CN107475300A - The construction method of Ifit3 eKO1 knock out mice animal models and application - Google Patents

The construction method of Ifit3 eKO1 knock out mice animal models and application Download PDF

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CN107475300A
CN107475300A CN201710839228.3A CN201710839228A CN107475300A CN 107475300 A CN107475300 A CN 107475300A CN 201710839228 A CN201710839228 A CN 201710839228A CN 107475300 A CN107475300 A CN 107475300A
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ifit3
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mice
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李冬
吴军录
权文强
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Shanghai Tongji Hospital
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Abstract

The present invention relates to a kind of method for establishing Ifit3 eKO1 gene knock-out mice models, belongs to biological technical field, and described method comprises the following steps：Step 1: specific target sites sgRNA1, the sgRNA2 of Ifit3 eKO1 mouse gene to be knocked out are determined, and with Cas9 nucleases in-vitro transcription into mRNA；Step 2: active sgRNA and Cas9RNA microinjections are entered in mouse fertilized egg, Ifit3 eKO1 knock out mice is obtained.Its advantage is shown：The present invention uses CRISPR/Cas9 gene Knockouts, establishes the mouse model of Ifit3 eKO1 gene knockouts first, and convenient, reliable, economic animal model is provided for effects of the research Ifit3 in tumorigenesis.

Description

The construction method of Ifit3-eKO1 knock out mice animal models and application
Technical field
The present invention relates to biological technical field, is a kind of Ifit3-eKO1 knock out mice animal model specifically Construction method and application.
Background technology
Ifit3 genes, also known as RIG-G genes (Induced by Retinoic Acid gene G, retinoic acid.induced gene G) cloned from acute promyelocytic leukemia (acute promyelocytic leukemia, APL) cell line NB4 One arrived can be by the gene of ATRA (all-trans retinoic acid, ATRA) induced expression, positioned at 10 Number chromosome q24, one protein containing 490 amino acid residues of coding, molecular weight is about 60000.
Although Ifit3 is initially obtained by being cloned in acute promyelocytic leukemia cell line NB4, biological information It is a member in interferon-induced gene family to learn analysis shows Ifit3.Studies have reported that Ifit3 is except the energy in NB4 cells Enough by beyond the notable induced expressions of ATRA, moreover it is possible to the quick induced expression of element, including NB4 are disturbed in kinds of tumor cells, The myelogenous leukemia cell lines such as HL-60, U937 and cervical cancer tumer line Hela, Lines H460, A549 With the solid tumor cell such as neck squamous cancer cell.At present, the most directly effective method of relevant gene functional research is to establish to turn base Cause and (or) Gene Knock-Out Animal Model model.
Short palindrome repetitive sequence (CRISPR/Cas9) technology in rule cluster interval is based on to exempting from bacterium and archeobacteria The epidemic disease system reform and establish, mediate endonuclease Cas9 albumen to carry out target dna sequence identification by being oriented to RNA (sgRNA) And cause DNA double chain to be broken, promote to repair damaged dna in a manner of homologous recombination or non-homologous end joining, so as to target position Point realizes that the fixed point of gene is knocked out, knocked in and a variety of modifications such as gene amendment.
Because it has the characteristics of specificity is high, and molecule construction is simple, and flow is short, CRISPR/Cas9 technologies obtain in recent years Obtained fast development.Carrying out gene knockout using CRISPR/Cas9 technologies needs two key factors, is effective first The presence of sgRNA homing sequences, besides Cas9 albumen.With Zinc finger nuclease (ZFN) technology and class transcription sample effector core Sour enzyme (TALEN) technology is compared, because the simplicity of the specificity of its targeting editor's target gene, high efficiency and design etc. is many excellent Point obtains more and more extensive application, is all shown in bacterium, mammalian cell and zebra fish, mouse, rat etc. very strong Genome editor activity.
Therefore this research obtains Cas9mRNA and sgRNA structures by way of CRISPR/Cas9 technologies are by in-vitro transcription Stable knockout Ifit3-eKO1 genetic mouse models are built, the function for further research Ifit3 is had laid a good foundation.
