WO2019222974A1 - A method for knocking out mouse ebln1 gene by using crispr/cas9 system - Google Patents

Abstract

Provided is a method for knocking out the mouse EBLN1 gene by using a CRISPR/Cas9 system. The method comprises: firstly, obtaining the DNA sequence for the gRNA recognition region of the mouse EBLN1 gene; then, designing gRNAs that target the DNA sequence and constructing a T7-promoter-containing gRNA in vitro transcription vector for the EBLN1 gene; and finally, using Cas9 and the gRNA in vitro transcription vector to obtain Cas9 mRNA and gRNA, wherein same can be used for producing transgenic mice with EBLN1 gene knockout.

Description

 Method for knocking out mouse EBLN1 gene using CRISPR / Cas9 system

 Technical field

 [0001] The present invention belongs to the field of animal genetic engineering and genetic modification, and particularly relates to a method for knocking out mouse EBLN1 gene by using CRISPR / Cas9 system.

 Background technique

 [0002] Borna disease virus (BDV) is a non-segment single-stranded negative-stranded RNA virus first discovered in Germany in 1885. It has strict neuropathy, and its genome encodes six viral proteins. Nucleoprotein (N) is the main infection marker and pathogenic protein. Studies have shown that similar fragments to the Bornavirus N gene (encoding nucleoprotein) are found in a variety of mammalian genomes, including humans, non-human primates, rodents, and elephants, and are named endogenous Endogenous Boma-like N Element (EBLN).

 Summary of invention

 technical problem

 [0003] According to research, the EBLN1 gene may be a potential susceptible gene that causes depression, which encodes a protein or participates in the pathophysiology of depression, and conducts in-depth systematic research on it, and further clarifies the molecular genetic basis of depression It is of great significance to search for drug treatment targets, but the lack of E BLN1 gene knockout mice in the prior art has caused certain obstacles to the progress of related research.

 Problem solution

 Technical solutions

 [0004] The present invention provides a method for using a CRISPR / Cas9 system to knock out mouse EBLN 1 gene.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is: A method for knocking out mouse EBLN1 gene by using CRISPR / Cas9 system, which is characterized in that it includes a sequence specific to a mouse EBLN1 gene target site The base sequence is shown in FIG. 1; the gRNA oligonucleotide sequence that specifically targets the EBLN1 gene is shown in FIG. 2. [0006] An in vitro transcription vector comprising the gRNA.

 [0007] The in vitro transcription vector of gRNA is pMD19T-T7-gRNA, and the in vitro transcription vector of Cas9 protein is p ST1374-NLS-flag-linker-Cas9.

 [0008] A method for knocking out a mouse EBLN1 gene using the CRISPR / Cas9 system includes the following steps

[0009] S1. Construct an in vitro transcription vector that specifically targets the gRNA of the EBLN1 gene; obtain gRNA that targets the EBLN 1 gene by in vitro transcription;

 [0010] S2. In vitro transcription vector of Cas9 protein in vitro, to obtain Cas9 mRNA;

 [0011] S3. The gRNA and Cas9 mRNA of the above steps are injected into the cytoplasm of mouse fertilized eggs, cultured in vitro and transplanted into the fallopian tubes of adult female mice for generating transgenic mice containing EBLN1 gene mutations.

 [0012] After gRNA and Cas9 mRNA are mixed in step S3, the final concentration is: Cas9 mRNA 30

ng / _[i l, gRNA 12 ng / _j xl.

 The beneficial effects of the invention

 Beneficial effect

 (1) gRNA and Cas9

 The mRNA is transfected into the cell at the same time, and the EBLN1 gene is knocked out for studying the function of the EBLN1 gene;

 [0014] (2) gRNA and Cas9 mRNA are injected into a fertilized egg at the same time, and then used to produce transgenic mice that knock out EBLN 1 by embryo transfer;

 [0015] (3) gRNA and Cas9 mRNA are transfected into cells at the same time, and positive cells are used as donor cells after screening

Transgenic mice containing EBLN1 gene mutations were generated by nuclear transfer.

 Brief description of the drawings

 BRIEF DESCRIPTION OF THE DRAWINGS

 [0016] FIG. 1 is a sequence diagram of a mouse EBLN1 gene-specific target site;

 [0017] FIG. 2 is a gRNA sequence diagram designed to target the EBLN1 gene.

 BRIEF DESCRIPTION OF THE DRAWINGS

 [0018] FIG. 1 is a sequence diagram of a mouse EBLN1 gene-specific target site;

[0019] FIG. 2 is a gRNA sequence diagram designed to target the EBLN 1 gene. Invention Examples

 Embodiments of the invention

 [0020] The present invention is described in further detail below with reference to the embodiments and the accompanying drawings, but the embodiments of the present invention are not limited thereto.

