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Gene knock-in composition and use method and application thereof

A gene knock-in and composition technology, applied in the field of gene knock-in composition, can solve problems such as low efficiency

Inactive Publication Date: 2015-09-30
SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, the efficiency of genome precise fixed-point modification by manufacturing DSB is low

Method used

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  • Gene knock-in composition and use method and application thereof
  • Gene knock-in composition and use method and application thereof
  • Gene knock-in composition and use method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0191] Example 1. Localization of potential nicks at the rat Bassoon integration target site

[0192] The goal of rat Bassoon genetic modification is to construct a transgenic rat strain that expresses Basson-EOS3.2 fusion protein at the endogenous Bassoon gene locus without changing other genome sequences. The operation of the corresponding transgene is integrated into the EOS3.2 DNA coding frame (ORF) within the front frame (in-frame) of the stop codon (stop codon) of Bassoon.

[0193] First, determine the integration site in the target site area, that is, near the stop codon of Bassoon, that is, select the single-strand gap (nick) site, and the integration site can be selected at the nick site or at a specific site between the cooperative nick sites. on the nucleotides.

[0194] This embodiment adopts Cas9 that has been genetically engineered, that is, Cas9-D10A and Cas9-H840A, hereinafter referred to as D10A and H840A, and the supporting sgRNA (upstream chain PAM: CCT T...

Embodiment 2

[0201] Example 2. Design of Exogenous Donor DNA Templates

[0202] Such as image 3 The donor DNA template was designed as shown, with 1 kb of homologous sequence upstream of the upstream nick (upstream homology arm) and 1 kb of homologous sequence downstream of the downstream nick (downstream homology arm). The EOS3.2 coding sequence was added at the integration site, and a linker region was added at the junction of EOS3.2 and Bassoon gene to reduce the effect of fluorescent protein on the function of Bassoon gene protein. This element comprising the upstream and downstream homology arms and the EOS3.2 coding frame (ORF) is molecularly cloned into the pBluescript II vector (Agilent's plasmid vector), and the plasmid is amplified and extracted to become an exogenous donor DNA (nucleotide sequence See SEQ ID NO: 15).

[0203] Donor DNA Construction:

[0204] 1kb upstream homology arm - CCTCA* + coding region of polyG linker + EOS3.2 coding region containing TAA stop codon *T...

Embodiment 3

[0205] Example 3. Preparation of recombinant enzyme complex

[0206] Four recombination factors derived from Escherichia coli (E.coli), collectively called OFAR (RecO, RecF, RecA, RecR) reading frame sequences were cloned from the genome of DH5alpha strain. After sequencing verification, the mammalian codons were optimized, and the nuclear localization sequence was added respectively. Then the SP6 promoter sequence (ATTTA GGTGA CACTA TAGAA, SEQ ID NO: 16) was added upstream of the entire sequence, and the in vitro transcription kit (mMESSAGE mMACHINE T7 / SP6kit, Life Technologies) and transcription vector pSP73 (Promega, USA) were used to obtain Capped and polyA-added mRNAs were mixed and frozen at -80°C with a working concentration of 100ng / ul.

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Abstract

The invention provides a gene knock-in composition containing a single incision enzyme or polynucleotide coding the same, recombinase or polynucleotide coding the same, and an optional donor DNA containing an exogenous gene. The invention also provides a corresponding gene knock-in method and a method for preparing a transgenic non-human mammal model. The composition and the methods provided by the invention can be used for realizing efficient homologous recombination integration, and meanwhile various defects of insertion or deletion, genome instability and the like which are generated easily in the prior art can be avoided.

Description

technical field [0001] The present invention relates to the field of biotechnology. Specifically, the present invention relates to a gene knock-in composition for accurately and efficiently introducing exogenous DNA fragments into mammalian genome, and its use method and application. Background technique [0002] At present, the basic principle of genome targeted modification is to use the spontaneous or induced DNA double-strand break (double-strain break, DSB) in the target site region, the double-strand break will activate the DNA repair mechanism in the cell to carry out genome modification, such as non- homologous region end joining (NHEJ) or homologous recombination (HR) ( figure 1 shown). [0003] In cells, the optimistic estimate of the probability of spontaneous DSB generation is less than 10 4 , if I-SceI, I-AniI and other homing endonucleases (homing endonuclease), FoxI, Cas9 and other nucleases are used to induce DSB through genetic engineering, the efficiency...

Claims

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Application Information

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IPC IPC(8): C12N15/09A01K67/027C12N5/10
Inventor 杨宇丰仇子龙孙强
Owner SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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