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Cytochrome P450 enzyme humanized rat model as well as construction method and application thereof

A rat model and construction method technology, applied in the field of biomedicine, can solve the problem that the CYP humanized rat animal model has not been widely used, and achieve the effect of eliminating species differences.

Active Publication Date: 2021-10-29
EAST CHINA NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is currently no mature method for constructing CYP humanized rat models in the world, and CYP humanized rat animal models have not been widely used

Method used

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  • Cytochrome P450 enzyme humanized rat model as well as construction method and application thereof
  • Cytochrome P450 enzyme humanized rat model as well as construction method and application thereof
  • Cytochrome P450 enzyme humanized rat model as well as construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0097] Embodiment 1 target design

[0098] Check the gene sequence and splicing isoforms of the target gene through the website http: / / www.ensembl.org / index.html. Select the gene sequence ending with NGG (N stands for any base) as the target, and if a gene has multiple splicing isoforms, select the expression exons shared by several isoforms. If the first expression exon is too small (only a few tens of bp), the target can be found on the next expression exon. The CRISPR / Cas9 gene knockout system can recognize the DNA sequence ending in NGG corresponding to its guide RNA in the genome, so the selected target of the present invention is a 18bp nucleotide at the 5' end of the NGG sequence in the target gene of the rat sequence; because the CRISPR / Cas9 system for gene knockout utilizes: the introduction of mutated bases in the repair of DNA in non-homologous recombination, which causes a frameshift mutation of the 3' end sequence behind the mutated site, therefore, the selected ...

Embodiment 2

[0112] Example 2 Synthesis and transcription of sgRNA template

[0113] In the present invention, humanization is carried out by co-injecting sgRNA, Cas9 mRNA and homologous recombination plasmids into fertilized rat eggs. First synthesize a 60bp oligonucleotide sequence containing the T7 promoter sequence, target sequence and sgRNA 5' end sequence; then synthesize the complete sgRNA template sequence by overlapping PCR, with a total length of 125bp; through the T7 in vitro transcription kit The template sequence was transcribed in vitro; the purified target sgRNA co-transcription product was obtained after extraction with phenol and chloroform. The corresponding oligonucleotide sequence of 60bp synthesized by the present invention is as follows:

[0114] The complete sequence of sgRNA is as follows:

[0115]

[0116] The highlighted part (·) is the T7 promoter sequence; the sequence N indicates that it will be replaced with the target sequence; the rest is the sgRNA back...

Embodiment 3

[0155] Example 3 Homologous Recombination Plasmid Construction

[0156] Take the humanization of CYP1A2 as an example. The purpose of this example is to insert human CYP1A2 into the rat Cyp1a2 gene to construct a rat with inactivated rat Cyp1a2 and stable expression of human CYP1A2. When constructing the donor gene template, the gene sequences on both sides of the cut in the Cyp1a2 gene in the CRISPR / Cas9 system of the present invention respectively take 800bp as the left and right homology arms, and by overlapping the PCR method, the left homology arm sequence (LHA) , CYP1A2 gene CDS sequence, Flag tag sequence, 6X His sequence and right homology arm sequence (RHA) are connected sequentially to form a donor plasmid for rat CYP1A2 humanization. Schematic diagram of homologous recombination plasmid figure 1 Shown (taking CYP1A2 as an example):

[0157] The construction process and method of CYP3A4 humanized homologous recombination plasmid are similar to those of CYP1A2.

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Abstract

The invention belongs to the technical field of biological medicine, and discloses a construction method of a CYP enzyme humanized rat animal model for drug metabolism research and application of the CYP enzyme humanized rat animal model. The construction method comprises genotype identification, humanized gene expression identification and humanized gene function verification. The method comprises the following steps of firstly, constructing a CYP enzyme humanized rat by utilizing a CRISPR system, including selection of a gene editing target spot, in-vitro synthesis and transcription of sgRNA and Cas9 mRNA, construction of a homologous recombination template, preparation of a pseudopregnant female rat, in-vitro microinjection and transplantation of a single-cell embryo and reproduction of the rat, finally obtaining a homozygote CYP gene humanized rat, then performing CYP enzyme expression detection on the homozygous humanized rat from a protein level, and performing metabolic function verification to prove that the CYP humanized rat is successfully constructed. The CYP1A2 humanized rat has metabolic characteristics more similar to those of people, the species difference of CYP1A2 mediated drug metabolism between the rat and the people is eliminated, and a new rat animal model is provided for research on CYP mediated drug metabolism.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to cytochrome P450 enzyme humanized rats and its construction method and application. Background technique [0002] The marketing of a new drug usually goes through a long process, generally divided into the research stage and the development stage, which mainly includes the process of target establishment, compound activity screening, lead compound optimization, preclinical research and clinical research. Statistics show that 40% of drugs in clinical research are forced to stop the research due to their poor pharmacokinetic properties. Therefore, evaluating the metabolism and pharmacokinetic properties of compounds at the early stage of compound screening is beneficial to reduce the investment and risk of failure in later research, among which drug screening based on cytochrome P450 enzymes (CYP enzymes) and related metabolic studies It plays an important role in t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/90C12N15/53C12N15/89A01K67/027C12N15/113
CPCC12N9/0081C12Y114/15006C12N15/907C12N15/89C12N15/113A01K67/0276C12N2310/20A01K2217/075A01K2227/106A01K2267/03A01K2267/0393Y02A50/30
Inventor 王昕鲁健张远金刘明耀
Owner EAST CHINA NORMAL UNIV
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