A method and application of mdr1a/1b double gene knockout

A gene knockout and dual gene technology, applied in the field of biomedicine, can solve the problem of late mice in the knockout rat model, and achieve the effects of low cost, large blood volume and simple operation.

Active Publication Date: 2022-07-08
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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  • A method and application of mdr1a/1b double gene knockout
  • A method and application of mdr1a/1b double gene knockout
  • A method and application of mdr1a/1b double gene knockout

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

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

Embodiment 2

[0058] Example 2 Synthesis and extraction of sgRNA

[0059]First, an Oligo fragment with a length of 60 bp was synthesized, including the sequence of the knockout target and the T7 promoter; then, using the Oligo fragment as a template, a complete sgRNA double-stranded template with a length of 130 bp was synthesized by overlapping PCR reaction. 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 sequence of Oligo fragment containing Mdr1a gene knockout target is 5'-GATCACTAATACGACTCACTATAGG AGATAGCTTTGCAAATGT GTTTTAGAGCTAGAAAT-3' (SEQ ID NO. 3); Oligo fragment sequence containing Mdr1b gene knockout target is 5'-GATCACTAATACGACTCACTATAGG CCTCCTGATGCTGGTGTT GTTTTAGAGC...

Embodiment 3

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

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

[0063] (2) Collection and microinjection of fertilized eggs. Robust, 6-7 week old female SD rats were selected for superovulation and then mated with robust, normally fertile male rats. The next day of mating, the female mice were sacrificed, and the fertilized eggs were taken for use. Before microinjection, collect the fertilized eggs 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 injected into the cytoplasm of fertilized eggs. Among them, the concentration of both sgRNAs was 25ng / mL, and the concentration of Cas9...

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Abstract

The present invention discloses a method for Mdr1a / 1b double gene knockout, and successfully constructs a Mdr1a / 1b gene knockout rat model, in order to study the function of P-glycoprotein (P-gp), such as mediating Drug delivery provides a new animal model. The present invention utilizes CRISPR / Cas9 technology for the first time to carry out gene knockout of rat P-gp, through the process of Mdr1a and Mdr1b target design, in vitro synthesis and transcription of sgRNA, preparation of pseudopregnant mice, in vitro microinjection of fertilized eggs and embryo transfer, etc. The F0 generation chimeric mouse was obtained, and after two generations of breeding and screening, the Mdr1a / 1b double gene knockout homozygous rat was finally obtained. As verified by T7EI endonuclease, there is no off-target phenomenon in the obtained knockout rats.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and particularly relates to a method for Mdr1a / 1b double gene knockout and its application in the construction of an Mdr1a / 1b gene knockout rat model. Background technique [0002] In 1976, when Juliano RL and LingV et al. screened colchicine-resistant Chinese hamster ovary cells, they found that a glycoprotein on the cell membrane could make the cells cross-drug resistant. ), so it was named P-glycoprotein (P-gp). [0003] P-gp is the first 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%, each subunit includes 1 Transmembrane domain (TMD) and 1 nucleic acid binding domain (NBD). Among them, TMD is composed of 6 α-helices, which is ...

Claims

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Application Information

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Patent Type & Authority Patents(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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