CN112662669A - Il21 gene knockout mouse model and construction method and application thereof - Google Patents

Abstract

The invention discloses an Il21 gene knockout mouse model and a construction method and application thereof, wherein the construction method comprises the following steps: s1, determining a knockout region of an Il21 gene based on a CRISPR/Cas9 technology, designing sgRNA1 and sgRNA2 of specific target sites according to the determined knockout region, wherein gene sequences of gRNA1 and gRNA2 are respectively shown as SEQ ID No.1 and SEQ ID No. 2; s2, microinjecting active gRNA1, gRNA2 and cas9 protein into mouse fertilized eggs, transplanting the fertilized eggs surviving after injection into a pseudopregnant female mouse, and obtaining an F0-generation mouse after the pregnant female mouse farves; s3, mating the F0 generation mouse obtained in the step S2 with a wild type mouse to obtain an F1 generation heterozygote; s4, inbreeding the F1 generation heterozygote obtained in the step S3 to obtain an F2 generation mouse which is an IL21 gene knockout mouse model. Based on CRISPR/Cas9 technology, the invention establishes a mouse animal model by designing reasonable heterologous target site gRNA1 and gRNA2 gene sequences and knocking out the exon3-exon5 region in ENSMUST00000029273.7 transcript of Il21 gene for the first time, and can be applied to related research after tumor healing.

Description

Il21 gene knockout mouse model and construction method and application thereof

Technical Field

The invention relates to the technical field of construction of gene knockout mouse models, in particular to an Il21 gene knockout mouse model and a construction method and application thereof.

Background

IL-21, interleukin-21 (IL-21), is a secreted protein secreted by activated CD4+ T cells and has a molecular structure similar to IL-15, belonging to the IL-15/IL-21 family, which binds to a complex receptor consisting of a proprietary receptor (IL21R) and the usual cytokine receptor chain (gamma (C)). IL-21 affects a variety of cell types including CD8+ memory T cells, NK cells, and CD4 memory T cell subsets. Its receptors are widely distributed on lymphohematopoietic cells. IL-21 promotes the anti-tumor activity of CD8+ t cells and NK cells by enhancing the antigen-specific response of immune cells, and acts by binding to the specific type I cytokine receptor IL-21R. It has been shown that IL-21 depletion results in CD8+ T cell dysfunction.

The existing research on the effect of IL-21 in the tumor healing process mainly uses a method for injecting exogenous IL-21 into tumor tissues, the method is limited by factors such as the type of a carrier of an injection, the source of the exogenous IL-21, related side effects and the like, and the research on the IL-21 in a knockout mouse can effectively avoid related adverse factors and reduce the cost.

The gene knockout animal model can be constructed by a gene targeting technology and a CRISPR/Cas9 technology. The gene targeting technology is a molecular biology technology established on the basis of ES cell and DNA homologous recombination and other technologies. The gene of a specific site of the ES cell is modified by utilizing a homologous recombination technology, and the ES cell after genetic modification is introduced into a recipient embryo by a microinjection or embryo fusion method. The genetically modified ES cells still maintain totipotency of differentiation and can develop into germ cells of chimeric animals, such that the modified genetic information is inherited by the germ line to obtain mice derived from ES cells. Therefore, gene editing can be performed on ES cells to obtain genetically modified mice.

The ES cell targeting technology obtains chimeric mice from the construction of homologous recombination vectors, targeting and positive clone screening of ES cells, and blastocyst injection of ES cells, which takes about 4-6 months. The CRISPR/Cas9 technology abandons the work, and the fertilized eggs of the mice after direct transplantation and injection are obtained for F0 generation, which only needs about 1 month, thus greatly saving the development period and the cost. On the other hand, genetically modified mice obtained by ES cell targeting technology have genetic background from the ES cells we have selected, whereas mature, gene-targeted ES cell lines generally have only a limited number of strain sources: the hybrid F1 lines of C57BL/6N, C57BL/6J, 129S3 and C57 and 129. If other backgrounds are needed, the strain needs to be obtained by backcrossing with wild-type mice of the target strain, and the number of backcrossing is more than 10 generations. The CRISPR/Cas9 technology breaks the restriction of mouse strains, and can realize genetic modification on different strains and even immunodeficient strains, such as: BALB/c, FVB, Nod-scid, etc.

