阅读说明：本技术 Gpr21基因敲除小鼠模型及其建立方法 (GPR21 gene knockout mouse model and establishment method thereof ) 是由 谢欣 李静 赵婷婷 李艳利 于 2021-05-10 设计创作，主要内容包括：本发明提供了一种GPR21基因敲除小鼠模型及其建立方法,具体涉及用于GPR21基因敲除的sgRNA、载体及其构建方法和检测方法,以及F0、F1代小鼠基因型检测方法。实施方法为利用CRISPRCas9基因敲除技术,靶向敲除GPR21基因第2个外显子。有研究表明GPR21是2型糖尿病的新靶标,GPR21敲除小鼠显示出更好的葡萄糖耐量和胰岛素反应。本发明GPR21基因敲除小鼠模型为进一步研究糖尿病的发病机制以及基因治疗提供经济、简单、可靠的动物模型,具有良好的推广应用价值。(The invention provides a GPR21 gene knockout mouse model and an establishment method thereof, and particularly relates to sgRNA for GPR21 gene knockout, a vector, a construction method and a detection method thereof, and F0 and F1 mouse genotype detection methods. The implementation method is to target knock out the 2 nd exon of GPR21 gene by using CRISPRCs 9 gene knock-out technology. There are studies showing that GPR21 is a new target for type 2 diabetes, and GPR21 knockout mice show better glucose tolerance and insulin response. The GPR21 gene knockout mouse model provides an economic, simple and reliable animal model for further researching the pathogenesis and gene therapy of diabetes, and has good popularization and application values.)

1. A GPR21 gene knockout mouse model is characterized in that the model is an F1 generation mouse model with GPR21 gene knockout, which is obtained by knocking out a No. 2 exon in a mouse GPR21 gene.

2. The method for constructing the model mouse according to claim 1, which comprises the following steps:

a. designing a gene knockout strategy aiming at a mouse GPR21 gene, wherein a knockout region is selected as a No. 2 exon;

b. obtaining an F0 generation mouse by using a CRISPR-Cas9 gene editing and oocyte injection method;

c. after breeding positive F0 generation mice and C57BL/6J, a positive F1 generation mouse model with a GPR21 gene knockout is obtained.

3. The method according to claim 2, characterized in that the CRISPR-Cas9 gene editing method of step b comprises the following specific steps:

a. design of sgRNA: the sgRNA is 80 nucleotides in length and comprises two regions: the first 20 nucleotides of the 5' end of the sgRNA correspond to the target DNA, and the remaining nucleotides form a hairpin structure, and the sequence condition of the sgRNA combined with the target DNA is as follows: 20 bp; complete complementation with target DNA; ③ the corresponding end of the target DNA and the inserted base sequence is any nucleotide sequence (N) + two cytosine nucleotides (-NCC) at the 5' end;

construction of sg RNA-PX330-mCherry plasmid: after digestion of the vector with Bbs1, the sgRNA annealing product was ligated and transformed. Selecting clone, carrying out PCR verification of bacterial liquid, and extracting plasmid for later use.

c. Construction of a validated target-GFP reporter plasmid: the cleavage site (80bp) including sgRNA was added with BamHI and HindIII ends before and after, Target: ATGAACTCCA CCTGGGATGG TAATCAGAGC AGCCATCCTT TCTGTCTTCT GGCACTGGGC TACTTGGAAA CTGTCAGGTT;

cutting the vector by BamHI and HindIII, then carrying out enzyme digestion on the GFP reporter vector, connecting the vector with a target annealing product, and transforming; selecting clone, carrying out bacteria liquid PCR verification to obtain correct bacteria liquid, and extracting plasmid for later use;

sgrnas effect validation: the sg RNA/PX330-mCherry plasmid and the target-GFP reporter plasmid are co-transferred into HEK293T cells, if the target sequence in the target-GFP reporter plasmid transferred into the cells has double-strand breaks, the cells express GFP, pictures are taken at 48h after the transfection, and the sgRNA cutting efficiency is seen.

