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Construction method of Slco1b2 gene knockout rat and application thereof

A technology of gene knockout and construction method, which is applied in the field of biomedicine and can solve the problems of high cost, long experiment cycle, and difficulty in obtaining

Pending Publication Date: 2019-02-01
EAST CHINA NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The existing Slco1b2 (homologous to SLCO1B1 / SLCO1B3 in humans) gene knockout mouse model is based on the traditional principle of homologous recombination, which is complicated to operate, low in efficiency, long in the experimental cycle, and high in cost
Due to technical limitations such as the difficulty in obtaining and culturing rat embryonic stem cells, there is no report on the Slco1b2 gene knockout rat model

Method used

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  • Construction method of Slco1b2 gene knockout rat and application thereof
  • Construction method of Slco1b2 gene knockout rat and application thereof
  • Construction method of Slco1b2 gene knockout rat and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Example 1 Selection of Slco1b2 Knockout Target

[0068] First, the rat (Rattus norvegicus (Norway rat)) Slco1b2 gene sequence was searched through the NCBI database, and then the start codon and stop codon were found in the gene sequence, and the exon regions were marked in sequence. In order to completely knock out the target gene, the selected target of the present invention is at the first exon of the rat Slco1b2 gene, and the first exon sequence of the Slco1b2 gene is input into the online target prediction website https: http: / / benchling.com, get the target sequence shown in SEQ ID NO.1 with a length of 18bp.

[0069] And because the CRISPR / Cas9 gene knockout system recognizes the DNA sequence ending in NGG (PAM site) corresponding to its gRNA in the genome, a 21bp sequence is obtained, including 18bp of the target site and 3bp of the PAM site.

[0070] The online target prediction website selected by the present invention is: https: / / benchling.com.

[0071] The ...

Embodiment 2

[0072] Example 2 In vitro sgRNA template synthesis and transcription

[0073] The sgRNA template is a 60 bp Oligo fragment comprising the T7 promoter sequence and the 18 bp target sequence obtained in Example 1. After the fragment is synthesized in vitro, use it as a template to synthesize a complete sgRNA double-stranded template by overlapping PCR reaction, extract and separate the double-stranded template, and then use the T7 in vitro transcription kit to transcribe the sgRNA double-stranded template in vitro. The product was extracted and separated to obtain the sgRNA of the Slco1b2 gene.

[0074] Among them, the 60bp Oligo fragment sequence containing the Slco1b2 gene knockout target is:

[0075] 5’-GATCACTAATACGACTCACTATAGG GCAAACAAGGTTCTGCGA GTTTTAGAGCTAGAAAT-3' (SEQ ID NO. 2). The underlined area is the target sequence (SEQ ID NO.1).

Embodiment 3

[0076] Embodiment 3 embryo microinjection

[0077] Firstly, pseudopregnant female rats are prepared, and healthy adult male rats (over 8 weeks old) are sterilized, and then placed in cages with healthy adult female rats (over 8 weeks old). The female rats with vaginal plugs were successfully mated and were the pseudopregnant female rats required for subsequent experiments.

[0078] Then superovulate the normal adult female mice, put the female mice in a cage with normal male mice, and make them mate and conceive normally. The mated female mice were sacrificed, fertilized eggs were taken out, and the fertilized eggs were kept in vitro at 37°C CO 2 Incubate with embryo medium for 2 hours in the incubator.

[0079] Using the microinjection technique, the sgRNA of the Slco1b2 gene obtained in Example 2 and the Cas9 mRNA were co-injected into the fertilized egg cytoplasm. Wherein, the sgRNA injection concentration is 25ng / ml, and the Cas9 mRNA injection concentration is 50ng / ml....

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Abstract

The invention discloses a construction method of Slco1b2 gene knockout rat and an application thereof. According to the invention, construction of Slco1b2 gene knockout rat is carried out by using a CRISPR / Cas9 system, which includes selection of Slco1b2 knockout targets, in-vitro synthesis and transcription of sgRNA and Cas9 mRNA, preparation of pseudopregnant mother rats, in-vitro microinjectionand transplantation of single cell embryos, rat breeding and screening, and obtaining of homozygote Slco1b2 gene knockout rat. Through verification by T7EI endonuclease, the gene knockout rat prepared by the method of the invention does not generate off-target phenomenon, and in the F2-generation knockout rat of about 4 weeks, total bilirubin, direct bilirubin and indirect bilirubin content are significantly higher than that of wild type rats. The invention also proposes an application of the construction method for hyperbilirubinemia research and drug development. The method provides an effective method for researching diseases such as increased bilirubin, and provides an effective experimental tool for drug research and development, and has broad application prospects.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a construction method and application of a Slco1b2 gene knockout rat. Background technique [0002] Organic anion-transporting polypeptides (OATPs) are 12 transmembrane proteins encoded by soluble carrier (Solute Carrier, SLC) genes, which mediate the entry of important endogenous substances and exogenous drugs into cells. OATPs include six subfamilies, of which the OATP1B subfamily plays a vital role. The OATP1B subfamily includes two family members, OATP1B1 and OATP1B3, which are encoded by the SLCO1B1 (SLC21A6) and SLCO1B3 (SLC21A8) genes, respectively, and are mainly expressed in liver tissue. and some tumor tissues. The substrates of OATP1B1 and OATP1B3 cover a wide range, including endogenous substances such as bilirubin, bile acids, and steroid hormones, as well as clinically commonly used statins for regulating blood lipids and reducing blood clots, and s...

Claims

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Application Information

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IPC IPC(8): C12N15/90A01K67/027C12N15/11
CPCA01K67/0276C12N15/907C07K14/47A01K2267/03A01K2217/075A01K2227/105
Inventor 王昕马新润覃璇尚旭阳刘明耀
Owner EAST CHINA NORMAL UNIV
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