At present, no case of successfully constructing an IL-21 gene knockout mouse model by adopting a CRISPR/Cas9 technology exists.

Disclosure of Invention

The invention aims to provide a construction method of an Il21 gene knockout mouse model, and the construction method not only can successfully construct the Il21 gene knockout mouse model, but also has the advantages of short period and no limitation of mouse strains. .

In addition, the invention also provides an Il21 gene knockout mouse model constructed by the construction method and application of the Il21 gene knockout mouse model.

The invention is realized by the following technical scheme:

a method for constructing an Il21 gene knockout mouse model comprises the following steps:

s1, determining a knockout region of an Il21 gene based on a CRISPR/Cas9 technology, designing sgRNA1 and sgRNA2 of specific target sites according to the determined knockout region, wherein gene sequences of gRNA1 and gRNA2 are respectively shown as SEQ ID No.1 and SEQ ID No. 2;

s2, microinjecting active gRNA1, gRNA2 and cas9 protein into mouse fertilized eggs, transplanting the fertilized eggs surviving after injection into a pseudopregnant female mouse, and obtaining an F0-generation mouse after the pregnant female mouse farves;

s3, mating the F0 generation mouse obtained in the step S2 with a wild type mouse to obtain an F1 generation heterozygote;

s4, inbreeding the F1 generation heterozygote obtained in the step S3 to obtain an F2 generation mouse which is an IL21 gene knockout mouse model.

The difficulty in constructing an Il21 gene knockout mouse model is the design of heterologous target site gRNA1 and gRNA2 and the gene determination of F0 generation mice.

Based on CRISPR/Cas9 technology, an Il21 gene knockout mouse model is successfully constructed by designing reasonable heterologous target site gRNA1 and gRNA2 gene sequences, and the method can be applied to related research after tumor healing; the construction method has the advantages of short period and no limitation of mouse strains.

Furthermore, the genotype of the F0 generation mouse is determined, and the heterozygote mouse is selected to be mated with the wild type mouse, so that the F1 generation heterozygote is obtained.

Further, the genotype determination process of F0 generation mice was as follows:

extracting genome DNA of F0 mouse, PCR amplifying and sequencing identifying to confirm genotype; the primers for PCR amplification comprise F1, R1, F2 and R2, wherein the gene sequences of F1, R1, F2 and R2 are respectively shown as SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO. 6; the gene sequence of the sequencing primer is shown as SEQ ID NO. 7.

Another difficulty or key point in constructing the Il21 knockout mouse model is the gene determination of the F0 generation mouse and F1 generation heterozygote.

According to the invention, by reasonably designing a PCR amplification sequence and a sequencing sequence, genes of F0 generation mice and F1 generation heterozygotes can be successfully determined, and the success rate of constructing an Il21 gene knockout mouse model is further improved.

Further, the obtained F1 generation heterozygotes comprise 5 types, namely a knockout type 1, a knockout type 2, a knockout type 3, a knockout type 4 and a knockout type 5, wherein 6281 base pairs are deleted for the knockout type 1, 6282 base pairs are deleted for the knockout type 2, 6272 base pairs are deleted for the knockout type 3, 6283 base pairs are deleted for the knockout type 4, 6279 base pairs are deleted for the knockout type 5, and F1 generation heterozygotes of the knockout type 1 are inbred to obtain F2 generation mice.

Further, the type determination process of heterozygote of the F1 generation is as follows:

extracting genome DNA of F1 mouse, PCR amplifying and sequencing identifying to confirm genotype; the primers for PCR amplification comprise F1, R1, F2 and R2, the gene sequences of F1, R1, F2 and R2 are respectively shown as SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6, and the gene sequence of the sequencing primer is shown as SEQ ID NO. 7.