4. The method of claim 3, wherein the expression of the mouse GPR21 gene, GenBank:

and (b) an NM-005294.3 sequence, wherein the sgRNA target is designed and constructed based on a CRISPR-Cas9 system, and the sequence of the sgRNA target designed in the step a is as follows:

sg1 GCTCTGATTACCATCCCAGG sg2 GGGATGGTAATCAGAGCAGC

sg3 GTGCCAGAAGACAGAAAGGA。

5. the method according to claim 2, wherein the oocyte injection method of the step b comprises the following specific steps: and uniformly mixing the sgRNA target and Cas9 protein, injecting the sgRNA target and the Cas9 protein into cytoplasm of a single-cell fertilized egg of a C57BL/6 mouse, transplanting the fertilized egg surviving after injection into a pseudopregnant mother mouse, and generating a mouse by a surrogate pregnant mouse, namely an F0-generation mouse.

6. Use of a GPR21 gene knockout mouse model according to claim 1 for the study of the onset of diabetes.

Technical Field

The invention relates to a GPR21 gene knockout mouse model and an establishment method thereof.

Technical Field

Diabetes is a metabolic disorder syndrome mainly characterized by long-term hyperglycemia, is caused by absolute deficiency or relative deficiency of insulin caused by genetic factors, environmental factors, immune dysfunction or microbial infection and the like, and mainly comprises type 1 diabetes and type 2 diabetes.

The G protein-coupled receptor (GPCR) superfamily includes nearly thousand seven transmembrane receptors that participate in a variety of physiological and pathological functions. Approximately 36% of the commercially available drugs target human GPCRs. Over 30 GPCRs have been implicated in the development of beta cell dysfunction, insulin resistance, obesity and type 2 diabetes drugs. Among them, the GPR21 receptor is likely one of the relevant receptors for insulin resistance caused by obesity to trigger diabetes.

The orphan receptor GPR21 was originally isolated from the brain and GPR21 was located in the region of human chromosome 9, q33 and mouse chromosome 2 by Northern blot analysis of human RNA with the highest identity (71%) to GPR 52.

The physiological role of GPR21 remains unknown. There are studies that suggest GPR21 is a novel target for type 2 diabetes. Insulin resistance caused by obesity is a major factor in the etiology of type 2 diabetes, and the prevalence of these diseases is on the rise worldwide. In recent years, chronic low grade inflammation has become an important factor in the development of insulin resistance. The inflammatory response caused by obesity promotes infiltration of macrophages and other immune cells into adipose tissue and the liver. Thus, obesity and inflammation are both key causes of insulin resistance states, including type 2 diabetes. The orphan receptor GPR21 is highly expressed in the hypothalamus and macrophages of mice, and systemic knock-out of this receptor results in improved glucose tolerance, insulinemia and systemic insulin sensitivity. The data suggest that GPR21 may be a novel control point for coordinating macrophage pro-inflammatory activity in the context of obesity-induced insulin resistance, a potential role that GPR21 may play in the development of type 2 diabetes. Pharmacological GPR21 inhibition may prevent the negative effects of a high-sugar, high-fat diet by increasing energy expenditure and improving insulin sensitivity, thereby reducing insulin resistance and the development of type 2 diabetes.

Clustered regularly interspaced short palindromic repeats/Clustered regularly interspaced short palindromic repeats (CRISPR/Cas) technology is a new tool for editing fixed-point genomes, and has the advantages of flexibility, high efficiency, low cost, easiness in operation and the like. By using a CRISPR/Cas9 technology, specific DNA recognition can be realized by designing specific sgRNA, cutting is completed at a target position, and then the repair of a fracture is completed through a DNA repair mechanism of a cell, so that the target gene is edited. According to the experimental requirements, one or more specific genes of the mouse are knocked out, and the cultured mouse becomes an animal model suitable for drug experiments.

Disclosure of Invention

One of the purposes of the invention is to provide a GPR21 gene knockout mouse model.

The second purpose of the invention is to provide a method for constructing the GPR21 gene knockout mouse model.

In order to achieve the purpose, the invention adopts the following technical scheme:

a GPR21 gene knockout mouse model is characterized in that the model is an F1 generation mouse model with GPR21 gene knockout, which is obtained by knocking out a No. 2 exon in a mouse GPR21 gene.

