Construction method for gene site-specific mutagenesis in embryonic cell of mouse

A gene-directed mutagenesis, mouse embryo technology, applied to other methods of inserting foreign genetic materials, cells modified by introducing foreign genetic materials, using micro-injection methods, etc., can solve the problem of heavy workload, high difficulty, long cycle, etc. problems, to achieve low toxicity, avoid long cycle times, and reduce construction costs

Inactive Publication Date: 2012-12-26
EAST CHINA NORMAL UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The purpose of the present invention overcomes the disadvantages of the existing technology in the construction of gene knockout mice, such as long cycle, heavy workload, high difficulty, low efficiency, etc. TALEN messenger RNA of the target gene, a method for rapid construction of gene knockout mice

Method used

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  • Construction method for gene site-specific mutagenesis in embryonic cell of mouse
  • Construction method for gene site-specific mutagenesis in embryonic cell of mouse
  • Construction method for gene site-specific mutagenesis in embryonic cell of mouse

Examples

Experimental program
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Embodiment 1

[0061] Example 1 Construction of Lepr gene knockout in mouse cells

[0062] 1. Prediction of TALE target sites, that is, determination of the target target sequence

[0063] The mouse target genome sequence in this example is the Lepr gene. Log in to the webpage TAL Effector Nucleotide Targeter 2.0 (https: / / boglab.plp.iastate.edu / node / add / talen-old), and follow the instructions to insert the third exon of the target gene Lepr (due to failure in Lepr Find the target site that meets the conditions in the first two exons of ) and paste the sequence into the corresponding text box, and check the selection box of the relevant parameters. Set the range of the number of bases (Repeat Array Length) to be recognized by the TALE module, preferably, set to 15-20 in this embodiment; set the number of bases in the interval region (Spacer), preferably, in this embodiment Set to 15-20. After screening, several candidate target sites were obtained. Further, the target site sequence (SEQ I...

Embodiment 2

[0097] Example 2 Construction of Paklip1 gene knockout and knockin in mouse cells

[0098] 1. Construction and verification of TALEN plasmid targeting Paklip1 knock-in site

[0099] The Paklip1 gene is used as the mouse target genome sequence in this example. Log in to the web page TAL EffectorNucleotide Targeter 2.0 (https: / / boglab.plp.iastate.edu / node / add / talen-old), follow the instructions, paste the first exon sequence of the target gene Paklip1 into the corresponding text box, Tick ​​the checkboxes for the relevant parameters. Set the range of the number of bases (Repeat Array Length) to be recognized by the TALE module, preferably, set to 15-20 in this embodiment; set the number of bases in the spacer region (Spacer), preferably, in this embodiment Set to 15-20. Due to the short length of the first exon of Paklip1, no target sites meeting the conditions were found in it, so the second exon sequence was submitted, and several candidate target sites were obtained after ...

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Abstract

The invention discloses a construction method for gene site-specific mutagenesis in an embryonic cell of a mouse. A gene site-specific mutagenesis mouse is constructed by introducing a specific nucleotide sequence into the embryonic cell of the mouse. The construction method comprises the following steps: confirming a target sequence in a target genome sequence of the mouse; constructing a TALEN nucleotide sequence capable of identifying and cutting a target gene, according to the sequence of the target sequence; introducing the TALEN nucleotide sequence into the embryonic cell of the mouse; and applying the obtained embryonic cell of the mouse to culture in vitro or directly implanting to a female mouse, expressing the TALEN and cutting the target genome, thereby selecting the gene site-specific mutagenesis mouse. The construction method provided by the invention has the advantages that a homologous recombined target carrier need not be constructed, the ES target cell selection is unnecessary, the mutant proportion is high, the operation is convenient, the test cost and period are greatly reduced, and the like.

Description

technical field [0001] The invention relates to a method for rapid gene editing by using a transcription activator-like nuclease, in particular to a method for constructing gene site-directed mutation in mouse cells. Background technique [0002] With the completion of the Human Genome Project, biological research in the post-genome era urgently needs effective means to analyze gene functions. Due to the high consistency with humans at the physiological and genetic levels, gene knockout mouse models have become a powerful tool for deciphering human gene functions and finding treatments for human diseases. [0003] Gene targeting technology is the main traditional means of constructing gene knockout mice. Gene targeting is a technology that integrates an exogenous gene into a certain site on the target cell genome through homologous recombination, so as to achieve the purpose of site-specific modification and transformation of a certain gene on the chromosome. [0004] One ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/89A01K67/027
Inventor 李大力邱中伟刘明耀
Owner EAST CHINA NORMAL UNIV
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