Further, the knockout region determined in step S1 is exon3-exon5 of Il21-201(ENSMUST00000029273.7) transcript.

The Il21 gene knockout mouse model constructed by the construction method is adopted.

A cell with IL21 gene knocked out.

An Il21 gene knockout mouse model comprising a cell in which the Il21 gene is knocked out.

Application of an Il21 gene knockout mouse model in related researches after tumor healing.

Compared with the prior art, the invention has the following advantages and beneficial effects:

1. based on CRISPR/Cas9 technology, the invention establishes a mouse animal model by designing reasonable heterologous target site gRNA1 and gRNA2 gene sequences and knocking out the exon3-exon5 region in ENSMUST00000029273.7 transcript of Il21 gene for the first time, and can be applied to related research after tumor healing.

2. According to the invention, by reasonably designing a PCR amplification sequence and a sequencing sequence, genes of F0 generation mice and F1 generation heterozygotes can be successfully determined, and the success rate of constructing an Il21 gene knockout mouse model is further improved.

3. The construction method has the advantages of short period and no limitation of mouse strains.

Drawings

The accompanying drawings, which are included to provide a further understanding of the embodiments of the invention and are incorporated in and constitute a part of this application, illustrate embodiment(s) of the invention and together with the description serve to explain the principles of the invention. In the drawings:

FIG. 1 is a schematic diagram of the strategy for constructing specific target sites in example 1;

FIG. 2 is a graph showing the results of the identification of F0 mouse in example 1;

FIG. 3 is a graph showing the result of the identification of F1 mouse in example 1;

FIG. 4 is a graph of the sequencing peak of # 8 in the F1 mouse assay;

FIG. 5 is a graph showing the result of identification of F2 mouse;

FIG. 6 is a schematic diagram of an authentication strategy.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to examples and accompanying drawings, and the exemplary embodiments and descriptions thereof are only used for explaining the present invention and are not meant to limit the present invention.

Example 1:

a method for constructing an Il21 gene knockout mouse model comprises the following steps:

s1, RISPR/Cas9 technology, using exon3-exon5 of an Il21-201(ENSMUST00000029273.7) transcript as a knockout region according to Il21 gene information, wherein the knockout region comprises a 258bp coding sequence; determining specific target sites gRNA1 and gRNA2 to be knocked out of the IL10 gene of a C57BL/6J mouse according to the region, wherein the gene sequences of the gRNA1 and the gRNA2 are shown as SEQ ID No.1 and SEQ ID No.2 respectively or shown as Table 1;

TABLE 1 gRNA sequence information

sgRNA name	gRNA sequence(5’*3’)	PAM
			sgRNA1	TGCCTATGTATGTGTCGTCC	TGG
sgRNA2	TGGAGTGCCCACCTATCTGA	TGG

S2, microinjecting active gRNA1, gRNA2 and cas9 proteins into mouse fertilized eggs, transplanting the fertilized eggs surviving after injection into a pseudopregnant female mouse, obtaining an F0-generation mouse after the pregnant female mouse is pregnant, extracting genomic DNA of the F0-generation mouse, and sequentially carrying out PCR amplification and sequencing identification to confirm the genotype; the primers for PCR amplification comprise F1, R1, F2 and R2, wherein the gene sequences of F1, R1, F2 and R2 are respectively shown as SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO. 6; the gene sequence of the sequencing primer is shown as SEQ ID NO.7 or shown as Table 2, and the F0 mouse identification result is shown as figure 2:

TABLE 2 primer information

The PCR amplification reaction system is shown in Table 3:

TABLE 3 reaction System

Seg.	reaction component	Volume(μl)
			1	2×Taq Master Mix，Dye Plus，(Vazyme P112-03)	12.5
2	ddH2O	9.5
			3	Primer A(10pmol/μl)	1
4	Primer B(10pmol/μl)	1
			5	Template(≈100ng/μl)	1