The method for constructing the mouse model is characterized by comprising the following specific steps:

a. designing a gene knockout strategy for a mouse GPR21 gene, and selecting a knockout region as a No. 2 exon;

b. obtaining an F0 generation mouse by using a CRISPR-Cas9 gene editing and oocyte injection method;

c. after breeding positive F0 generation mice and C57BL/6J, a positive F1 generation mouse model with a GPR21 gene knockout is obtained.

The CRISPR-Cas9 gene editing method in the step b comprises the following specific steps:

a. design of sgRNA: the sgRNA is 80 nucleotides in length and comprises two regions: the first 20 nucleotides of the 5' end of the sgRNA correspond to the target DNA, and the remaining nucleotides form a hairpin structure, and the sequence condition of the sgRNA combined with the target DNA is as follows:

①20bp；

complete complementation with target DNA;

③ the corresponding end of the target DNA and the inserted base sequence is any nucleotide sequence (N) + two cytosine nucleotides (-NCC) at the 5' end;

construction of sg RNA-PX330-mCherry plasmid: after the vector is cut by Bbs1 enzyme, the vector is connected with the sg RNA annealing product and transformed; selecting clone, carrying out PCR verification of bacterial liquid, and extracting plasmid for later use.

c. Construction of a validated target-GFP reporter plasmid: the cleavage site (80bp) including sgRNA was added with BamHI and HindIII ends before and after, Target: ATGAACTCCA CCTGGGATGG TAATCAGAGC AGCCATCCTT TCTGTCTTCT GGCACTGGGC TACTTGGAAA CTGTCAGGTT;

cutting the vector by BamHI and HindIII, then carrying out enzyme digestion on the GFP reporter vector, connecting the vector with a target annealing product, and transforming; selecting clone, carrying out bacteria liquid PCR verification to obtain correct bacteria liquid, and extracting plasmid for later use;

sgrnas effect validation: the sg RNA/PX330-mCherry plasmid and the target-GFP reporter plasmid are co-transferred into HEK293T cells, if the target sequence in the target-GFP reporter plasmid transferred into the cells has double-strand breaks, the cells express GFP, pictures are taken at 48h after the transfection, and the sgRNA cutting efficiency is seen.

According to the mouse GPR21 gene, GenBank: and (b) an NM-005294.3 sequence, wherein the sgRNA target is designed and constructed based on a CRISPR-Cas9 system, and the sequence of the sgRNA target designed in the step a is as follows:

sg1 GCTCTGATTACCATCCCAGG sg2 GGGATGGTAATCAGAGCAGC

sg3 GTGCCAGAAGACAGAAAGGA。

the oocyte injection method of the step b comprises the following specific steps: and (3) uniformly mixing the sgRNA target point with the C as9 protein, injecting the mixture into the cytoplasm of a C57BL/6 mouse single-cell fertilized egg, transplanting the fertilized egg surviving after injection into a pseudopregnant mother mouse, and generating a mouse, namely an F0-generation mouse, from a surrogate pregnant mouse.

The GPR21 gene knockout mouse model is applied to the research of the onset of diabetes.

There are studies showing that GPR21 is a new target for type 2 diabetes, and GPR21 knockout mice show better glucose tolerance and insulin response. The GPR21 gene knockout mouse model provides an economic, simple and reliable animal model for further researching the pathogenesis and gene therapy of diabetes, and has good popularization and application values.

Drawings

FIG. 1 is a schematic diagram of a GPR21 knock-out mouse model constructed in the present invention;

fig. 2 is a graph showing the validation of the knockdown efficiency of sgrnas designed in the present invention;

FIG. 3 is a genotype identification chart of F0 mouse in the invention.

Detailed Description

The first embodiment is as follows:

1) designing a gene knockout strategy for a mouse GPR21 gene, and selecting a knockout region as a No. 2 exon;

2) design of sgRNA of GPR21 Gene

Designing sgRNA based on CRISPR-Cas9 system according to mouse GPR21 gene (GenBank: NM-005294.3) sequence; the sgRNA sequence of GPR21 is shown in table 1.

