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Method for introducing mutated gene, gene having mutation introduced therein, cassette for introducing mutation, vector for introducing mutation, and knock-in non-human mammal

A gene knock-in, mammalian technology, applied in vectors, nucleic acid vectors, genetic engineering, etc., can solve problems such as low probability of homologous recombination, not improving the probability of homologous recombination

Inactive Publication Date: 2011-09-28
FUKUOKA UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is a big disadvantage in the method of making this gene knock-in animal: using the above-mentioned homologous recombination, the probability of homologous recombination of the desired exogenous gene DNA into the gene DNA of the target gene is very low, about 10 -5 the following
However, none of these existing technologies is a method for improving the probability of homologous recombination, and is no different from the conventional method of introducing foreign DNA through homologous recombination with a very low probability of homologous recombination

Method used

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  • Method for introducing mutated gene, gene having mutation introduced therein, cassette for introducing mutation, vector for introducing mutation, and knock-in non-human mammal
  • Method for introducing mutated gene, gene having mutation introduced therein, cassette for introducing mutation, vector for introducing mutation, and knock-in non-human mammal
  • Method for introducing mutated gene, gene having mutation introduced therein, cassette for introducing mutation, vector for introducing mutation, and knock-in non-human mammal

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0156] (1) Sequence structure of target recombinant vector

[0157] The target recombination vector consists of a part derived from a plasmid, a negative selection marker cassette, a partial sequence of the KCNQ2 gene as the first homologous recombination DNA sequence region (long arm region), and lox71 as the first mutant lox sequence from the 5'-end. sequence, a positive selection marker DNA sequence, a lox2272 sequence as the second mutant lox sequence, a second homologous recombination DNA sequence region (short arm region), and a sequence structure composed of a restriction endonuclease site for linearization of the vector composition.

[0158]The target recombination vector (pTgKCNQ2) was constructed based on the pBluescript II SK+ plasmid, with a full-length DNA sequence of 14,164 bp (SEQ ID NO: 5). Its sequence structure is as follows, and Figure 4 Represents a plasmid map.

[0159] Base number 1-673: part derived from pBluescript II SK+

[0160] Base number 674-2...

Embodiment 2

[0200] This example relates to a mutation-introducing vector used in homologous recombination with the target recombination vector prepared in Example 1.

[0201] The KCNQ2 mutation introduction vector (pMtKCNQ2YC and pMtKCNQ2AT) is used to introduce the KCNQ2 gene such as Tyr 284 →Cys or Ala 306 →Two types of vectors for nucleotide replacement of Thr. The loxKR3 sequence is set at the 5' end of the 570bp KCNQ2 gene fragment (exon 6 and before and after) containing any of the above-mentioned arbitrary nucleotide substitutions, and the puromycin resistance gene (PuroR) and lox2272 sequence are set at the 3' end. When these vectors are introduced into the recipient ES cells together with the Cre recombinase expression vector, the region between lox71-lox2272 on the KCNQ2 gene of the recipient ES cells will be efficiently replaced with the mutant KCNQ2 sequence through the action of Cre recombinase . To confer puromycin resistance on the replaced cells, positive selection can ...

Embodiment 3

[0230] This example relates to the construction of recipient ES cells capable of introducing mutations into the KCNQ2 gene. The construction of the recipient ES cells is carried out in the following manner (refer to Figure 7 ).

[0231] The mouse ES cells were subcultured in 2 culture dishes, and the semi-confluent (semi-confluent) mouse ES cells (KPTU strain) in a semi-confluent state were detached from the culture dishes by trypsin treatment, and placed in PBS Suspended in medium to a total of 1.6ml. 20 μg of the linearized target recombinant vector prepared in Example 1 was added thereto, and after cooling on ice for 10 minutes, the aliquots were transferred to two cuvettes for electroporation. ), each discharge once under the conditions of 0.8kV and 3.0μF, and carry out the introduction of DNA.

[0232] As described above, the target recombination vector was electroporated to introduce DNA into mouse ES cells, and the above-mentioned sequence factors were introduced in...

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Abstract

Disclosed is a method for introducing a mutation into a gene, which comprises the following steps: a homologous recombination step of carrying out the homologous recombination between a target gene into which the mutation is to be introduced and a target recombinant vector, thereby substituting an exon in the target gene into which the mutation is to be introduced by a target DNA sequence in the target recombinant vector; and a mutation introduction step of carrying out the specific recombination between the target DNA sequence in the resulting recombined target gene and a mutation introduction cassette of a mutation introduction vector carrying a mutated DNA sequence containing a mutated exon by the intervening action of Cre recombinase to substitute the target DNA sequence by the mutated DNA in the mutation introduction cassette, thereby producing a mutation-introduced gene into which the mutant DNA sequence has been introduced. The method enables the production of a knock-in non-human animal, such as a knock-in mouse, which carries the mutation-introduced gene.

Description

technical field [0001] The invention relates to a mutation gene introduction method, a mutation introduction gene, a mutation introduction cassette, a mutation introduction carrier and a gene knock-in non-human mammal. More specifically, the present invention relates to a method for introducing a mutant gene, a gene for introducing a mutation and a method for preparing the same, a cassette for introducing a mutation, and a carrier for introducing a mutation, and Knock-in of non-human mammals with mutations introduced into genes. Background technique [0002] In organisms, it is not an exaggeration to say that genes, a "design map" that holds genetic information, are used to control all the functions used to maintain their life. The entire genetic information of these genes is controlled by a combination of base pairs consisting of 4 bases called DNA. If the combination of the base pair changes abnormally for some reason, resulting in a gene mutation, the genetic informatio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09A01K67/027C12N5/10
CPCC12N15/8509A01K2227/105A01K67/0275A01K2217/072C12N15/907C12N2800/30C12N2800/107A01K2267/0306C07K14/705
Inventor 广濑伸一弟子丸正伸荒木喜美
Owner FUKUOKA UNIV
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