Establishment of carrier based on fish CRISPR/Cas9 system by using gene knockout method ad establishing method of carrier

A carrier and division technology, which is applied in the field of fish genetic engineering, can solve the problems of high technical requirements, high cost, and long test cycle, and achieve the effects of simple operation, high targeting accuracy, and short test cycle

Inactive Publication Date: 2014-12-24
HEILONGJIANG RIVER FISHERY RES INST CHINESE ACADEMY OF FISHERIES SCI
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  • Abstract
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Problems solved by technology

[0007] The purpose of the present invention is to solve the problem of introducing exogenous DNA into cells by means of microinjection, electroporation, sperm carrier method, etc. in the existing transgenic methods, and randomly integrating it. It is difficult to determine the position in the genome where the transgene is inserted, and there are Potential safety hazards, and the existing gene knockout methods have cumbersome procedures, high technical requirements, high costs, long test cycles, and success rates are limited by many factors. Vector of CRISPR / Cas9-like system and construction method thereof

Method used

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  • Establishment of carrier based on fish CRISPR/Cas9 system by using gene knockout method ad establishing method of carrier
  • Establishment of carrier based on fish CRISPR/Cas9 system by using gene knockout method ad establishing method of carrier
  • Establishment of carrier based on fish CRISPR/Cas9 system by using gene knockout method ad establishing method of carrier

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specific Embodiment approach 1

[0038] Specific embodiment one: a kind of carrier of this embodiment adopts gene knockout method to construct based on fish CRISPR / Cas9 system, and described carrier comprises backbone carrier, MSTN a fragment and MSTN b fragment; The first exon of MSTNa The nucleotide sequence of the first exon of MSTN b is shown in the sequence table Seq ID No: 1 (the size is 176bp) as shown in the sequence table Seq ID No: 1 (the size is 180bp) .

specific Embodiment approach 2

[0039] Embodiment 2: This embodiment is different from Embodiment 1 in that: the backbone vector is pX330 vector. Others are the same as in the first embodiment.

specific Embodiment approach 4

[0040] Specific embodiment four: A kind of adopting gene knockout method of this embodiment to construct the method based on fish CRISPR / Cas9 system vector, it is carried out according to the following steps:

[0041] 1. Design crRNAs based on the first exon of carp MSTN a and MSTN b, synthesize MSTN a crRNA primers and MSTN bcrRNA primers respectively, treat in boiling water for 15 minutes, then cool down naturally and anneal overnight, and obtain two viscosities with BbsI enzyme cutting respectively. DNA double-stranded annealing product at the end;

[0042] 2. Use BbsI enzyme to perform single enzyme digestion on the pX330 vector, and then use SAP phosphatase to perform a dephosphorylation reaction to recover the linearized pX330 vector;

[0043] 3. Using T4 ligase, ligate the linearized pX330 vector recovered in step 2 with the two annealed products obtained in step 1 to obtain the ligation product;

[0044] 4. Transfer the connection product of step 3 into competent cell...

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Abstract

The invention discloses establishment of a carrier based on a fish CRISPR/Cas9 system by using a gene knockout method ad an establishing method of the carrier, and relates to a carrier based on the fish CRISPR/Cas9 system and an establishing method of the carrier, so as to solve the problems that in a conventional transgenosis method the position that transgenosis is inserted into genome is hard to confirm and potential safety hazard can be generated when external DNA is led into cells and is randomly integrated by using a microscopic injection method, an electroporation method, a sperm carrier method and the like, and the problems that a conventional gene knockout method is complex in process, high in requirement on techniques, high in expense and long in experiment period, and the success rate is limited by multiple factors. By co-ejecting two non-integrated knockout carriers, namely, pX330-MSTN a and pX330-MSTN b, into zygotes of fishes in the 1-2 cell stages, high-efficiency and rapid gene knockout can be achieved.

Description

technical field [0001] The invention relates to a fish CRISPR / Cas9 system-based vector and a construction method thereof, in particular to a construction method of carp MSTN a and MSTN b gene knockout vectors. The invention belongs to the field of fish genetic engineering. Background technique [0002] my country is a big fishery country, and it is also the first country in the world to carry out research on genetically modified aquatic products, and has developed the world's first genetically modified fish. In 1984, Chinese scholar Zhu Zuoyan established the first theoretical model of transgenic fish for the first time. The Heilongjiang Fisheries Research Institute has carried out research on fast-growing transgenic carp by using genetic engineering technology, and prepared a salmon growth hormone gene "super carp", whose weight at the third age is 1.8 times that of the control fish, and its rapid growth characteristics are suitable for northern The needs of the carp bree...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12N15/66
Inventor 闫学春于泽张旭赵翊丞滕春波
Owner HEILONGJIANG RIVER FISHERY RES INST CHINESE ACADEMY OF FISHERIES SCI
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