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Targeting vector and reconstituted cell for Cas9-mediated site-specific integration of FABP4 (adipose fatty acid-binding protein) gene and MSTN (myostatin) gene point mutation

A FABP4, site-specific integration technology, applied in genetically modified cells, cells modified by introducing foreign genetic material, genetic engineering, etc., can solve problems such as low fat content and affect meat quality, shorten breeding time, and improve transgenic efficiency. , the effect of improving the screening efficiency

Pending Publication Date: 2019-01-01
NORTHWEST A & F UNIV
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Problems solved by technology

However, animals with a "double-muscle" phenotype have lower levels of intramuscular fat, which seriously affects meat quality

Method used

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  • Targeting vector and reconstituted cell for Cas9-mediated site-specific integration of FABP4 (adipose fatty acid-binding protein) gene and MSTN (myostatin) gene point mutation
  • Targeting vector and reconstituted cell for Cas9-mediated site-specific integration of FABP4 (adipose fatty acid-binding protein) gene and MSTN (myostatin) gene point mutation
  • Targeting vector and reconstituted cell for Cas9-mediated site-specific integration of FABP4 (adipose fatty acid-binding protein) gene and MSTN (myostatin) gene point mutation

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Embodiment Construction

[0035] The present invention will be described in further detail below in conjunction with the accompanying drawings and embodiments.

[0036] The present invention first constructs the Cas9 eukaryotic expression vector pSpCas9-sgRNA targeting MSTN gene and the targeting vector pLR-P2A-FABP4-PolyA-SE for site-specific integration of FABP4 gene and MSTN gene point mutation, and then utilizes the method of electroporation to transform Cas9 The nuclear expression vector pSpCas9-sgRNA and the targeting vector pLR-P2A-FABP4-PolyA-SE were co-transfected into Luxi cattle fetal fibroblasts, cloned cells were obtained by Puro screening, and positive clones for precise targeting were obtained by Junction PCR screening and verification cell. Finally, the positive cloned cells were used as nucleated cells to transfer into enucleated bovine oocytes to obtain transgenic cloned embryos.

[0037] (1) Preparation of reagents and solutions

[0038] 1. Reagents

[0039] Restriction endonuclea...

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Abstract

The invention discloses a targeting vector and a reconstituted cell for the Cas9-mediated site-specific integration of FABP4 (adipose fatty acid-binding protein) gene and MSTN (myostatin) gene point mutation. The invention utilizes an electroporation method to co-transfect a Cas9 eukaryotic expression vector and a targeting vector targeting the MSTN gene to Luxi bovine fetal fibroblasts, consequently, gene targeting for the Luxi bovine fetal fibroblasts is realized, and bovine fetal fibroblasts with FABP4 gene and MSTN gene point mutation knocked in the MSTN site are obtained. By Junction of PCR (Polymerase Chain Reaction) screening and verification, targeted positive cloned cells are obtained. The positive cloned cells as donor cells are transplanted into denucleated bovine oocytes, so that a transgenic cloned embryo can be obtained, and thereby a solid foundation is laid for the rapid and efficient development of high-quality new genetically modified beef varieties.

Description

technical field [0001] The invention belongs to the technical field of transgenic cloning animals, and relates to a CRISPR / Cas9-mediated site-directed integration of FABP4 gene and MSTN gene point mutation targeting vector and recombinant cells. Background technique [0002] Myostatin (MSTN), also known as growth differentiation factor 8 (GDF-8), is a member of the transforming growth factor β (transforming growth factor β, TGF-β) superfamily. MSTN is a negative regulator of skeletal muscle growth and development, and inhibition of its activity leads to muscle overgrowth and increased carcass leanness. Inactivation of the MSTN gene is the molecular cause of the double-muscle phenotype in beef cattle, for example, Belgian blue and Piedmont cattle. Compared with wild-type mice, mice homozygous for the mutant MSTN gene gained more than 30 percent of their body weight and their muscle fibers had a larger cross-section (hypertrophy) and a greater number of muscle fibers (hypertr...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/90C12N5/10C12N15/877
CPCC12N15/85C07K14/47C07K14/475C12N15/8771C12N15/907C12N2310/20C12N2510/00
Inventor 张涌康健权富生谢晓刚葛陆星董翔宸王丰瑜孙洪政韩成全
Owner NORTHWEST A & F UNIV
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