The PCR amplification reaction procedure is shown in table 4:

TABLE 4 reaction procedure

S3, mating the sexually mature positive F019 # mouse obtained in the step S2 with a wild type mouse to breed a first generation, wherein the gene sequence of the wild type C57BL/6J mouse is shown as SEQ ID No.8, the gene sequence of gRNAs is shown as SEQ ID No.9, the gene sequence of a region where a knockout coding region is located is shown as SEQ ID No.10, and the born F1 mouse cuts the tail and numbers of the toes in 5-7 days, extracts genome DNA for PCR identification and confirms the genotype; there were 5 types of heterozygote mice obtained at the F1 generation, which were: knock-out type 1, deletion of 6281 base pairs; knock-out type 2, deletion of 6282 base pairs; knock-out type 3, deletion 6272 base pairs; knock-out type 4, deletion of 6283 base pairs; knock-out type 5, deletion of 6279 base pairs; the identification method is the same as that of F0 mouse, and the genotype identification result of F1 is shown in figure 3;

according to the PCR strategy diagram, as shown in FIG. 1, through PCR and sequencing confirmation,

1#, 8 #: 6281bp, E3- -E5 complete deletion, KO positive

2 #: 6282bp, E3- -E5 complete deletion, KO positive

5 #: 6272bp, E3- -E5 complete deletion, KO positive

9 #: 6283bp, E3- -E5 complete deletion, KO positive

10 #: 6279bp, E3- -E5 complete deletion, KO positive;

according to the PCR strategy diagram, through PCR and sequencing confirmation,

the sequencing peak pattern of # 8 is shown in figure 4: sequencing double peaks, and recommending breeding;

s4, inbreeding the F1 generation heterozygous mouse which is obtained in the step S3 and lacks 6281 base pairs to obtain an F2 generation homozygous mouse, namely the model of the Il21 gene knockout mouse constructed in the embodiment.

The results of the identification are shown in FIG. 5: 33# and 39# are homozygous (ko/ko) mice.

FIG. 6 is a schematic diagram of an authentication strategy:

wild type: PCR reaction objects a single WT band; secondly, PCR reaction objects a single WT band (wild type: firstly, a WT band is obtained by PCR reaction and secondly, a WT band is obtained by PCR reaction).

Heterozygate: PCR reaction objects a WT band and a KO band; ② PCR reaction objects a WT band (heterozygote: firstly, PCR reaction obtains a WT and a KO band; and secondly, PCR reaction obtains a WT band).

Homozygote: (i) PCR reaction objects a single KO band; (S) PCR reaction with product (homozygote: a KO band is obtained by PCR reaction; and a band is absent by PCR reaction).

The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are merely exemplary embodiments of the present invention, and are not intended to limit the scope of the present invention, and any modifications, equivalent substitutions, improvements and the like made within the spirit and principle of the present invention should be included in the scope of the present invention.

Sequence listing

<110> Chengdu Yaokang Biotechnology Ltd

<120> Il21 gene knockout mouse model, and construction method and application thereof

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acatacacac attcacattc aactccacaa ggcttgtacc cactgcttca acctcagagt 300

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ctgtcttttg ctgagtttaa gtggggatat gaccagacct aatggagccc ctaaaaaaat 480

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caatgttcat taacatatgc taattatctt taaggtcatt aacatatgct aattgtctct 600

