scholarly journals Effective MSTN Gene Knockout by AdV-Delivered CRISPR/Cas9 in Postnatal Chick Leg Muscle

2020 ◽  
Vol 21 (7) ◽  
pp. 2584 ◽  
Author(s):  
Ke Xu ◽  
Cheng Xiao Han ◽  
Hao Zhou ◽  
Jin Mei Ding ◽  
Zhong Xu ◽  
...  
Keyword(s):  
Growth And Development ◽  
Gene Editing ◽  
Gene Knockout ◽  
Muscle Growth ◽  
Meat Production ◽  
Wild Type ◽  
Knock Out ◽  
Mstn Gene ◽  
Differentiation And Proliferation ◽  
Transcriptomic Changes

Muscle growth and development are important aspects of chicken meat production, but the underlying regulatory mechanisms remain unclear and need further exploration. CRISPR has been used for gene editing to study gene function in mice, but less has been done in chick muscles. To verify whether postnatal gene editing could be achieved in chick muscles and determine the transcriptomic changes, we knocked out Myostatin (MSTN), a potential inhibitor of muscle growth and development, in chicks and performed transcriptome analysis on knock-out (KO) muscles and wild-type (WT) muscles at two post-natal days: 3d (3-day-old) and 14d (14-day-old). Large fragment deletions of MSTN (>5 kb) were achieved in all KO muscles, and the MSTN gene expression was significantly downregulated at 14d. The transcriptomic results indicated the presence of 1339 differentially expressed genes (DEGs) between the 3d KO and 3d WT muscles, as well as 597 DEGs between 14d KO and 14d WT muscles. Many DEGs were found to be related to cell differentiation and proliferation, muscle growth and energy metabolism. This method provides a potential means of postnatal gene editing in chicks, and the results presented here could provide a basis for further investigation of the mechanisms involved in muscle growth and development.

Transcriptomic Analysis of MSTN Knockout in the Early Differentiation of Chicken Fetal Myoblasts

Genes ◽  
2021 ◽  
Vol 13 (1) ◽  
pp. 58
Author(s):  
Ke Xu ◽  
Hao Zhou ◽  
Chengxiao Han ◽  
Zhong Xu ◽  
Jinmei Ding ◽  
...  
Keyword(s):  
Signaling Pathways ◽  
Molecular Mechanism ◽  
Growth And Development ◽  
Biological Effects ◽  
Muscle Growth ◽  
Negative Regulator ◽  
Myoblast Differentiation ◽  
Fatty Acid Binding ◽  
Knock Out ◽  
Early Differentiation

In mammals, Myostatin (MSTN) is a known negative regulator of muscle growth and development, but its role in birds is poorly understood. To investigate the molecular mechanism of MSTN on muscle growth and development in chickens, we knocked out MSTN in chicken fetal myoblasts (CFMs) and sequenced the mRNA transcriptomes. The amplicon sequencing results show that the editing efficiency of the cells was 76%. The transcriptomic results showed that 296 differentially expressed genes were generated after down-regulation of MSTN, including angiotensin I converting enzyme (ACE), extracellular fatty acid-binding protein (EXFABP) and troponin T1, slow skeletal type (TNNT1). These genes are closely associated with myoblast differentiation, muscle growth and energy metabolism. Subsequent enrichment analysis showed that DEGs of CFMs were related to MAPK, P13K/AKT, and STAT3 signaling pathways. The MAPK and P13K/AKT signaling pathways are two of the three known signaling pathways involved in the biological effects of MSTN in mammals, and the STAT3 pathway is also significantly enriched in MSTN knock out chicken leg muscles. The results of this study will help to understand the possible molecular mechanism of MSTN regulating the early differentiation of CFMs and lay a foundation for further research on the molecular mechanism of MSTN involvement in muscle growth and development.

scholarly journals Inactivation of porcine interleukin-1β results in failure of rapid conceptus elongation

