A vector for site-directed mutation of mstn and simultaneous site-directed integration of pparγ

A technology of MRF-PPAR and site-directed mutagenesis, which is applied in vectors, nucleic acid vectors, DNA preparation, etc., can solve the problems of skeletal muscle increase, MSTN inactivity, and destruction of three-dimensional structure stability and function, so as to improve accuracy and efficiency, and improve transgenic Efficiency, the effect of shortening the breeding time

CN109679998BActive Publication Date: 2020-04-28NORTHWEST A & F UNIV
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Patent Information

Authority / Receiving Office
CN · China
Patent Type
Patents(China)
Current Assignee / Owner
Publication Date
2020-04-28

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Abstract

The invention discloses a vector for carrying out site-specific mutagenesis on MSTN (Myostatin) and site-specific integration on PPARgamma (Peroxisome Proliferator-activated Receptor gamma) at the same time, and recombinant cells. The vector comprises a Cas9 cutting expression vector and a targeting vector. According to the vector disclosed by the invention, a recombinant cell strain can be constructed by carrying out co-transfection on host cells by the Cas9 cutting expression vector and the targeting vector, the recombinant cell strain can be used as nuclear transfer donor cells for producing transgenic cloned embryo, function exertion of the MSTN can be inhibited, and meanwhile, specific expression of the PPARgamma in skeletal muscle cells can be realized, so that support is provided for quickly and efficiently developing high-quality transgenic beef new products.
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Description

technical field

[0001] The present invention relates to the technical field of transgenic cloning animals, and more specifically relates to a vector and a recombinant cell for targeting site-directed mutation of MSTN gene through miRNA and simultaneously site-specific integration of PPARγ gene. Background technique

[0002] Myostatin (MSTN) is also called growth differentiation factor (GDF8). As a negative regulator of muscle growth and differentiation, it can inhibit the transcriptional activity of the positive regulator - myogenic determinant factor (MoyD). Inhibits proliferation and differentiation of myoblasts. Mature MSTN inhibited the DNA and protein synthesis of C2C12 myogenic cells cultured in vitro in a dose-sensitive manner, and could inhibit the mitotic transition from G1 phase to S phase and from G2 phase to M phase of C2C12 myogenic cells, as well as induce C2C12 myogenic cells. The autophagy of myogenic cells thus inhibits the proliferation of C2C12 myogenic c...

Examples

Embodiment 1

[0056] Example 1 Construction of CRISPR / Cas9 Cutting Expression Vector

[0057] 1) Targeting site design and cutting expression vector construction

[0058] In order to realize the specific transcription and expression of PPARγ gene in bovine skeletal muscle cells, the g+6354G-A point mutation was introduced into the 3'UTR region of MSTN gene (GenBank: AF019761.1) to create bta-mir-1 / 206 target site, resulting in decreased expression of MSTN protein. The specific expression of PPARγ gene (GenBank: NM_181024.2) in bovine skeletal muscle was achieved by using the bovine skeletal muscle-specific promoter MRF, and it was inserted into the 3'UTR region of the bovine MSTN gene, and the bovine MSTN gene was introduced with the help of the 5' homology arm Therefore, the 3'UTR region of MSTN (chr2:6219940-6220590) is selected as the target region with a total of 650bp, see figure 1 .

[0059] Using the online software http: / / chopchop.cbu.uib.no / search.php, CCTop-CRISPR / Cas9target on...

Embodiment 2

[0071] Example 2 Construction of site-directed integration of PPARγ gene and MSTN gene point mutation targeting vector pLR-MRF-PPARγ-PloyA-SE

[0072] Site-directed integration of PPARγ gene and MSTN gene point mutation targeting vector contains 5'arm, 3'arm, PPARγ gene CDS region, LoxP-EF1α-EGFP-P2A-Puro-PolyA-LoxP screening marker, in which the upstream of PPARγ gene CDS region and skeletal muscle Specific promoter MRF linked, downstream and screening markers first LoxP sequence separated by PloyA, see Figure 5 .

[0073] The primer sequences of each element are as follows:

[0074]

[0075]

[0076] Among them, the bases in the underlined part are restriction sites; the bases in the box are point mutations introduced by the upstream homology arm.

[0077] The cultured Luxi yellow cattle fetal fibroblasts (collected in Xi'an, Shaanxi Province in January 2016) were digested and then the genome was collected and extracted. Using the extracted genome as a template, the...

Embodiment 3Ca

[0086] Example 3 Cas9 cutting expression vector and site-directed integration of PPARγ gene and MSTN gene point mutation targeting vector co-transfected Luxi yellow cattle bovine fetal fibroblasts to construct nuclear donor cells

[0087] In the present invention, the method of electroporation is used to co-transfect the fetal fibroblasts of Luxi yellow cattle with the Cas9 cutting expression vector targeting MSTN gene and the targeting vector, so as to realize the gene targeting of the fetal fibroblasts of Luxi yellow cattle and obtain the MSTN gene g+6354G - Bovine fetal fibroblasts with A point mutation and MSTN site-directed knock-in in the CDS region of the PPARγ gene.

[0088] 1) Bovine fetal fibroblast culture

[0089] Take out the frozen primary Luxi cattle fetal fibroblasts from the liquid nitrogen tank, thaw at 37°C, and centrifuge; discard the waste liquid, add 1ml of DMEM containing 10% FBS to resuspend, inoculate into 60mm cell culture dishes, and place in CO 2 C...