Chinese patent literature CN107043787A discloses a kind of small based on CRISPR/Cas9 acquisition MARF1 rite-directed mutagenesises The construction method of mouse model.Chinese patent literature CN104293831A discloses one kind and is based on CRISPR/Cas9 gene knockout skills The method that art establishes hypertension mouse model.Chinese patent literature CN105950639A discloses a kind of staphylococcus aureus The preparation method of CRISPR/Cas9 systems and its application in genetic modification mouse model is built.Chinese patent literature CN106172238A discloses one kind and establishes miR-124 knock out mice animals using Crispr/cas9 gene Knockouts The method of model.But the construction method on Ifit3-eKO1 knock out mice animal models yet there are no report.
The content of the invention
The purpose of the present invention is to be directed to deficiency of the prior art, there is provided a kind of Ifit3-eKO1 knock out mice animal The construction method of model.
Second object of the present invention is to provide a kind of construction method of Ifit3-eKO1 knock out mice animal model Application.
Third object of the present invention is to provide a kind of cell of knockout Ifit3-eKO1 genes.
Fourth object of the present invention is to provide a kind of based on CRISPR/Cas9 gene Knockouts structure Ifit3- The sgRNA of eKO1 gene knock-out mice models.
The 5th purpose of the present invention is to provide above-mentioned sgRNA application.
To achieve the above object, the present invention adopts the technical scheme that：One kind establishes Ifit3-eKO1 knock out mice The method of model, described method are based on CRISPR/Cas9 gene Knockouts, and described method comprises the following steps：
Step 1: specific target sites sgRNA1, the sgRNA2 of Ifit3-eKO1 mouse gene to be knocked out are determined, and with Cas9 nucleases in-vitro transcription is into mRNA；
Step 2: active sgRNA and Cas9RNA microinjections are entered in mouse fertilized egg, Ifit3-eKO1 is obtained Knock out mice.
Further, sgRNA1 such as SEQ ID NO:2, sgRNA 2 such as SEQ ID NO:Shown in 3.
Further, step 2 is following steps in described method：
(1), mouse ovulation induction and in vitro fertilization, cultivation embryonated egg；
(2), by active sgRNA and Cas9RNA microinjections into mouse fertilized egg；
(3), the cultivation of embryonated egg in vitro culture, implantation acceptor and target gene modification animal.
Further, described method comprises the following steps：
(1) Ifit3-eKO1 genes target spot to be knocked out, is determined, and by sgRNA and Cas9 nuclease mRNA in-vitro transcriptions；
(2), mouse ovulation induction, in vitro fertilization, embryonated egg microinjection；
(3), taking after injection the zygote transplation survived, output mouse, as F0 is for mouse in false pregnancy rat body；
(4) F0, is extracted to expand for mouse tail DNA, PCR and send product to sequencing；
(5), positive mice is mated with wild type opposite sex mouse and obtains F1 generation hybrid mice；
(6), F1 generation hybrid mice is hybridized and obtains F2 for homozygote mouse, as mouse model.
Further, described method also includes step 3, and step 3 is：Identification Ifit3-eKO1 knock out mice moves Thing model.
Further, step 3 is following steps in described method：
Taking after injection the zygote transplation survived, output mouse, as F0 is for mouse in false pregnancy rat body；
F0 is extracted to expand for mouse tail DNA, PCR and send product to sequencing；
Positive mice is mated with wild type opposite sex mouse and obtains F1 generation hybrid mice；
F1 generation hybrid mice is hybridized and obtains F2 for homozygote mouse, as mouse model.
To realize above-mentioned second purpose, the present invention adopts the technical scheme that：Described method is in tumor research Using.
To realize above-mentioned 3rd purpose, the present invention adopts the technical scheme that：The mouse species that described method obtains The cell of the knockout Ifit3-eKO1 genes of model.
To realize above-mentioned 4th purpose, the present invention adopts the technical scheme that：One kind is based on CRISPR/Cas9 clpp genes Except the sgRNA of technique construction Ifit3-eKO1 gene knock-out mice models, sgRNA1 such as SEQ ID NO:2, sgRNA2 such as SEQ ID NO:Shown in 3.