2017 ◽  
Vol 115 (2) ◽  
pp. 307-312 ◽  
Author(s):  
Jeffrey J. Whyte ◽  
Ashley E. Meyer ◽  
Lee D. Spate ◽  
Joshua A. Benne ◽  
Raissa Cecil ◽  
...  
Keyword(s):  
Gene Editing ◽  
Mammalian Species ◽  
Interleukin 1Β ◽  
Corpora Lutea ◽  
Morphological Transition ◽  
Wild Type ◽  
Morphological Transformation ◽  
Proinflammatory Response ◽  
Knock Out ◽  
Estrogen Synthesis

Conceptus expansion throughout the uterus of mammalian species with a noninvasive epitheliochorial type of placentation is critical establishing an adequate uterine surface area for nutrient support during gestation. Pig conceptuses undergo a unique rapid morphological transformation to elongate into filamentous threads within 1 h, which provides the uterine surface to support development and maintain functional corpora lutea through the production of estrogen. Conceptus production of a unique interleukin 1β, IL1B2, temporally increases during the period of trophoblast remodeling during elongation. CRISPR/Cas9 gene editing was used to knock out pig conceptus IL1B2 expression and the secretion of IL1B2 during the time of conceptus elongation. Trophoblast elongation occurred on day 14 in wild-type (IL1B2+/+) conceptuses but did not occur in ILB2-null (IL1B2−/−) conceptuses. Although the morphological transition of IL1B2−/− conceptuses was inhibited, expression of a number of conceptus developmental genes was not altered. However, conceptus aromatase expression and estrogen secretion were decreased, indicating that IL1B2 may be involved in the spatiotemporal increase in conceptus estrogen synthesis needed for the establishment of pregnancy in the pig and may serve to regulate the proinflammatory response of endometrium to IL1B2 during conceptus elongation and attachment to the uterine surface.

scholarly journals The Scaffold Derived from GGTA1 and CMAH Gene Knockout Pigs Generated by Gene Editing Provoked Little Inflammatory Responses in a Humanized Pig Model

2020 ◽  
Author(s):  
chon-ho yen ◽  
Hao-Chih Tai ◽  
Su-Hei Peng ◽  
Tien-Shuh Yang ◽  
Ching-Fu Tu
Keyword(s):  
Gene Editing ◽  
Inflammatory Responses ◽  
Gene Knockout ◽  
Giant Cells ◽  
Small Intestinal Submucosa ◽  
Wild Type ◽  
Porcine Small Intestinal Submucosa ◽  
Small Intestinal ◽  
Intestinal Submucosa ◽  
Made In

Abstract BackgroundThe porcine small intestinal submucosa ECM (SIS-ECM) has been used as a supportive scaffold for healing in a variety of tissues. However, the outcomes of its application are far from satisfactory.ResultsThe possibility of generating a porcine small intestinal submucosa extracellular matrix (SIS-ECM) that is fully biocompatible was investigated. Samples of SIS-ECM were prepared from either domestic wild-type (WT) or double-gene knockout (dKO) pigs without antigenic responses to α-Gal and N-glycolylneuraminic acid (Neu5Gc). The scaffolds, which were sutured into cuts made in the longissimus muscle of the dKO pigs, were expected to exhibit xeno-reactions through natural antibodies as in a human response. A process was established to manufacture ameliorating acellular porcine SIS-ECM implants with consistent quality characteristics, which were assured by analyzing the level of residual DNA, the glycosaminoglycan content, and histochemical stains. Once implanted, the acellular SIS-ECM from the WT pigs caused a significant increase in serum IL-6 levels in the dKO recipient pigs, indicating a host defense through immune reactions. The levels remained unchanged when preparations from the dKO pigs were used. The pathological score of the multinuclear giant cells of the dKO (WT) group (2.3±0.5) was significantly greater than that of the dKO (dKO) group (0.7±0.3).ConclusionThe IL-6 levels and pathological evidences suggested that dKO pigs without α-Gal or Neu5Gc antigenic causing lower inflammatory response can serve as biocompatible SIS-ECM donors or as animal models for testing anthropomorphic immune responses to biomedical devices.

