Mdr1a/1b double-gene knockout method and application

A gene knockout and double gene technology, applied in the field of biomedicine, can solve the problem of late emergence of mice in gene knockout rat models

Active Publication Date: 2019-03-05
EAST CHINA NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to technical limitations, gene knockout rat models appeared later than mice, and so far, no Mdr1a / 1b gene knockout rat models have emerged

Method used

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  • Mdr1a/1b double-gene knockout method and application
  • Mdr1a/1b double-gene knockout method and application
  • Mdr1a/1b double-gene knockout method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1 Selection of Mdr1a and Mdr1b targets

[0056] The Mdr1a and Mdr1b gene sequences of the rat (Rattus norvegicus (Norway rat)) were respectively found in the NCBI database, and then the start codon and stop codon of the gene were found, and the exon region was marked. Since the first two exon sequences of Mdr1a and Mdr1b genes are short (the number of bases is less than 70bp), the target point is selected on the third exon. The third exon sequences of the two genes were successively input into the online target prediction website, and two targets with a length of 18bp were obtained.

[0057] Among them, the online target prediction website is http: / / zifit.partners.org / ZiFiT / ChoiceMenu.aspx; the Mdr1a gene target sequence is 5'-AGATAGCTTTGCAAATGT-3' (SEQ ID NO.1); the Mdr1b gene target sequence is 5'-CCTCCTGATGCTGGTGTT-3' (SEQ ID NO. 2).

Embodiment 2

[0058] Synthesis and extraction of embodiment 2 sgRNA

[0059]First, synthesize a 60bp Oligo fragment, which includes the knockout target and T7 promoter sequence; then, using the Oligo fragment as a template, synthesize a complete sgRNA double-stranded template with a length of 130bp by overlapping PCR reaction, and pass The sgRNA double-stranded template was extracted and separated by the method of phenol-chloroform extraction; finally, the sgRNA double-stranded template was transcribed in vitro with the T7 in vitro transcription kit, and the transcription product was extracted and separated by the method of phenol-chloroform extraction to obtain sgRNA.

[0060] Among them, the Oligo fragment sequence containing the Mdr1a gene knockout target is 5'-GATCACTAATACGACTCACTATAGG AGATAGCTTTGCAAATGT GTTTTAGAGCTAGAAAT-3'(SEQ ID NO.3); the sequence of the Oligo fragment containing the Mdr1b gene knockout target is 5'-GATCACTAATACGACTCACTATAGG CCTCCTGATGCTGGTGTT GTTTTAGAGCTAGAAAT-3'...

Embodiment 3

[0061] Example 3 sgRNA and Cas9 mRNA co-injection and embryo transfer

[0062] (1) Preparation of pseudopregnant mice. Select robust male SD rats over 8 weeks old for sterilization, and then select robust female SD rats 7-8 weeks old to mate with the above-mentioned sterile male mice. After mating, the female mouse will show signs of pregnancy, which is the desired pseudopregnant mouse.

[0063] (2) Collection and microinjection of fertilized eggs. Strong, 6-7-week-old female SD rats were selected for superovulation, and then mated with healthy, normal-born male rats. On the second day of mating, the female mice were sacrificed, and the fertilized eggs were collected for later use. Before microinjection, the collected fertilized eggs were incubated in embryo medium at 37°C CO 2 After culturing in an incubator for 3-4 hours, the sgRNAs of Mdr1a and Mdr1b were mixed with Cas9 mRNA and then injected into the cytoplasm of fertilized eggs. Among them, the concentration of the ...

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Abstract

The invention discloses an Mdr1a/1b double-gene knockout method. An Mdr1a/1b double-gene knockout rat model is successfully constructed, and a new animal model is provided for research of functions ofP-glycoprotein (P-glycoprotein, P-gp) such as transfer of mediated medicines. According to the method, gene knockout of rat P-gp is firstly implemented by the aid of CRISPR/Cas9 technique, an F0-generation chimeric rat is acquired by processes such as design of Mdr1a and Mdr1b target spots, sgRNA in-vitro synthesis and transcription, preparation of false pregnancy rats, oosperm in-vitro micro-injection and embryo transplantation, and breeding and screening of two generations are implemented to obtain Mdr1a/1b double-gene knockout homozygous rats. Off-target conditions in the acquired gene knockout rats are omitted by verification of T7EI endonuclease.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a method for Mdr1a / 1b double gene knockout and its application in the construction of Mdr1a / 1b gene knockout rat model. Background technique [0002] In 1976, when Juliano RL and Ling V et al. were screening colchicine-resistant Chinese hamster ovary cells, they found that a glycoprotein on the cell membrane could cause cells to produce cross-resistance, because the protein and the permeability of the cell membrane (Permeability ), so it was named P-glycoprotein (P-glycoprotein, P-gp). [0003] P-gp is the earliest discovered and the most thoroughly studied ABC transporter, also known as ABCB1 (or MDR1). P-gp is a single-chain protein with about 1280 amino acids and a molecular weight of about 170kD. It is a 12-time transmembrane protein located on the cell membrane. It consists of two subunits with a similarity of 43%, and each subunit includes one Transmembran...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/89A01K67/027C12N15/113C12N15/10C12N15/11
CPCA01K67/0276C12N15/113C12N15/89C12Q1/68C07K14/47C12N2310/10C12N2800/80C12N2810/10A01K2227/105A01K2217/075A01K2267/03
Inventor 王昕赵军芳鲁健刘明耀
Owner EAST CHINA NORMAL UNIV
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