To realize above-mentioned 5th purpose, the present invention adopts the technical scheme that：Described sgRNA is establishing gene defect Application in mouse
The invention has the advantages that：
The present invention uses CRISPR/Cas9 gene Knockouts, establishes the mouse of Ifit3-eKO1 gene knockouts first Animal model, the effect of the successful implementation of project for research Ifit3 in tumorigenesis provide convenient, reliable, economical Animal model.
Brief description of the drawings
Accompanying drawing 1 is that CRISPR/Cas9 gene knock-out mice models establish schematic diagram.
Accompanying drawing 2 is CRISPR/Cas9 gene knockout layout strategy schematic diagrames.
Accompanying drawing 3 is in-vitro transcription Cas9, sgRNA electrophoresis result.
Accompanying drawing 4 is sequencing result comparison before and after the type mice gene knockouts of F1 generation 1.
Accompanying drawing 5 is sequencing result comparison before and after the type mice gene knockouts of F1 generation 2.
Accompanying drawing 6 is that F2 identifies electrophoretic band figure for mouse PCR.
Accompanying drawing 7 is F2 for mouse PCR sequencing identification figures.
Embodiment
Embodiment provided by the invention is elaborated below in conjunction with the accompanying drawings.
First, this implementation of class is related to one kind and based on CRISPR/Cas9 gene Knockouts establishes Ifit3-eKO1 gene knockouts The method of mouse model, its technology path are as shown in Figure 1.
2nd, the essential information of gene is knocked out really
1. knock out Gene Name (No. MGI)：Ifit3(1101055)
2. knock out gene M GI website links：http://www.informatics.jax.org/marker/MGI: 1101055
3. knock out Gene Name (Ensembl)：Ifit3(ENSMUSG00000074896)
4. knock out gene Ensembl website links：http://asia.ensembl.org/Mus_musculus/Gene/ SummaryG=ENSMUSG00000074896；R=19:34583531-34588731；T=ENSMUST00000102825
5. knock out the transcript (No. Ensembl) being directed to：Ifit3-001(ENSMUST00000102825.3)
Knock out the exon being directed to：exon 2
3rd, it is CRISPR/Cas9 gene knockout layout strategy schematic diagrames, as shown in Figure 2.
4th, gene knockout site upstream and downstream sequence information is confirmed, such as SEQ ID NO:Shown in 1.
5th, specific target sites sgRNA1, the sgRNA2 of Ifit3-eKO1 mouse gene to be knocked out are determined, is linearized and pure Change DNA and with Cas9 nucleases in-vitro transcription into mRNA；The purity that purifying sgRNA injects to suitable transgenosis.The sgRNA1 Sequence such as SEQ ID NO:Shown in 2；The sequence of the sgRNA2 such as SEQ ID NO:Shown in 3；In-vitro transcription Cas9, sgRNA Electrophoresis result is as shown in Figure 3.
SEQ ID NO:2：GGGTCATGGGTATAGAACCGG
SEQ ID NO:3：CTAGGAGAGGCCAAGAAATCAGG
6th, by after sgRNA the and Cas9 nuclease mRNA in-vitro transcriptions described in step 5, it is expelled in embryonated egg, takes note For the zygote transplation survived after penetrating in false pregnancy rat body, the mouse birth of embryo transfer is F0 for mouse.
7th, cut tail extraction DNA after mouse is born 3 weeks to go forward side by side performing PCR amplification, product connects through T-vector, is sequenced, sun Property F0 is for positive mice：No. 22 (the genome sequence such as SEQ ID NO after mutation:And No. 42 (genome sequences after mutation 4) Row such as SEQ ID NO:5).
