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Method of target-integrating foreign DNA (Deoxyribonucleic Acid) sequence to Rosa26 sites of rat and mouse as well as application thereof

A DNA sequence, exogenous technology, applied in the direction of DNA/RNA fragments, recombinant DNA technology, biochemical equipment and methods, etc., to achieve the effect of clear genetic background, accurate biological function, and shortened screening period

Active Publication Date: 2014-01-29
OBIO TECH SHANGHAI CORP LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The present invention utilizes TALEN technology to solve the defect of above-mentioned traditional method

Method used

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  • Method of target-integrating foreign DNA (Deoxyribonucleic Acid) sequence to Rosa26 sites of rat and mouse as well as application thereof
  • Method of target-integrating foreign DNA (Deoxyribonucleic Acid) sequence to Rosa26 sites of rat and mouse as well as application thereof
  • Method of target-integrating foreign DNA (Deoxyribonucleic Acid) sequence to Rosa26 sites of rat and mouse as well as application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] 1. Rat and mouse TALENs recognize the Rosa26 site

[0041] After research, it was found that a TALEN that can be used for targeted integration of foreign DNA sequences into rats and mice recognizes the Rosa26 site. The upstream and downstream recognition sequences of the TALEN that recognizes the Rosa26 site are as follows:

[0042]Upstream recognition sequence: 5'-agccaataatcaaatt-3' (as shown in SEQ ID NO.1)

[0043] Downstream recognition sequence: 5'-agttccttggtaacatt-3' (as shown in SEQ ID NO.2).

[0044] where the details are available as figure 1 shown.

[0045] 2. Construction of a pair of TALEN plasmids that recognize the above sites

[0046] 1. TALEN recognizes the upstream and downstream TALEN sequence synthesis and digestion of the Rosa26 site

[0047] The following upstream and downstream TALEN sequences (CDS coding regions) were chemically synthesized with BsmB I sites at both ends. For the synthesized sequence, the enzyme BsmB I was used to digest, a...

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Abstract

The invention discloses a method of target-integrating a foreign DNA (Deoxyribonucleic Acid) sequence to Rosa26 sites of rats and mice as well as application thereof. According to the invention, a stable cell line can be obtained for expressing various foreign genes through constructed TALEN plasmid, rat homologous recombination plasmid and mouse homologous recombination plasmid by means of respectively transferring corresponding plasmids to cells of rats and mice. The stable cell line obtained can be stably handed from generation to generation with clear genetic background, so that the biological function of the foreign gene is accurately represented.

Description

technical field [0001] The present invention relates to a method and application of integrating exogenous DNA sequence, in particular to a method and application of targeted integration of exogenous DNA sequence into rat or mouse Rosa26 site. Background technique [0002] TALEN (Transcription Activator-like Effector Nuclease) technology is a new molecular biology tool. Utilizing that the amino acid sequence of the nucleic acid binding domain of the TAL protein has a constant correspondence with the nucleic acid sequence of its target site, it can be assembled into a modular protein that specifically binds to any DNA sequence, and by adding an endonuclease domain, the TALEN protein can target Cut genomic DNA [Cermak T, Doyle EL, Christian M, et al., Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Nucleic Acids Res. 2011.39(12):e82.]. In the presence of a homologous template (Donor plasmid), cells will undergo homologou...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12N15/85C12N5/10C12Q1/02A01K67/027
Inventor 杨兴林陈国泽彭娟潘讴东王富杰夏清梅殷珊
Owner OBIO TECH SHANGHAI CORP LTD
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