cctttcaggg gcactgtgag catgcagctt ttgcctgttt tcagaaggcc aaactcaagc 660

catcaaaccc tggaaacaat aagacattca tcattgacct cgtggcccag ctcaggagga 720

ggctgcctgc caggagggga ggaaagaaac agaagcacat agctgtaaga ttgtcctctg 780

tcatctttct tgttcgcagt tatcactagc atggagcatt tagttattaa gataagtctt 840

acccgttagt tgtaacatac tatgcttcac acatccacac agctggagcg tctcccacct 900

tcttgcagag ttactccata caaggtctac tttgaaaagt ggtgtcacct ccaataagtc 960

tttactgtct gtctagaaac cctactagaa ctggccacca ctccctcggc agtatgttaa 1020

cagaagtttc tgggggtgaa gaagccatta ggaagagaat gtgttagacg ttacaagata 1080

ttttcccttt tgcccatgtg ctggccatag cttgtgtcat ctttatcggg gactagagct 1140

cagctttctg acaagaaaag agagcttaca ataccaagat caatctatta ctgtcccagg 1200

caaaagcaga gtcaatgatg aatttaaaag ggtgtacctt tttatggggc tgaaaaagtc 1260

caagttcctg tttctttggg accctgaatt ttcttgtata ttttctttgg ccaacttatg 1320

tgcaattcag ggactcaaga atcaactcat tcaaacatta atcagaacct gcagagagga 1380

cagaaaagaa gtttctctgt catattcatt ggttgtctgt ccttcaagaa atgccacatg 1440

gacaaaaatg aaatcataat tacattatga aaaggagtct tgtttcttct gattgcttgg 1500

ccaggttatt atgtgaaaat gtccctctga aactttcttc acaatgaaaa gaactgccta 1560

ggaaggtagt gtgtccctgt gaggtcagca ccaggaagct gaggcaggag gattgagact 1620

tccaggacag ctgggttatc tgggtgagac cttgttcagg aagctttgaa gaaggaagta 1680

ggggaggagg cagggaggga gagaaggaaa ggaaggaaag gaaggaaagg aaggaaagga 1740

aggaaaggaa ggaaaggaag gaaggaagga aggaaggaag gaacaaagga aggaaggaga 1800

aatggagggg gtggtggtga caaattgacc acagctatga aactcagaaa aacttacgaa 1860

atggtgctgt gtactgtaga cctggaaccc aactcttcta caccaagctc ctttgtgtag 1920

acatgaagaa agtgagaact gtaaagaggc caactttctc caggtttcac aaacagctgt 1980

gtcaggatga agaagaacat agatgccatg ctgcctccag atgttctcac cgtgatacca 2040

tgtgactgag cattaaaaag cagccagttg ttttggtatt ggaacaggaa tgagcataag 2100

actggaggag aaagggggga gggagactga tgaagaggga ggaaggtcag ccacattcga 2160

acatttggaa catttgctca gtacccctaa taatcaagtg aatggaagtt aaaagaggaa 2220

atcctgtctt gtaattgtgg aaacagtgcc caatagtggt aagactcgtc gaagcacaaa 2280

gaggaaacat gttttatgat atatagtcct tgaggtattt atccctttaa tctacttgat 2340

aagatctaga aatttgatat atatgaaaaa attatctgcc caggtgatct ctatagactt 2400

gtacataaaa gctagatacc acttctgtat ctaaaaatag taagtgctcc atctgtgcag 2460

ggtgggggtg ggtgggaggg ggagcgacag agcagccacc acaaatcctg ccctgaaagc 2520

atatctaaag cccaggaaac ccttgtaatt ggttaaaaac taacagcaaa cagcatataa 2580

caaccagggt aacaagaatg cagaggagga ggagggcgag gaagaggaag aggaagaaag 2640

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ctagaaagac taaaatggct ccttcaaaag gtatgcacct taaatgcatt tctttcactt 3000

ccatgttgtg tccgggtacc tcctgtgccc agtgactcat aggctctcgt tcccacagat 3060

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ttgcctcttg ggattgcaag tggagaagta cgatacgtta tgataagaac aactcagaaa 3240

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<213> 10 (Artificial sequence 10)

