain Proteins/metabolism , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Membrane Proteins/genetics , Membrane Proteins/metabolism , Morpholinos/genetics , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Neural Stem Cells/metabolism , Receptor, Notch1/metabolism , Receptor, Notch3 , Receptors, Notch/metabolism , Signal Transduction/genetics , Spinal Cord/cytology , Ubiquitin-Protein Ligases/metabolism , Zebrafish Proteins/genetics , Zebrafish Proteins/metabolismABSTRACT
Zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) are new tools for producing gene knockout (KO) animals. The current study reports produced genetically modified pigs, in which two endogenous genes were knocked out. Porcine fibroblast cell lines were derived from homozygous α1,3-galactosyltransferase (GalT) KO pigs. These cells were subjected to an additional KO for the cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH) gene. A pair of ZFN-encoding mRNAs targeting exon 8 of the CMAH gene was used to generate the heterozygous CMAH KO cells, from which cloned pigs were produced by somatic cell nuclear transfer (SCNT). One of the cloned pigs obtained was re-cloned after additional KO of the remaining CMAH allele using the same ZFN-encoding mRNAs to generate GalT/CMAH-double homozygous KO pigs. On the other hand, the use of TALEN-encoding mRNAs targeting exon 7 of the CMAH gene resulted in efficient generation of homozygous CMAH KO cells. These cells were used for SCNT to produce cloned pigs homozygous for a double GalT/CMAH KO. These results demonstrate that the combination of TALEN-encoding mRNA, in vitro selection of the nuclear donor cells and SCNT provides a robust method for generating KO pigs.
Subject(s)
Animals, Genetically Modified/genetics , Galactosyltransferases/genetics , Gene Knockout Techniques/veterinary , Mixed Function Oxygenases/genetics , Sus scrofa/genetics , Alleles , Animals , Animals, Genetically Modified/metabolism , Animals, Newborn , Cell Line , Cloning, Organism/veterinary , Embryo Transfer/veterinary , Exons , Female , Galactosyltransferases/antagonists & inhibitors , Galactosyltransferases/metabolism , Homozygote , Japan , Male , Mixed Function Oxygenases/antagonists & inhibitors , Mixed Function Oxygenases/metabolism , Nuclear Transfer Techniques/veterinary , RNA/metabolism , RNA Interference , RNA, Messenger/metabolism , Sus scrofa/metabolismABSTRACT
BACKGROUND: Pig erythrocytes are potentially useful to solve the worldwide shortage of human blood for transfusion. Domestic pig erythrocytes, however, express antigens that are bound by human preformed antibodies. Advances in genetic engineering have made it possible to rapidly knock out the genes of multiple xenoantigens, namely galactose α1,3 galactose (aGal) and N-glycolylneuraminic acid (Neu5Gc). We have recently targeted the GGTA1 and CMAH genes with zinc finger endonucleases resulting in double knockout pigs that no longer express aGal or Neu5Gc and attract significantly fewer human antibodies. In this study, we characterized erythrocytes from domestic and genetically modified pigs, baboons, chimpanzees, and humans for binding of human and baboon natural antibody, and complement-mediated lysis. METHODS: Distribution of anti-Neu5Gc IgG and IgM in pooled human AB serum was analyzed by ELISA. Erythrocytes from domestic pigs (Dom), aGal knockout pigs (GGTA1 KO), aGal and Neu5Gc double knockout pigs (GGTA1/CMAH KO), baboons, chimpanzees, and humans were analyzed by flow cytometry for aGal and Neu5Gc expression. In vitro comparative analysis of erythrocytes was conducted with pooled human AB serum and baboon serum. Total antibody binding was accessed by hemagglutination; complement-dependent lysis was measured by hemolytic assay; IgG or IgM binding to erythrocytes was characterized by flow cytometry. RESULTS: The pooled human AB serum contained 0.38 µg/ml anti-Neu5Gc IgG and 0.085 µg/ml anti-Neu5Gc IgM. Both Gal and Neu5Gc were not detectable on GGTA1/CMAH KO erythrocytes. Hemagglutination of GGTA1/CMAH KO erythrocytes with human serum was 3.5-fold lower compared with GGTA1 KO erythrocytes, but 1.6-fold greater when agglutinated with baboon serum. Hemolysis of GGTA1/CMAH KO erythrocytes by human serum (25%) was reduced 9-fold compared with GGTA1 KO erythrocytes, but increased 1.64-fold by baboon serum. Human IgG binding was reduced 27-fold on GGTA1/CMAH KO erythrocytes compared with GGTA1 KO erythrocytes, but markedly increased 3-fold by baboon serum IgG. Human IgM binding was decreased 227-fold on GGTA1/CMAH KO erythrocytes compared with GGTA1 KO erythrocytes, but enhanced 5-fold by baboon serum IgM. CONCLUSIONS: Removal of aGal and Neu5Gc antigens from pig erythrocytes significantly reduced human preformed antibody-mediated cytotoxicity but may have complicated future in vivo analysis by enhancing reactivity from baboons. The creation of the GGTA1/CMAH KO pig has provided the xenotransplantation researcher with organs and cells that attract fewer human antibodies than baboon and our closest primate relative, chimpanzee. These finding suggest that while GGTA1/CMAH KO erythrocytes may be useful for human transfusions, in vivo testing in the baboon may not provide a direct transplantation to the clinic.
