Method for deleting sheep Myostatin gene locus by zinc finger nuclease

A technology of myostatin and zinc finger nuclease, which is applied in the direction of introducing foreign genetic material using vectors, recombinant DNA technology, artificial cell constructs, etc., and can solve problems such as methods for not disclosing sheep myostatin genes

Active Publication Date: 2013-09-11
新疆维吾尔自治区畜牧科学院中国-澳大利亚绵羊育种研究中心
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Problems solved by technology

[0005] In the prior art, although Chinese patent applications CN102296073A and CN102260711A also disclose the t

Method used

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  • Method for deleting sheep Myostatin gene locus by zinc finger nuclease
  • Method for deleting sheep Myostatin gene locus by zinc finger nuclease
  • Method for deleting sheep Myostatin gene locus by zinc finger nuclease

Examples

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Embodiment 1Z

[0015] Construction and screening of embodiment 1 ZFN expression vector

[0016] 1. Design and synthesis of zinc finger nuclease for targeted deletion of Myostatin gene

[0017] According to the sheep Myostatin gene sequence (DQ530260.1) obtained from the NCBI website, the ZFNs design was completed by Sigma Company, and the specific target site sequence was determined by bioinformatics methods:

[0018] ATACCCATCTTGTGCACC aagcaa ACCCCAAAGGTTCAG ; The middle part sequence (aagcaa) is the FokI endonuclease cleavage site, that is, the target site sequence for ZFN specific knockout. The corresponding expression vector is PZFN1 / PZFN2-set1.

[0019] 2. Screening of ZFNs

[0020] In a sheep fibroblast cell line, it is detected whether the expression vector can cut the corresponding genomic DNA sequence of the cell. Primers were designed on both sides of the ZFNs site, set1-Forward: 5'-GTGTCAGGCATTCAGATATTC-3'; set1-Reverese: 5'-GCTTGTGCTTAAGTGACTGTAGC-3'.

[0021] Two pairs of...

Embodiment 2

[0022] Example 2 Obtaining of Single Cell Clones and Identification of Gene Knockout Clones

[0023] 1. Zinc finger nuclease-mediated establishment of MSTN gene deletion cell lines

[0024] (1) Isolation and culture of sheep primary fibroblasts

[0025] The ear tissues of 2 robust, well-developed, newly born Dorset rams were collected, immediately placed in D-Hanks solution containing 3% double antibody, and brought back to the laboratory for ultra-clean bench processing. In a 35mm petri dish, add 3% double antibody D-Hanks to wash 8-10 times, remove fat and connective tissue, cut the tissue pieces with ophthalmic scissors, add 100 μL serum to wet the tissue pieces, and use elbow forceps to remove the tissue pieces ( About 2-3mm) evenly attached to the bottle wall, placed in a 37°C, 5% CO2 incubator for cultivation. On the next day, a small amount of culture medium (4-5 mL) containing 20% ​​fetal bovine serum was added to the culture bottle, and the culture was cultured in a...

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Abstract

The invention discloses a method for deleting sheep Myostatin gene locus by zinc finger nuclease, and the method comprises the following steps: designing a sequence which targets a sheep Myostatin gene exon 3 and can recognize a specific target site of 39bp, constructing a zinc finger nuclease plasmid, transfecting a sheep fibroblast by the plasmid, obtaining a monoclonal cell with deleted diallele with 95bp and 113bp. The invention demonstrates that the method can accurately delete Myostatin gene exon 3 and separate cells whose Myostatin gene locus is deleted, thereby confirming the effectiveness of the sequence, and the obtained monoclonal cell with deletion is not need to be screened by antibiotic. The invention has two advantages of genome targeting modification and safety, and has important meanings for cultivating transgenic sheep without resistance marker genes and with safety and high efficiency.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for point-directed deletion of a specific target site sequence of a sheep myostatin (Myostatin) gene by a zinc finger nuclease. Background technique [0002] Targeted genetic modification of animal and plant genes is of great significance for the analysis of gene function, the study of disease treatment or pathogenesis, and the establishment of transgenic animals and plants of high economic value. Traditional animal gene-targeted genetic modification technology is based on homologous recombination or nuclear transfer in embryonic stem cells (ES cells). It is difficult to establish ES cell lines. Currently, the ES cell lines that can be used for targeted genetic modification of animals are limited to rats and mice. In addition, the success rate of animal-targeted genetic modification by nuclear transfer is also low, and the specificity is not high, so the application of thes...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N5/071
Inventor 刘明军张雪梅李文蓉张宁贺三刚刘晨曦玛依拉
Owner 新疆维吾尔自治区畜牧科学院中国-澳大利亚绵羊育种研究中心
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