A method for constructing gene site-directed mutagenesis

A technology of gene site-directed mutation and construction method, which is applied in the field of gene site-directed mutagenesis in rat embryonic cells, which can solve the problems of low frequency of ideal target sites, harsh culture conditions, and great difficulty, and achieve fast and efficient gene site-specification The effect of mutation, shortening the experiment cycle, and reducing the cost of the experiment

Active Publication Date: 2015-10-28
BIORAY LABORATORIES INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It was not until 2008 that the isolation and culture of rat embryonic stem cells in the true sense, as well as the successful report of germline integration and passage of chimeric rats, were reported. However, due to the complex and harsh culture conditions and the influence of resistance selection on stemness maintenance, gene targeting in rat embryonic stem cells is still difficult.
Similar to that in mice, homologous recombination-based gene targeting in rat embryonic stem cells must be subject to the following constraints: 1. The construction process of targeting vectors is long and difficult
2. Since the occurrence rate of spontaneous homologous recombination in animal cells is only 10 -5 -10 -6 , after electrotransferring the targeting vector into rat embryonic stem cells, it takes a lot of time, manpower and material resources to screen the embryonic stem cells that undergo the targeted homologous recombination
3. ES cells have strict requirements on the culture conditions in the in vitro culture process, a little carelessness will cause their irreversible differentiation, and make them lose the ability of germline transfer, and cannot produce mutant progeny in subsequent operations
[0009] However, in the construction process of TALEN, about 20 highly repetitive small fragments of DNA need to be assembled into a size of about 2kb. The process is still time-consuming and cumbersome, and there are certain unstable factors.
In addition, because TALEN requires the 5' end of the target to be adjacent to the site, and the base of the 3' end of the target itself must be T, plus the requirement for the appropriate length of the TALEN target itself and the spacer (Spacer) (15-20bp), in practice The frequency of ideal target sites in the operation is low, which limits the flexibility of targeting site selection to some extent

Method used

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  • A method for constructing gene site-directed mutagenesis
  • A method for constructing gene site-directed mutagenesis
  • A method for constructing gene site-directed mutagenesis

Examples

Experimental program
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Effect test

Embodiment 1

[0075] Example 1 Construction of Mc4r Gene Knockout Rats by Injecting RNA in Rat Cells

[0076] 1. Construction of NLS-hCas9-NLS in vitro transcription vector

[0077] Such as figure 2 As shown, the Cas9 protein expression module consists of the SP6 promoter sequence from the 5' end to the 3' end, the N-terminal nuclear localization signal (Nuclear localization sequence, NLS), the humanized Cas9 coding DNA sequence, the C-terminal nuclear localization signal, Polyadenylic acid (polyA) composition. The specific construction strategy is, on the basis of the vector pX260 (Addgene plasmid #42229), introduce the SP6 promoter and the Kozak sequence through the NcoI restriction endonuclease site overlapping with the start codon of the NLS-hCas9-NLS coding sequence. That is, synthesize single-stranded oligonucleotides P1 (SEQ ID NO.1) and P2 (SEQ ID NO.2), use PNK phosphatase to add 5' phosphate groups to the above-mentioned single-stranded oligonucleotides, and anneal them Ligate...

Embodiment 2

[0102] Example 2 Construction of Mc3r / Mc4r knockout rats by injecting CAS protein and gRNA in rat cells

[0103] 1. Construction of CAS nuclease prokaryotic expression vector PET-28a-H6-3FLAG-NLS-CAS9-NLS

[0104] Such as Image 6 As shown, the prokaryotic expression vector of Cas9 fusion protein consists of T7 promoter sequence, His6Tag, 3×Flag, N-terminal nuclear localization sequence (NLS1), and humanized Cas9 encoding DNA from the 5' end to the 3' end. Sequence, C-terminal nuclear localization signal NLS2 constitutes. First, based on the NLS-hCas9-NLS in vitro transcription vector, His6 and 3×FLAG tags, as well as a new N-terminal NcoI restriction enzyme site and a C-terminal EcoRI restriction enzyme site were introduced by overlapping PCR. A new H6-3FLAG-NLS-CAS9-NLS junction was inserted through the NcoI and EcoRI restriction sites on PET-28a and used as translation initiation for protein expression through the start codon contained in the NcoI restriction site site. ...

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Abstract

The invention discloses a method for constructing gene site-directed mutation. The method comprises the following steps: determining a target sequence which is in a rat target genome sequence and can be identified by an artificial modified CRISPR-Cas (clustered regularly interspaced short palindromic repeats-associated protein) system; constructing a gRNA (guide ribonucleic acid) which can identify a CAS protein and guide the CAS protein to a target gene target sequence; introducing the gRNA sequence and a CAS protein coded deoxyribonucleic acid sequence or the gRNA sequence and an in-vitro expressed CAS protein mixture into a rat embryonic cell. According to the method, a homologous recombined targeting vector does not need to be constructed, ES (embryonic stem) targeting cell screening is not needed, a mutant has a high proportion, the operation is convenient, simultaneous multi-site knockout can be realized, the experiment cost can be greatly reduced, and the experiment cycle can be shortened.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a method for constructing gene site-directed mutation in rat embryonic cells. Background technique [0002] Rats have been used as animal models in the fields of physiology, pharmacology, toxicology, nutrition, behavior, immunology, and oncology for more than 150 years. Similar to mice, rats have the advantages of small size, short intergenerational time, multiple births per litter, and low feeding costs. At the same time, compared with mice, rats have a larger body size, which is convenient for observation and surgical operations; the diet of rats is closer to that of humans, which is convenient for nutritional research; rats are more sensitive to toxic substances and are suitable for use in medicines. Toxicology experiment. At present, each drug needs to be tested in rats for toxicology before it goes on the market. Due to inherent genetic reasons, rat disease mo...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/85C12N5/10
Inventor 李大力刘明耀邱中伟
Owner BIORAY LABORATORIES INC
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