Skeletal muscle specific miRNA expression vector and reconstitution cell of target Myostatin gene

A skeletal muscle-specific, expression carrier technology, applied to cells modified by introducing foreign genetic material, genetic engineering, plant genetic improvement, etc., can solve the problem of myostatin gene deletion or mutation

Inactive Publication Date: 2014-04-09
NORTHWEST A & F UNIV +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] 1) Deletion or mutation of Myostatin gene

Method used

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  • Skeletal muscle specific miRNA expression vector and reconstitution cell of target Myostatin gene
  • Skeletal muscle specific miRNA expression vector and reconstitution cell of target Myostatin gene
  • Skeletal muscle specific miRNA expression vector and reconstitution cell of target Myostatin gene

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Embodiment Construction

[0042] The present invention first constructs the skeletal muscle-specific miRNA expression vector pmiR-MNM2 containing the pre-miRNA targeting Myostatin gene and the MYL1 gene promoter, and then introduces the exogenous expression vector pmiR-MNM2 into bovine skeletal muscle cells by liposome method. Muscle cells, the expression of the marker gene EmGFP was observed with a fluorescence microscope, and the expression level of Myostatin mRNA in the transfected group and the non-transfected group was detected by RT-PCR, and the expression level of Myostatin mRNA in the transfected group and the non-transfected group was detected by western blot. The level of Myostatin protein expression in skeletal muscle myoblasts in the stained group. Then, the exogenous expression vector pmiR-MNM2 was introduced into red Angus neonatal bovine fibroblasts by electroporation, and positive cells were obtained by G418 screening. PCR identification was performed to confirm that the target gene targ...

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Abstract

The invention discloses a skeletal muscle specific miRNA expression vector and a reconstitution cell of target Myostatin gene. Transgenic cloned embryos are prepared by the following steps: firstly constructing a skeletal muscle specific miRNA expression vector pmiR-MNM2 containing pre-miRNA and MYL1 gene promoters of the target Myostatin gene; then guiding the exogenous expression vector pmiR-MNM2 into red Augus neonatal bovine fibroblasts by an electric dye-transfer method, obtaining positive cells through G418 screening, and performing PCR (Polymerase Chain Reaction) identification to verify that the target gene is integrated into the genome of bovine embryonic fibroblasts; transferring bovine embryonic fibroblasts subjected to pmiR-MNM2 dye transfer, which are taken as donor cells, into bovine enucleated oocytes to finally obtain the transgenic cloned embryos which are transplanted into wombs of acceptor cows. The invention lays a solid foundation for production of red Augus cloned cattle with a dual-muscle characteristic.

Description

technical field [0001] The invention belongs to the technical field of transgenic cloning animals, and relates to the construction of nuclear donor cells of transgenic cloned embryos, in particular to a skeletal muscle-specific miRNA expression vector targeting Myostatin gene and recombinant cells. Background technique [0002] Myostatin is a negative regulator of skeletal muscle growth and development. The natural mutation of Myostatin gene (MSTN gene) causes the skeletal muscles of beef cattle, pigs, sheep and other animals to exhibit an overdeveloped double-muscle phenotype. The muscle mass of Myostatin-knockout mice is 2-3 times that of wild-type mice (McPherron, Lawler,.et al.Regulation of skeletal muscle mass in mice by a new TGF-β superfamily member[J].Nature,1997, 387:83-90.), therefore, it is possible to produce transgenic livestock with high-yielding meat traits by inactivating the Myostatin gene. Existing inactivation methods include the following: [0003] 1) D...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/85C12N5/10C12N15/873
Inventor 张涌郑艳玲权富生王勇胜刘军
Owner NORTHWEST A & F UNIV
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