 Example 1: Construction of CRISPR / Cas9 System Targeting EBLN1 Gene

 [0022] The sequence of the mouse EBLN1 gene was found in GenBank and the gRNA was designed. The target site sequence is shown in FIG. 1 and the gRNA sequence is shown in FIG. 2.

 [0023] Construction of a pMD19T-T7-gRNA vector containing a specific gRNA sequence:

 [0024] (1) designing and synthesizing gRNA that recognizes the EBLN1 gene;

 [0025] (2) annealing the synthesized gRNA oligonucleotide in vitro;

 (3) pass gRNA through Kpn I and Sph

 I site was digested, ligated, and inserted into pMD19T-T7-gRNA.

 [0027] The CRISPR / Cas9 system for the EBLN1 gene is:

 In vitro transcription vectors pMD19T-T7-gRNA and pST1374-NLS-flag-linker-Cas9.

 [0028] Example 2: In vitro transcription

 [0029] Using the constructed vector pMD 19T-T7-gRNA and Cas9

 The in vitro transcription vector pST1374-NLS-flaglinker-Cas9 performs in vitro transcription mediated by the T7 promoter. First use pMD19T-T7-gRNA with EcoR

 V linearization, pST1374-NLS-flag-linker-Cas9 was linearized with Age I; then TMD in vitro transcription kit (Biomax) was used to perform pMD19T-T7-gRNA, pST1374-NLS-flag-linker-Cas9 Transcription; Finally, MEGA Clear kit (Ambion) was used to purify gRNA and Cas9 mRNA.

 Example 3: Application of CRISPR / Cas9 system targeting mouse EBLN1 gene to generate transgenic mice containing EBLN1 gene mutation

 [0031] The embryos at the single-cell stage were collected in a naturally mated donor female mouse by surgery, and the premixed gRNA and Cas9 mRNA (the final concentration after mixing was Cas9 mRNA 30 using a microinjector).

ng / ^ il, gRNA 12 ng / ^ il) is injected into the cytoplasm of fertilized eggs. The injected fertilized eggs were transferred to Quinn's Advantage Cleavage Medium, cultured in vitro at 37 ° C for 24 hours, and then transplanted into recipient female mice to generate transgenic mice containing EBLN1 gene mutations. Industrial applicability

 (1) gRNA and Cas9

 The mRNA is transfected into the cell at the same time, and the EBLN1 gene is knocked out for studying the function of the EBLN1 gene;

 (2) gRNA and Cas9 mRNA are injected into a fertilized egg at the same time, and then used to produce transgenic mice that knock out EBLN 1 by embryo transfer;

 (3) gRNA and Cas9 mRNA are transfected into cells at the same time, and positive cells are used as donor cells after screening

Transgenic mice containing EBLN1 gene mutations were generated by nuclear transfer.

Claims

Claim  [Claim 1] A method for knocking out mouse EBLN1 gene by using CRISPR / Cas9 system, characterized in that: it comprises a mouse EBLN1 gene specific target site sequence, and its base sequence is shown in Figure 1; A gRNA oligonucleotide sequence that specifically targets the EBLN1 gene is shown in FIG. 2.  [Claim 2] A method for knocking out a mouse EBLN1 gene using the CRISPR / Cas9 system according to claim 1, comprising: an in vitro transcription vector comprising the gRNA. [Claim 3] The method for knocking out mouse EBLN1 gene using the CRISPR / Cas9 system according to claim 1, characterized in that: the in vitro transcription vector of the gRNA is pMD19 T-T7-gRNA, Cas9 The in vitro transcription vector of the protein is pST1374-NLS-flag-linker-Ca s9.  [Claim 4] The method for knocking out the mouse EBLN1 gene by using the CRISPR / Cas9 system according to claim 1, comprising the following steps:  51. Construct an in vitro transcription vector that specifically targets gRNA of the EBLN1 gene; obtain gRNA that targets EBLN1 by in vitro transcription;  52. An in vitro transcription vector for Cas9 protein in vitro, to obtain Cas9 mRNA;  53. Inject the gRNA and Cas9 mRNA from the above steps into the cytoplasm of fertilized oocytes of mice, culture them in vitro and transplant them into the fallopian tubes of adult female mice to generate transgenic mice containing EBLN1 gene mutations.  [Claim 5] The method for knocking out mouse EBLN1 gene by using the CRISPR / Cas9 system according to claim 4, characterized in that: after the gRNA and Cas9 mRNA are mixed in step S3, the final concentration is: Cas9 mRNA 30 ng / [il, gRNA 12 ng / [il.