Table 1: sgRNA sequence of mGPR21

Target numbering	Target sequence
		sg1	GCTCTGATTACCATCCCAGG
sg2	GGGATGGTAATCAGAGCAGC
		sg3	GTGCCAGAAGACAGAAAGGA

。

Construction of a validated target-GFP reporter plasmid: the cleavage site (80bp) including sgRNA was added with BamHI and HindIII ends before and after, Target: ATGAACTCCA CCTGGGATGG TAATCAGAGC AGCCATCCTT TCTGTCTTCT GGCACTGGGC TACTTGGAAA CTGTCAGGTT;

cutting the vector by BamHI and HindIII, then carrying out enzyme digestion on the GFP reporter vector, connecting the vector with a target annealing product, and transforming; selecting clone, carrying out bacteria liquid PCR verification to obtain correct bacteria liquid, and extracting plasmid for later use;

2) verification of sgRNA cleavage efficiency

3 sgrnas were constructed into PX330-mCherry vector, which contains s.pyogenes Cas9 sequence and expresses mCherry red fluorescent protein. The Target-GFP-reporter vector and the sgRNA-PX330-mCherry vector are co-transferred into HEK293T cells, and after the inserted fragment is cut by the sgRNA, the wrong GFP sequence can be recombined into GFP capable of being normally expressed by the homologous recombination principle, and green fluorescence is emitted (figure 2). The third sgRNA was relatively efficient in vitro cleavage and was subsequently selected for microinjection experiments.

3) Oocyte microinjection

And (3) uniformly mixing the sgRNA and the Cas9 protein, injecting the mixture into the cytoplasm of a C57BL/6 mouse single-cell fertilized egg by using a microinjection instrument, transplanting the fertilized egg surviving after injection into a pseudopregnant mother mouse, and generating a mouse, namely an F0 mouse, from the pseudopregnant mouse.

4) Genotype identification of F0 mouse

Microinjection experiments were carried out on 21F 0 mice, and genomic DNA from the toes of the mice was extracted by phenol-chloroform extraction and diluted to 20 ng/. mu.L for use. Selecting a sequence of about 500bp before and after the target gene for PCR, connecting a PCR product into a PMD-19T vector, transforming and coating a plate, and selecting 6 clones from each sample for sequencing identification (Table 2). Sequencing results showed that one chromosome of #17 mouse knocked out 10 bases near the sgRNA target and made a frameshift mutation in GPR21 gene (fig. 3).

Table 2: genotype identification of F0 mouse

Numbering	Genotype(s)	Numbering	Genotype(s)
				1♂	KO 4bp(2/4)	12♀	WT
2♂	WT	13♀	WT
				3♂	KO 2bp(2/5)	14♀	KO 3bp(2/4)
4♂	WT	15♀	WT
				5♂	WT	16♀	KO 12bp(1/4)
6♂	WT	17♂	KO 10bp(6/6)
				7♂	WT	18♀	WT
8♂	KO 12bp(4/5)	19♀	WT
				9♂	WT	20♀	KO 7bp(2/5)
10♀	WT	21♀	WT
				11♀	KO 4bp(3/5)

5) Propagation and genotype identification of F1-generation mice

After 7 weeks of age of the #17 male mice of the F0 generation were mated with wild-type heteromouse F1 generation, mice were genotyped 20 days after birth (table 3), and if mice born with 10 bases knocked out near the sgRNA target were born, it was indicated that the transgene had integrated into the germ cell.

Table 3: genotype identification of F1 mouse

<110> Shanghai university, Shanghai institute for pharmaceutical science of Chinese academy of sciences

<120> GPR21 gene knockout mouse model and establishment method thereof

<160> 3

<210> 1

<211> 20

<212> DNA

<213> Gene sequence

<400> 1

GCTCTGATTACCATCCCAGG 20

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<211> 20

<212> DNA

<213> Gene sequence

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GGGATGGTAATCAGAGCAGC 20

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<211> 20

<212> DNA

<213> Gene sequence

<400> 1

GTGCCAGAAGACAGAAAGGA 20