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Method for conditional knockout of Lox13 gene of mouse and application

A gene knockout, conditional technology, applied in the field of genetic engineering

Active Publication Date: 2015-05-27
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no report on the knockout of the mouse Loxl3 gene

Method used

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  • Method for conditional knockout of Lox13 gene of mouse and application
  • Method for conditional knockout of Lox13 gene of mouse and application
  • Method for conditional knockout of Lox13 gene of mouse and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] The construction of embodiment 1Loxl3 conditional gene knockout vector

[0047] The design of the vector is to knock out the second exon (containing the promoter sequence) of the LOXL3 gene, so as to realize the inactivation of the entire gene. Firstly, six homologous recombination fragments (A, B, C, D, E and F) were amplified from BAC, and connected into pL253, pL452 and pL451 ( figure 1 ), through one-step retrieval and two-step homologous recombination, two loxP sites and Neo marker genes (two FRT sites on both sides) were introduced on both sides of the second exon of the LOXL3 gene to construct a LOXL3 conditional gene knockout Remove vector plasmid ( figure 2 ); then linearly transfected ES cells with the vector plasmid, and screened positive ES clones for homologous recombination; finally, injected ES clones into C57BL / 6 mouse blastocysts by microinjection technology, and obtained LOXL3 conditional gene knockout by PCR screening mice.

[0048] 1. Extraction...

Embodiment 2

[0205] Transfection and screening of embodiment 2Loxl3 conditional gene knockout vector

[0206] 1. Large-scale extraction of Loxl3 conditional gene knockout vector plasmid

[0207] (1) Inoculate the preserved strain DH5α containing the vector plasmid (Beijing Quanshijin Biotechnology Co., Ltd.) on ampicillin (50 μg / ml) and kana (50 μg / ml) resistant solid medium plates, and culture at 37 ° C 12h, pick a single colony and inoculate it into 5ml LB liquid medium, culture at 37°C, 200rpm shaking for 12-16h;

[0208] (2) Take 1ml of bacterial liquid and transfer it to 500ml LB liquid medium, shake and culture at 37°C and 200rpm for 12-16h;

[0209] (3) Centrifuge at 6000g for 10min at 4°C, discard the supernatant and collect the bacteria;

[0210] (4) Use QIAGEN's large extraction kit for extraction, add 4ml of reagent P1 to the collected bacteria, and suspend it;

[0211] (5) Add 4ml of reagent P2 to the tube, gently turn it upside down 4-6 times, and keep it at room temperatur...

Embodiment 3

[0253] Example 3 Using microinjection technology to make floxed mice that can be used for conditional gene knockout

[0254] The ES cells that were screened and identified correctly were injected into the blastocysts of C57BL / 6 mice through microinjection technology (see the mouse embryo operation guide), and the injected blastocysts were transferred to the uterus of pseudopregnant mother mice for implantation, giving birth to embedded embryos. Zygomatic mice, chimeric mice were mated with C57BL / 6 mice to produce brown and black offspring mice. The tails of the brown mice were cut off to extract DNA, and the positive mice were screened by the long PCR amplification described above, which were floxed mice with loxl3 conditional gene knockout. The mouse tail DNA extraction steps are as follows:

[0255] The DNA extraction steps are as follows:

[0256] (1) Cut the 0.3-0.5cm long mouse tail and put it into an EP tube. After shredding, rinse with normal saline or PBS, and centr...

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Abstract

The invention relates to a method for conditional knockout of Lox13 gene of a mouse and an application. The method comprises the steps that loxP loca of a Cre recombinase targeting sequence are added to the two ends of a key exon of an Lox13 gene; a conditional gene knockout carrier is constructed, and transfected with an embryonic stem cell (ESC); microinjection is conducted for making a floxed mouse capable of being used for conditional gene knockout; the floxed mouse is mated with a Cre mouse with corresponding tissue specificity; and the mouse of which an Lox13 gene can be knocked out at a specific tissue is obtained. The mouse obtained by the method serves as a model mouse for morbidity research on a cleft palate disease, so that the functions of the Lox13 gene in the tissue and the effects of the Lox13 gene in an embryonic development regulation mechanism can be illustrated in details. In addition, the mouse can serve as an animal model for researching the functions of the Lox13 gene, and has important application values.

Description

technical field [0001] The invention relates to a method and application of a mouse Loxl3 gene conditional knockout, belonging to the technical field of genetic engineering. Background technique [0002] Since the completion of the Human Genome Project, life science research has entered the post-genome era, and the core work of the post-genome era is to study the function of genes. At present, the most effective method for studying gene function is gene targeting (gene knockout and gene knockin) technology. Gene targeting technology is a new technology developed in the field of life science research in recent years. It is developed on the basis of embryonic stem cell (ES) technology and homologous recombination technology. , and then obtain information on gene function by observing and analyzing the phenotype of the animal with the altered gene. There is another important use of gene targeting technology—creating animal models of human diseases. [0003] Existing studies ...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12Q1/68
Inventor 张剑高建刚侯从哲杨瑞刘子熠
Owner SHANDONG UNIV
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