<400> 10

gggcactgtg agcatgcagc ttttgcctgt tttcagaagg ccaaactcaa gccatcaaac 60

cctggaaaca ataagacatt catcattgac ctcgtggccc agctcaggag gaggctgcct 120

gccaggaggg gaggaaagaa acagaagcac atagctatga ttcatcagca tctctcctag 180

aacacatagg acccgaagat tcctgaggat ccgagaagat tcccgaggac tgaggagacg 240

ccggacacta tagacgctca cgaatgcagg agtacatctt gcctcttggg attgcaagtg 300

gagaagtacg atacgttatg ataagaacaa ctcagaaaag ctataggtta agatcctttc 360

gcccattaac taagcagaca ttgtggttcc ctgcacagac tccatgctgt caacatggaa 420

aatctcaact caacaagagc ccagcttccc gtgtcaggga tttctggtgc ttctcaagct 480

gtggcttcat cttattgccc aactgtgaca ttctttgatt ggaaggggaa aactaaagct 540

tttagcaaaa atacagctag ggaatttgtc gatctgcgag agtaagacct cttatgatcc 600

taacggaatg atgtaagctg gaaataataa gcataagatg aaattgaaaa ttgaagtctt 660

tattctttaa gaaaaacttt gtacttgaaa gcatgtctga agagtttact cattaccaca 720

aacatctagc atattgataa ctaacatctt tatactctac aagagaggct ttccagatag 780

gtacagtttt tcttctctat taggtctatc aaaatttaac ctattatgag ggtcacccct 840

ggctttcact gtttttctaa agaggcaagg gtgtagtaag aagcaggctt aagttgcctt 900

cctcccaatg tcaagttcct ttataagcta atagtttaat cttgtgaaga tggcaatgaa 960

agcctgtgga agtgcaaacc tcactatctt ctggagccaa gtagaatttt caagtttgta 1020

gctctcacct caagtggtta tgggtgtcct gtgatgaatc tgctagctcc agcctcagtc 1080

tcctctccca catcctttcc tttctttcct ctttgaaact tctaagaaaa agcaatccaa 1140

acaagttcag cacttaagac acattgcatg cacacttttg ataagttaaa tccaaccatc 1200

tatttaaaat caaaatcagg agatgagcca agagaccaga ggttctgttc cagttttaaa 1260

cagactttta ctgaacatcc caatctttta accacagagg ctaaattgag caaatagttt 1320

tgccatttga tataatttcc aacagtatgt ttcaatgtca agttaaaaag tctacaaagc 1380

tattttccct ggagtggtat catcgctttg agaatttctt atggttaaaa tggatctgag 1440

atccaagcat ggcctggggg atggttttga tctaaggaaa aaggtgtctg tacctcacag 1500

tgcctttaaa acaagcagag atcccgtgta ccgccctaag atagcacaga ctagtgttaa 1560

ctgattccca gaaaagtgtc acaatcagaa ccaacgcatt ctcttaaact ttaaaaatat 1620

gtattgcaaa gaacttgtgt aactgtaaat gtgtgactgt tgatgacatt atacacacat 1680

agcccacgta agtgtccaat ggtgctagca ttggttgctg agtttgctgc tcgaaagctg 1740

aagcagagat gcagtccttc acaaagcaat gatggacaga gaggggagtc tccatgtttt 1800

attcttttgt tgtttctggc tgtgtaactg ttgacttctt gacattgtga tttttatatt 1860

taagacaatg tatttatttt ggtgtgttta ttgttctagc cttttaaatc actgacaatt 1920

tctaatcaag aagtacaaat aattcaatgc agcacaggct aagagcttgt atcgtttgga 1980

aaagccagtg aaggcttctc cactagccat gggaaagcta cgctttagag taaactagac 2040

aaaattgcac agcagtcttg aacctctctg tgctcaagac tcagccagtc ctttgacatt 2100

attgttcact gtgggtggga acacattgga cctgacacac tgttgtgtgt ccatgaaggt 2160

tgccactggt gtaagctttt tttggttttc attctcttat ctgtagaaca agaatgtggg 2220

gctttcctaa gtctattctg tattttattc tgaacttcgt atgtctgagt tttaatgttt 2280

tgagtactct tacaggaaca cctgaccaca cttttgagtt aaattttatc ccaagtgtga 2340

tatttagttg ttcaaaaagg gaagggatat acatacatac atacatacat acatacatat 2400

atatatatat atatacatat atatatatat atatatgtat atatatatat atatagagag 2460

agagagagag agagagagag aaagagagag aggttgttgt aggtcatagg agttcagagg 2520

aaatcagtta tggccgttaa tactgtagct gaaagtgttt tctttgtgaa taaattcata 2580

gcattattga tctatgttat tgctctgttt tatttacagt cacacctgag aatttagttt 2640

taatatgaat gatgtacttt ataacttaat gattatttat tatgtatttg gttttgaatg 2700

tttgtgttca tggcttctta tttaagacct gatcatatta aatgctaccc agtcagaa 2758

Claims (10)

1. A method for constructing an Il21 gene knockout mouse model is characterized by comprising the following steps:

