Method for constructing gene site-directed mutation

A technology of gene site-directed mutation and construction method, which is applied in the field of gene site-directed mutation construction in rat embryonic cells. Experimental cost, effect of high proportion of mutants

Active Publication Date: 2013-11-13
BIORAY LABORATORIES INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The technical effect of this patented method lies within three aspects - faster learning speed, lower resource consumption, easier handling compared to existing techniques such as phage display and random insertional clone rearrangement. Additionally, the use of direct injection without fusion cascade allows for quick creative cloning of wild ungulate models while maintaining their original functions like metabolism. Overall, these improvements make it safer and economically viable when studying rodents and mouse monoclonal antibodies against disease related proteins.

Problems solved by technology

Technologies aim towards improving the efficiency and versatility of modifying proteins involved in biological processes like metabolism, cellular signal processing, disease detection, etc., particularly when dealing with rare mutations present within these structures. Current approaches involve assembling multiple copies of cloned templates onto chromosomal elements, but they require extensive effort and may lead to instability issues. To overcome these challenges, we propose a method involving modulation and assembly techniques based upon molecular recognition functions generated by enzymatic domains associated with DNA recognition mechanisms.

Method used

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  • Method for constructing gene site-directed mutation
  • Method for constructing gene site-directed mutation
  • Method for constructing gene site-directed mutation

Examples

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Embodiment 1

[0075] Example 1 Construction of Mc4r Gene Knockout Rats by Injecting RNA in Rat Cells

[0076] 1. Construction of NLS-hCas9-NLS in vitro transcription vector

[0077] Such as figure 2 As shown, the Cas9 protein expression module consists of the SP6 promoter sequence from the 5' end to the 3' end, the N-terminal nuclear localization signal (Nuclear localization sequence, NLS), the humanized Cas9 coding DNA sequence, the C-terminal nuclear localization signal, Polyadenylic acid (polyA) composition. The specific construction strategy is, on the basis of the vector pX260 (Addgene plasmid #42229), introduce the SP6 promoter and the Kozak sequence through the NcoI restriction endonuclease site overlapping with the start codon of the NLS-hCas9-NLS coding sequence. That is, synthesize single-stranded oligonucleotides P1 (SEQ ID NO.1) and P2 (SEQ ID NO.2), use PNK phosphatase to add 5' phosphate groups to the above-mentioned single-stranded oligonucleotides, and anneal them Ligate...

Embodiment 2

[0102] Example 2 Construction of Mc3r / Mc4r knockout rats by injecting CAS protein and gRNA in rat cells

[0103] 1. Construction of CAS nuclease prokaryotic expression vector PET-28a-H6-3FLAG-NLS-CAS9-NLS

[0104] Such as Image 6 As shown, the prokaryotic expression vector of Cas9 fusion protein consists of T7 promoter sequence, His6Tag, 3×Flag, N-terminal nuclear localization sequence (NLS1), and humanized Cas9 encoding DNA from the 5' end to the 3' end. Sequence, C-terminal nuclear localization signal NLS2 constitutes. First, based on the NLS-hCas9-NLS in vitro transcription vector, His6 and 3×FLAG tags, as well as a new N-terminal NcoI restriction enzyme site and a C-terminal EcoRI restriction enzyme site were introduced by overlapping PCR. A new H6-3FLAG-NLS-CAS9-NLS junction was inserted through the NcoI and EcoRI restriction sites on PET-28a and used as translation initiation for protein expression through the start codon contained in the NcoI restriction site locati...

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Abstract

The invention discloses a method for constructing gene site-directed mutation. The method comprises the following steps: determining a target sequence which is in a rat target genome sequence and can be identified by an artificial modified CRISPR-Cas (clustered regularly interspaced short palindromic repeats-associated protein) system; constructing a gRNA (guide ribonucleic acid) which can identify a CAS protein and guide the CAS protein to a target gene target sequence; introducing the gRNA sequence and a CAS protein coded deoxyribonucleic acid sequence or the gRNA sequence and an in-vitro expressed CAS protein mixture into a rat embryonic cell. According to the method, a homologous recombined targeting vector does not need to be constructed, ES (embryonic stem) targeting cell screening is not needed, a mutant has a high proportion, the operation is convenient, simultaneous multi-site knockout can be realized, the experiment cost can be greatly reduced, and the experiment cycle can be shortened.

Description

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Claims

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Application Information

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Owner BIORAY LABORATORIES INC
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