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Construction method and application of plasmid composition, dnapk gene knockout rat model

A technology for constructing methods and compositions, applied in recombinant DNA technology, biochemical equipment and methods, genetic engineering, etc., can solve the problems of easy spontaneous tumor formation, low serum insulin content, and large limitations, and improve the success of model preparation. efficiency, simplifying preparation and screening processes

Active Publication Date: 2020-07-14
BIOCYTOGEN PHARMACEUTICALS (BEIJING) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It is known that F344 rats are widely used in the study of carcinogenesis due to their low serum insulin content, large pituitary gland, and prone to spontaneous tumor formation.

Method used

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  • Construction method and application of plasmid composition, dnapk gene knockout rat model
  • Construction method and application of plasmid composition, dnapk gene knockout rat model
  • Construction method and application of plasmid composition, dnapk gene knockout rat model

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Example 1 Design of rat DNAPK gene-specific TALEN sequence and construction of recognition module

[0066] 1) Screening of TALEN target sites

[0067] According to the sequence characteristics of the SD rat DNAPK gene, the sequence on the fourth exon was selected as the target sequence recognized by TALEN, and the nucleotide sequence of the fourth exon of the rat gene is shown in SEQ ID NO:1. In this experiment, the nucleotide sequence with the structure T(Nn)A is used as the TALEN recognition target sequence, where N is any base in A, G, T and C, and n is any number between 13 and 21 .

[0068] In SEQ ID NO:1, select a part of the sequence, such as the sequence shown in SEQ ID NO:2 as the upstream target sequence (sequence L) recognized by TALEN, and select the sequence shown in SEQ ID NO:3 as TALEN recognition The downstream target sequence (sequence R). In addition, there is a sequence gap shown in SEQ ID NO:4 between SEQ ID NO:2 and 3. That is, the target sequ...

Embodiment 2

[0071] Example 2 Microinjection and Embryo Transfer

[0072] The pronuclear fertilized eggs of SD rats were taken, and the in vitro transcription products of the premixed vector 1 and vector 2 plasmids were injected into the cytoplasm or nucleus of the rat fertilized eggs using a microinjector. Refer to the method in the "Mouse Embryo Operation Experimental Manual (Third Edition)" for microinjection of fertilized eggs. After injection, the fertilized eggs are transferred to the culture medium for short-term culture, and then transplanted to the oviduct of the recipient mother mouse to produce genes. knockout rats.

[0073] In vitro transcription process: first use the Ambion in vitro transcription kit (mMESSAGEm MACHINE T7Kit, purchased from Thermo Fisher, product number: AM1344) to transcribe into mRNA, and then use the Ambion tailing kit (poly(A)tailing kit, purchased from Thermo Fisher, product number: AM1350) added a tail to the 3' end of the mRNA sequence, and the oper...

Embodiment 3

[0074] Example 3 Identification of knockout rats

[0075] 1. Genotype identification

[0076] Apply the immunodeficiency rat that the method of the present invention obtains, utilize DNA sequencing technology to check whether the DNAPK gene target sequence in the somatic cells of the immunodeficiency rat is successfully knocked out, and the sequencing analysis result is as follows: image 3 . From the sequencing results, it can be seen that the selected DNAPK gene target site has mutations, including 18 base deletions and 4 base insertions. The loss of the function of the DNAPK gene in rats is caused by the frameshift mutation caused by the fragment deletion and base insertion of the DNA sequence. The sequence of the successfully mutated DNAPK gene is compared with the wild-type rat sequence in Table 1.

[0077] Table 1 Comparison of target sequences between DNAPK knockout rats and wild-type rats

[0078]

[0079] Note: WT is a wild-type SD rat, △18 is a deletion of 18 ...

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Abstract

The invention discloses a plasmid composition, a DNAPK gene knockout rat model building method and application. A pair of specific transcription activator like effectors for specifically recognizing two sections of adjacent nucleotide sequences on rat DNAPK genes are built; the pair of transcription activator like effectors are used for obtaining a pair of transcription activator like effector nucleases; the rat DNAPK genes are subjected to accurate and efficient targeting; the DNAPK gene knockout rat model is further obtained.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a plasmid composition, a method for constructing a DNAPK gene knockout rat model, and an application thereof. Background technique [0002] Research on the mechanism of disease treatment and drug development for different human diseases has never stopped all over the world. However, due to the limitations of human ethics and technical realization, many studies cannot be directly carried out on humans, but need to be carried out on animals similar to humans. Experiment on the body. As a rodent model animal, rats have become an indispensable animal model for basic research and drug development related to human diseases. In recent years, model rats with specific genotypes and phenotypes have become research hotspots by transforming specific genes of rats through genetic engineering techniques, and the establishment of immunodeficiency rat models is of great significance to human diseas...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/85A01K67/027
CPCA01K67/0276A01K2217/075A01K2227/105A01K2267/03C12N9/12C12N15/8509C12N2800/107C12N2800/80C12Y207/11
Inventor 沈月雷白阳张美玲周小飞姚佳维苏幼红余巧玲杜吉超赵会珍
Owner BIOCYTOGEN PHARMACEUTICALS (BEIJING) CO LTD
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