s1, determining a knockout region of an Il21 gene based on a CRISPR/Cas9 technology, designing sgRNA1 and sgRNA2 of specific target sites according to the determined knockout region, wherein gene sequences of gRNA1 and gRNA2 are respectively shown as SEQ ID No.1 and SEQ ID No. 2;

s2, microinjecting active gRNA1, gRNA2 and cas9 protein into mouse fertilized eggs, transplanting the fertilized eggs surviving after injection into a pseudopregnant female mouse, and obtaining an F0-generation mouse after the pregnant female mouse farves;

s3, mating the F0 generation mouse obtained in the step S2 with a wild type mouse to obtain an F1 generation heterozygote;

s4, inbreeding the F1 generation heterozygote obtained in the step S3 to obtain an F2 generation mouse which is an IL21 gene knockout mouse model.

2. The method for constructing the Il21 gene knockout mouse model according to claim 1, wherein the genotype of F0 generation mouse is determined, and heterozygote mouse is selected to mate with wild type mouse to obtain F1 generation heterozygote.

3. The method for constructing an Il21 gene knockout mouse model according to claim 2, wherein the genotype of F0 generation mouse is determined as follows:

extracting genome DNA of F0 mouse, PCR amplifying and sequencing identifying to confirm genotype; the primers for PCR amplification comprise F1, R1, F2 and R2, wherein the gene sequences of F1, R1, F2 and R2 are respectively shown as SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO. 6; the gene sequence of the sequencing primer is shown as SEQ ID NO. 7.

4. The method for constructing an Il21 gene knockout mouse model according to claim 2, wherein the obtained F1 generation heterozygotes comprise 5 types, which are a knockout type 1, a knockout type 2, a knockout type 3, a knockout type 4 and a knockout type 5, respectively, wherein the knockout type 1 lacks 6281 base pairs, the knockout type 2 lacks 6282 base pairs, the knockout type 3 lacks 6272 base pairs, the knockout type 4 lacks 6283 base pairs and the knockout type 5 lacks 6279 base pairs, and the F1 generation heterozygote of the knockout type 1 is inbred to obtain the F2 generation mouse.

5. The method for constructing an Il21 gene knockout mouse model according to claim 2, wherein the type determination process of the heterozygote of the F1 generation is as follows:

extracting genome DNA of F1 mouse, PCR amplifying and sequencing identifying to confirm genotype; the primers for PCR amplification comprise F1, R1, F2 and R2, the gene sequences of F1, R1, F2 and R2 are respectively shown as SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6, and the gene sequence of the sequencing primer is shown as SEQ ID NO. 7.

6. The method for constructing an Il21 gene knockout mouse model according to any one of claims 1 to 5, wherein the knockout region determined in step S1 is exon3-exon5 of an Il21-201(ENSMUST00000029273.7) transcript.

7. The Il21 gene knockout mouse model constructed by the method for constructing the Il21 gene knockout mouse model according to any one of claims 1 to 6.

8. A cell with IL21 gene knocked out.

9. An Il21 gene knockout mouse model comprising a cell in which an Il21 gene is knocked out.

10. Use of the Il21 knockout mouse model of claim 8 or 9 in studies relating to tumor prognosis.

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