Muscle growth after postdevelopmental myostatin gene knockout

2007 ◽  
Vol 292 (4) ◽  
pp. E985-E991 ◽  
Author(s):  
Stephen Welle ◽  
Kirti Bhatt ◽  
Carl A. Pinkert ◽  
Rabi Tawil ◽  
Charles A. Thornton
Keyword(s):  
Muscle Mass ◽  
Gene Knockout ◽  
Muscle Growth ◽  
Myosin Heavy Chain Isoforms ◽  
Tamoxifen Treatment ◽  
Wild Type ◽  
Pretreatment Level ◽  
Myostatin Gene ◽  
Expression Of Genes ◽  
Genes Encoding

Constitutive myostatin gene knockout in mice causes excessive muscle growth during development. To examine the effect of knocking out the myostatin gene after muscle has matured, we generated mice in which myostatin exon 3 was flanked by loxP sequences (Mstn[f/f]) and crossed them with mice bearing a tamoxifen-inducible, ubiquitously expressed Cre recombinase transgene. At 4 mo of age, Mstn[f/f]/Cre+ mice that had not received tamoxifen had a 50–90% reduction in myostatin expression due to basal Cre activity but were not hypermuscular relative to Mstn[w/w]/Cre+ mice (homozygous for wild-type myostatin gene). Three months after tamoxifen treatment (initiated at 4 mo of age), muscle mass had not changed from the pretreatment level in Mstn[w/w]/Cre+ control mice. Tamoxifen administration to 4-mo-old Mstn[f/f]/Cre+ mice reduced myostatin mRNA expression to less than 1% of normal, which increased muscle mass ∼25% over the following 3 mo in both male and female mice ( P < 0.005 vs. control). Fiber hypertrophy appeared to be sufficient to explain the increase in muscle mass. The pattern of expression of genes encoding the various myosin heavy-chain isoforms was unaffected by postdevelopmental myostatin knockout. We conclude that, even after developmental muscle growth has ceased, knockout of the myostatin gene induces a significant increase in muscle mass.

scholarly journals Altered Differentiation and Proliferation of Prostate Epithelium in Mice Lacking the Androgen Receptor Cofactor p44/WDR77

Endocrinology ◽  
2010 ◽  
Vol 151 (8) ◽  
pp. 3941-3953 ◽  
Author(s):  
Shen Gao ◽  
Hong Wu ◽  
Fen Wang ◽  
Zhengxin Wang
Keyword(s):  
Androgen Receptor ◽  
Growth And Development ◽  
Target Gene ◽  
Secretory Proteins ◽  
Wild Type ◽  
Altered Expression ◽  
Prostate Development ◽  
Differentiation And Proliferation ◽  
Prostate Epithelium ◽  
Prostate Growth

Although it has been observed that various cofactors modulate activity of the androgen receptor (AR), the specific relationship between AR cofactors and prostate development and functions has not been well studied. To determine whether AR cofactor p44/WDR77 is important in prostate growth and development, we examined prostate architecture in p44/WDR77-null mice and wild-type (WT) littermates. Prostate glands from p44/WDR77-deficient animals were not only smaller than those from WT mice but also had fewer branches and terminal duct tips and were deficient in production of secretory proteins. The p44/WDR77-null prostate tissue was less differentiated and hyperproliferative relative to WT littermates. In addition, the altered expression of androgen-regulated genes was observed in the p44/WDR77-null prostate. Thus, these results suggest that the AR cofactor p44/WDR77 plays important roles in prostate growth and differentiation by modulating AR-target gene expression.

scholarly journals New insights into the construction of wild-type Saba pig-derived Escherichia coli irp2 gene deletion strains

3 Biotech ◽  
2021 ◽  
Vol 11 (9) ◽  
Author(s):  
Bo Zhang ◽  
Hongdan Wang ◽  
Weiwei Zhao ◽  
Chunlan Shan ◽  
Chaoying Liu ◽  
...  
Keyword(s):  
Iron Uptake ◽  
Gene Editing ◽  
Gene Deletion ◽  
Growth Characteristics ◽  
Wild Type ◽  
Knock Out ◽  
E Coli ◽  
Functional Studies ◽  
Key Points ◽  
Type Strains