Subject(s)
Erythrocyte Transfusion/methods , Galactosyltransferases/deficiency , Gene Knockout Techniques/veterinary , Mixed Function Oxygenases/deficiency , Sus scrofa/blood , Sus scrofa/genetics , Transplantation, Heterologous/methods , Animals , Animals, Genetically Modified , Antibodies, Heterophile/blood , Erythrocyte Transfusion/adverse effects , Erythrocytes/immunology , Galactosyltransferases/blood , Galactosyltransferases/genetics , Humans , Immunity, Innate , Immunoglobulin G/blood , Immunoglobulin M/blood , Mixed Function Oxygenases/blood , Mixed Function Oxygenases/genetics , Models, Animal , Papio , Primates , Transplantation, Heterologous/adverse effectsABSTRACT
Protein arginine methyltransferase 5 (PRMT5), a type II arginine methyltransferase, controls arginine dimethylation of a variety of substrates. While many papers have reported the function of mammalian PRMT5, it remains unclear how PRMT5 functions in chicken cells. In this study, we found that chicken (ch) PRMT5 is widely expressed in a variety of chicken tissues and is distributed in both the cytoplasm and the nucleus. Ectopic expression of chPRMT5 significantly suppresses chIFN-ß activation induced by chMDA5. In addition, a prmt5 gene-deficient DF-1 cell line was constructed using CRISPR/Cas9. In comparison with the wild-type cells, the prmt5-/- DF-1 cells displays normal morphology and maintain proliferative capacity. Luciferase reporter assay and overexpression showed that prmt5-/- DF-1 cells had increased IFN-ß production. With identified chicken PRMT5 and CRISPR/Cas9 knockout performed in DF-1 cells, we uncovered a functional link of chPRMT5 in suppression of IFN-ß production and interferon-stimulated gene expression.
Subject(s)
Chickens , Interferons , Animals , Interferons/metabolism , Chickens/genetics , Chickens/metabolism , CRISPR-Cas Systems , Gene Knockout Techniques/veterinary , Cell Line , Mammals/metabolismABSTRACT
BACKGROUND: Nuclear transfer (NT) technologies offer a means for producing the genetically modified pigs necessary to develop swine models for mechanistic studies of disease processes as well as to serve as organ donors for xenotransplantation. Most previous studies have used commercial pigs as surrogates. METHOD AND RESULTS: In this study, we established a cloning technique for miniature pigs by somatic cell nuclear transfer (SCNT) using Nippon Institute for Biological Science (NIBS) miniature pigs as surrogates. Moreover, utilizing this technique, we have successfully produced an α-1, 3-galactosyltransferase knockout (GalT-KO) miniature swine. Fibroblasts procured from a NIBS miniature pig fetus were injected into 1312 enucleated oocytes. The cloned embryos were transferred to 11 surrogates of which five successfully delivered 13 cloned offspring; the production efficiency was 1.0% (13/1312). In a second experiment, lung fibroblasts obtained from neonatal GalT-KO MGH miniature swine were used as donor cells and 1953 cloned embryos were transferred to 12 surrogates. Six cloned offspring were born from five surrogates, a production efficiency of 0.3% (6/1953). CONCLUSIONS: These results demonstrate successful establishment of a miniature pig cloning technique by SCNT using NIBS miniature pigs as surrogates. To our knowledge, this is the first demonstration of successful production of GalT-KO miniature swine using miniature swine surrogates. This technique could help to ensure a stable supply of the cloned pigs through the use of miniature pig surrogates and could expand production in countries with limited space or in facilities with special regulations such as specific pathogen-free or good laboratory practice.