AbstractTo construct wild-type E. coli irp2 gene deletion strains, CRISPR/Cas9 gene editing technology was used, and the difficulty and key points of gene editing of wild-type strains were analyzed. Based on the resistance of the CRISPR/Cas9 system expression vector, 4 strains of 41 E. coli strains isolated from Saba pigs were selected as the target strains for the deletion of the irp2 gene, which were sensitive to both ampicillin and kanamycin. Then, CRISPR/Cas9 technology was combined with homologous recombination technology to construct recombinant vectors containing Cas9, sgRNA and donor sequences to knock out the irp2 gene. Finally, the absence of the irp2 gene in E. coli was further verified by iron uptake assays, iron carrier production assays and growth curve measurements. The results showed that three of the selected strains showed single base mutations and deletions (Δirp2-1, Δirp2-2 and Δirp2-3). The deletion of the irp2 gene reduced the ability of E. coli to take up iron ions and produce iron carriers, but not affect the growth characteristics of E. coli. It is shown that the CRISPR/Cas9 knock-out system constructed in this study can successfully knock out the irp2 gene of the wild-type E. coli. Our results providing new insights into genome editing in wild-type strains, which enable further functional studies of the irp2 gene in wild-type E. coli.

scholarly journals Proteomic Studies on the Mechanism of Myostatin Regulating Cattle Skeletal Muscle Development

2021 ◽  
Vol 12 ◽  
Author(s):  
Hui Sheng ◽  
Yiwen Guo ◽  
Linlin Zhang ◽  
Junxing Zhang ◽  
Manning Miao ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Extracellular Matrix ◽  
Focal Adhesion ◽  
Growth And Development ◽  
Muscle Growth ◽  
Differentially Expressed ◽  
Type I ◽  
Differentially Expressed Proteins ◽  
Mstn Gene ◽  
Bovine Skeletal Muscle

Myostatin (MSTN) is an important negative regulator of muscle growth and development. In this study, we performed comparatively the proteomics analyses of gluteus tissues from MSTN+/− Mongolian cattle (MG.MSTN+/−) and wild type Mongolian cattle (MG.WT) using a shotgun-based tandem mass tag (TMT) 6-plex labeling method to investigate the regulation mechanism of MSTN on the growth and development of bovine skeletal muscle. A total of 1,950 proteins were identified in MG.MSTN+/− and MG.WT. Compared with MG.WT cattle, a total of 320 differentially expressed proteins were identified in MG.MSTN cattle, including 245 up-regulated differentially expressed proteins and 75 down-regulated differentially expressed proteins. Bioinformatics analysis showed that knockdown of the MSTN gene increased the expression of extracellular matrix and ribosome-related proteins, induced activation of focal adhesion, PI3K-AKT, and Ribosomal pathways. The results of proteomic analysis were verified by muscle tissue Western blot test and in vitro MSTN gene knockdown test, and it was found that knockdown MSTN gene expression could promote the proliferation and myogenic differentiation of bovine skeletal muscle satellite cells (BSMSCs). At the same time, Co-Immunoprecipitation (CO-IP) assay showed that MSTN gene interacted with extracellular matrix related protein type I collagen α 1 (COL1A1), and knocking down the expression of COL1A1 could inhibit the activity of adhesion, PI3K-AKT and ribosome pathway, thus inhibit BSMSCs proliferation. These results suggest that the MSTN gene regulates focal adhesion, PI3K-AKT, and Ribosomal pathway through the COL1A1 gene. In general, this study provides new insights into the regulatory mechanism of MSTN involved in muscle growth and development.

scholarly journals Enhanced soluble sugar content in tomato fruit using CRISPR/Cas9-mediated SlINVINH1 and SlVPE5 gene editing

PeerJ ◽  
2021 ◽  
Vol 9 ◽  
pp. e12478
Author(s):  
Baike Wang ◽  
Ning Li ◽  
Shaoyong Huang ◽  
Jiahui Hu ◽  
Qiang Wang ◽  
...  
Keyword(s):  
Gene Editing ◽  
Single Gene ◽  
Tomato Fruit ◽  
Sugar Content ◽  
Soluble Sugar ◽  
Wild Type ◽  
Tomato Fruits ◽  
Knock Out ◽  
Soluble Sugar Content ◽  
Soluble Solid