Subject(s)
Cloning, Organism/veterinary , Galactosyltransferases/deficiency , Galactosyltransferases/genetics , Swine, Miniature/genetics , Animals , Animals, Genetically Modified , Cloning, Organism/methods , Embryo Transfer/veterinary , Female , Galactosyltransferases/immunology , Gene Knockout Techniques/veterinary , Male , Nuclear Transfer Techniques , Pregnancy , Swine , Swine, Miniature/immunology , Transplantation, HeterologousABSTRACT
The editing efficiency primarily hinders the utility of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology in poultry. For a better understanding of the factors that influence the efficiency of gene knockout mediated by CRISPR/Cas9 in chicken DF1 cells, the single or dual single guide RNA (sgRNA) targeted exon regions of genes (taking anti-Müllerian hormone, TGF-beta receptor type-2 and Peroxisome proliferator-activated receptor gamma as examples) were designed. The sgRNA-CRISPR/Cas9 vectors with corresponding reporter vectors were transfected into DF1 cells. T7 endonuclease 1 (T7E1) and amplicon sequencing assay were compared for evaluating genome editing efficiency and the indel profiles were analyzed based on the data of amplicon sequencing. Meanwhile, to evaluate the precision of Cas9 cleavage, we also analyzed the homology of small insertion with the nucleotides of upstream and downstream of cleave sties. The surrogate reporter systems showed strong enrichment function, and the indel percentages were increased after puromycin selection. The indel ratios of T7E1 assay were lower than amplicon sequencing assay, which indicated T7E1 isn't fit to be used as the sole evaluation criterion for the targeting efficiency of CRISPR/Cas9. Based on the amplicon sequencing analysis, the editing efficiency showed noticeable differences among cells treated with different sgRNAs. However, the variety of indel efficiencies was not related to the GC content of sgRNA or chromosome types of targeted genes. The results showed that the dual sgRNA might not raise the indel ratios compared with individual sgRNA, but they could increase the ratios of the fragment deletions. The present study suggested that the surrogate reporter was an effective method to promote the editing efficiencies of CRISPR/Cas9 in chicken cells. The dual sgRNA could increase the fragment deletions, and the sensitivity of amplicon sequencing to detect cleavage was higher than the T7 endonuclease 1 assay. These results are essential to improve the application of CRISPR/Cas9 technology in chicken cells.
Subject(s)
CRISPR-Cas Systems , RNA, Guide, CRISPR-Cas Systems , Animals , Gene Knockout Techniques/veterinary , Chickens/genetics , Endonucleases/geneticsABSTRACT
Studies of mammalian fertilization progressed very slowly in the beginning because of difficulties in obtaining a large quantity of fully mature eggs at one time. With progression of techniques to collect and handle eggs and spermatozoa, research in mammalian fertilization advanced rapidly. Today, far more papers are published on mammalian gametes and fertilization than those of all other animals combined. The development of assisted fertilization and related technologies revolutionized basic research as well as human reproductive medicine and animal husbandry. Reproduction is fundamental to human and animal lives. The author lists a few subjects of his personal interest for further development of basic and applied research of gametes and fertilization. Each reader will probably have more exciting subjects of future investigation.
Subject(s)
Reproductive Techniques, Assisted/veterinary , Animals , Animals, Genetically Modified , Female , Gene Knockout Techniques/methods , Gene Knockout Techniques/veterinary , Humans , Male , Oocyte Retrieval/methods , Oocyte Retrieval/veterinary , Sex Preselection/methods , Sex Preselection/veterinary , Sperm Retrieval/veterinaryABSTRACT
Myostatin (MSTN) is primarily expressed in skeletal muscle and plays an important role in the regulation of muscle growth and development as well as fat deposition; however, little is known about the molecular mechanism through which MSTN regulates body fat deposition. Therefore, in this study, we sought to identify the signaling pathways through which MSTN regulates fat accumulation in pigs. MSTN knockout (MSTN-/-) pigs showed increased muscle mass, decreased fat mass, and a leaner body composition. In this study, we found that the adipose tissue of MSTN-/- pigs exhibits the characteristics of beige adipose tissue, and the mRNA expression levels of beige adipose marker genes, including UCP3, Cidea, and CD137, were significantly increased. Remarkably, the observed beige phenotype was not adipocyte autonomous but rather caused by muscle-secreted myokine interleukin (IL)-6. This occurrence results in increased AMP-activated protein kinase (AMPK) phosphorylation in adipose tissue, which subsequently activates peroxisome proliferator-activated receptor gamma coactivator 1α and the conversion of white adipocytes to beige in pigs. Therefore, we concluded that MSTN deficiency leads to increased IL-6 secretion in skeletal muscle and activates AMPK in adipocytes, thereby increasing the beige adipose tissue in MSTN-/- pigs.