Soluble sugar is known to improve the sweetness and increase tomato sauce yield. Studies have focused on improving the content of soluble sugar in tomato fruits, usually by promoting functional genes. We studied two genes (SlINVINH1 and SlVPE5) that inhibited the accumulation of soluble sugar in tomato fruits and obtained two genes’ knocked-out lines (CRISPR-invinh1 or CRISPR-vpe5) using CRISPR/Cas9. Aggregated lines with CRISPR-invinh1 and CRISPR-vpe5 were gained by hybridization and self-pollination. Compared to wild-type lines, the glucose, fructose, and total soluble solid (TSS) contents of CRISPR-invinh1 and CRISPR-vpe5 increased significantly. Glucose, fructose, and TSS levels further improved simultaneously with CRISPR-invinh1 and CRISPR-vpe5 than with single gene knock-out lines. This indicates that these genes have a synergistic effect and will increase the soluble sugar content. Thus, the knock-out SlINVINH1 and SlVPE5 may provide a practical basis for improving the sweetness of tomato fruits and their processing quality.

scholarly journals CRISPR/Cas9-Mediated SlMYBS2 Mutagenesis Reduces Tomato Resistance to Phytophthora infestans

2021 ◽  
Vol 22 (21) ◽  
pp. 11423
Author(s):  
Chunxin Liu ◽  
Yiyao Zhang ◽  
Yinxiao Tan ◽  
Tingting Zhao ◽  
Xiangyang Xu ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Late Blight ◽  
Phytophthora Infestans ◽  
Gene Knockout ◽  
Disease Index ◽  
Oxygen Species ◽  
Wild Type ◽  
Pr Genes ◽  
Knock Out ◽  
Pathogenesis Related

Phytophthora infestans (P. infestans) recently caused epidemics of tomato late blight. Our study aimed to identify the function of the SlMYBS2 gene in response to tomato late blight. To further investigate the function of SlMYBS2 in tomato resistance to P. infestans, we studied the effects of SlMYBS2 gene knock out. The SlMYBS2 gene was knocked out by CRISPR-Cas9, and the resulting plants (SlMYBS2 gene knockout, slmybs2-c) showed reduced resistance to P. infestans, accompanied by increases in the number of necrotic cells, lesion sizes, and disease index. Furthermore, after P. infestans infection, the expression levels of pathogenesis-related (PR) genes in slmybs2-c plants were significantly lower than those in wild-type (AC) plants, while the number of necrotic cells and the accumulation of reactive oxygen species (ROS) were higher than those in wild-type plants. Taken together, these results indicate that SlMYBS2 acts as a positive regulator of tomato resistance to P. infestans infection by regulating the ROS level and the expression level of PR genes.

Advances of MSTN Genetic Markers in Domesticated Animals

2020 ◽  
Author(s):  
Cheng-Li Liu ◽  
Guang-Xin E ◽  
Wei-Wei Ni ◽  
Xiao Wang ◽  
Shu-Zhu Cheng ◽  
...  
Keyword(s):  
Growth And Development ◽  
Structural Characteristics ◽  
Muscle Growth ◽  
Animal Husbandry ◽  
Negative Regulator ◽  
Economic Traits ◽  
Animal Products ◽  
Mstn Gene ◽  
Animal Muscle

The myostatin (MSTN) gene is a negative regulator of animal muscle growth and development. This gene not only inhibits muscle cell growth and reduces fat accumulation but also exerts a significant effect on back fat thickness, birth weight and carcass traits. MSTN gene mutation, an important factor that influences economic traits, directly affects the growth and development of animals and consequently the quality of animal products. This paper reviews the structural and functional characteristics of the MSTN gene. The genetic variation of the MSTN gene is then compared among four domestic animals (cattle, sheep, goat and pig) and its correlation with important economic traits is analysed. The mechanism and structural characteristics of MSTN gene mutants are further discussed. This paper provides explication on the application of MSTN gene research in breeding and its importance to the advancement of animal husbandry.

Sign in / Sign up

Export Citation Format

Share Document