Subject(s)
Adipose Tissue, Beige , Myostatin , Adipose Tissue/metabolism , Adipose Tissue, Beige/metabolism , Adipose Tissue, White/metabolism , Animals , Gene Knockout Techniques/veterinary , Interleukin-6/genetics , Muscle, Skeletal/metabolism , Myostatin/genetics , SwineABSTRACT
The application of new technologies for gene editing in horses may allow the generation of improved sportive individuals. Here, we aimed to knock out the myostatin gene (MSTN), a negative regulator of muscle mass development, using CRISPR/Cas9 and to generate edited embryos for the first time in horses. We nucleofected horse fetal fibroblasts with 1, 2 or 5 µg of 2 different gRNA/Cas9 plasmids targeting the first exon of MSTN. We observed that increasing plasmid concentrations improved mutation efficiency. The average efficiency was 63.6% for gRNA1 (14/22 edited clonal cell lines) and 96.2% for gRNA2 (25/26 edited clonal cell lines). Three clonal cell lines were chosen for embryo generation by somatic cell nuclear transfer: one with a monoallelic edition, one with biallelic heterozygous editions and one with a biallelic homozygous edition, which rendered edited blastocysts in each case. Both MSTN editions and off-targets were analyzed in the embryos. In conclusion, CRISPR/Cas9 proved an efficient method to edit the horse genome in a dose dependent manner with high specificity. Adapting this technology sport advantageous alleles could be generated, and a precision breeding program could be developed.
Subject(s)
Animals, Genetically Modified/genetics , CRISPR-Cas Systems , Embryo, Mammalian/metabolism , Gene Editing , Gene Knockout Techniques/veterinary , Myostatin/genetics , Nuclear Transfer Techniques/veterinary , Animals , Base Sequence , Embryo, Mammalian/cytology , Female , Fibroblasts/cytology , Fibroblasts/metabolism , Horses , Mutation , Myostatin/antagonists & inhibitors , Sequence HomologySubject(s)
Animals, Genetically Modified/physiology , CRISPR-Cas Systems , Food, Genetically Modified , Genetic Engineering/veterinary , Meat , Sus scrofa/genetics , Animals , Gene Knock-In Techniques/veterinary , Gene Knockout Techniques/veterinary , Genetic Engineering/methods , Genetic Engineering/trends , Homologous Recombination , Sus scrofa/physiologyABSTRACT
Animals as a source of organs and tissues for xenotransplantation could become a backup solution for the growing shortage of human donors. The presence of human xenoreactive anti- bodies directed against Galα1,3Gal antigens on the cell surface of a pig donor triggers the activa- tion of the complement leading to a hyperacute reaction. The development of genetic engineer- ing techniques has enabled the modification of genomes by knocking in and/or knocking out genes. In this paper, we report the generation of modified pigs with ZFN mediated disruption of the GGTA1 gene encoding the enzyme responsible for synthesis of Galα1,3Gal antigens. ZFN plasmids designed to target the exon 9 region of the pig GGTA1 gene encoding the catalytic domain were injected into the pronuclei of fertilized egg cells. Among 107 piglets of the F0 gene- ration analyzed, one female with 9-nt deletion in exon 9 of the GGTA1 gene was found. 13 of 33 piglets of the F1 generation represented the +/- GGTA1 genotype and 2 of 13 F2 piglets repre- sented the -/- GGTA1 genotype. No changes in the animals' behavior, phenotype or karyotype were observed. Analysis confirmed heredity of the trait in all animals. A complex functional analysis of the modified animals, including flow cytometry, human serum cytotoxicity test and immunohistochemical detection, was performed to estimate the phenotype effect of genetic modification and this indicated an efficient GGTA1 knock-out in modified pigs.
Subject(s)
Galactosyltransferases/metabolism , Gene Knockout Techniques/veterinary , Swine/genetics , Animals , Base Sequence , Cell Survival , Disaccharides/metabolism , Embryo Transfer/veterinary , Female , Galactosyltransferases/genetics , Gene Deletion , Humans , Immunohistochemistry , Karyotype , Pregnancy , ZygoteABSTRACT
Rats are effective model animals and have contributed to the development of human medicine and basic research. However, the application of reproductive engineering techniques to rats is not as advanced compared with mice, and genome editing in rats has not been achieved using embryos obtained by in vitro fertilization (IVF). In this study, we conducted superovulation, IVF, and knock out and knock in using IVF rat embryos. We found that superovulation effectively occurred in the synchronized oestrus cycle and with anti-inhibin antiserum treatment in immature rats, including the Brown Norway rat, which is a very difficult rat strain to superovulate. Next, we collected superovulated oocytes under anaesthesia, and offspring derived from IVF embryos were obtained from all of the rat strains that we examined. When the tyrosinase gene was targeted by electroporation in these embryos, both alleles were disrupted with 100% efficiency. Furthermore, we conducted long DNA fragment knock in using adeno-associated virus and found that the knock-in litter was obtained with high efficiency (33.3-47.4%). Thus, in this study, we developed methods to allow the simple and efficient production of model rats.