8th, positive F0 is for mouse PCR authentication methods：
1. primer information：
Primer Sequence 5'-->3' Primer type
I CACCTGGCCCCTTCTGGATA Forward
II GCTGGCCTGCTGCTTACCTTA Reverse
2. reaction system：
Reactive component Volume (μ l)
ddH2O 31
PrimeStar GXL PCR Buffer 10
2.5mMdNTP 4
Primer I(20pmol/μl) 1
Primer II(20pmol/μl) 1
PrimeStar GXL DNA Polymerase* 2
Tail genomic DNA 1
It is total 50
* PrimeStar GXL (TaKaRa, Code No：R050A)
3. reaction condition：
Step Temperature (DEG C) Time Remarks
1 98 2 minutes -
2 98 10 seconds -
3 63 15 seconds -
4 68 4 minutes Step 2-4, repeat 35 times
5 68 5 minutes -
6 12 - Keep
9th, the acquisition of F1 generation mouse and genotype identification
Choose positive F0 to mate with wild type C57BL/6J mouse respectively for No. 22 and No. 42 for mouse, the F1 generation of acquisition is miscellaneous Zygote mouse has 2 types, is respectively：Class1 is knocked out, lacks 2513 base-pairs；Type 2 is knocked out, lacks 2527 bases It is right；Authentication method is identified with F0 for mouse.
Knock out Class1：Lack 2513 base-pairs
Mouse number Genotype Sex Date of birth Parental generation
26 He(-2513) ♀ 2017-1-9 22#
Knock out type 2：Lack 2527 base-pairs
Mouse number Genotype Sex Date of birth Parental generation
34 He(-2527) ♂ 2017-1-9 42#
37 He(-2527) ♀ 2017-1-9 42#
Tenth, gene knockout F1 generation murine genes type comparative analysis：
1. in the Strains of Mouse, before and after gene knockout, target gene exon2 is lacked entirely, so as to cause gene function to lack Lose；
2. sequence analysis after Class1 (2513 base-pairs of missing) genotype knocks out is knocked out, such as SEQ IDNO:Shown in 6.Strike Except rear sequencing result is as shown in Figure 4；
3. sequence analysis after type 2 (2527 base-pairs of missing) genotype knocks out is knocked out, such as SEQ IDNO:Shown in 7.Strike Except rear sequencing result is as shown in Figure 5；
4.Sbjct is wild-type genomic sequence, and Query is actual sequencing result.
11, F1 generation hybrid mice is hybridized and obtains F2 for homozygote mouse, as mouse model.
12, identifications of the F2 for homozygote mouse：
1. because gene Ifit3 and Ifit3b sequence and upstream and downstream sequence similarity are high, it is virtually impossible to individually amplify Ifit3 fragments.
2. qualification program is：Using primer I, II amplification mouse genomes, wild-type mice only has big band.Heterozygote and Homozygote mouse has the band of size two, and because deletion fragment reaches 2.5kb, the difference of size fragment is very big, as shown in Figure 6.
13, heterozygote and homozygote are distinguished, big band sequencing need to be reclaimed, the foundation of interpretation is sequencing result " peak Shape figure ", it is not the sequence information of sequencing result；Sequencing primer is CACAGCTGAAGTGCCATTTC, and sequencing result is single Ifit3b gene peak types for homozygote mouse；Sequencing result have Ifit3 genes and Ifit3b genes mixing peak type for heterozygosis Sub- mouse, as shown in Figure 7.
Described above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art Member, on the premise of the inventive method is not departed from, can also make some improvement and supplement, and these are improved and supplement also should be regarded as Protection scope of the present invention.
SEQUENCE LISTING
<110>Shanghai Tongji Hospital
<120>The construction method of Ifit3-eKO1 knock out mice animal models and application
<130> /
<160> 7
<170> PatentIn version 3.3
<210> 1
<211> 2907
<212> DNA
<213>Mouse（Mus musculus）
<400> 1
gtcctcttct ttctgaggtg cacctgccac cttgtaaatt agaacaggga taaagaatga 60
caacctctga aattatacta tgtttctgtc ctgaatcata agcagctttt gcagatgatt 120
aaagtgcaaa attgcaggga attcaagatt cttgaatcaa taaatctaag gcaaggctaa 180
gaagggtcat gggtatagaa ccggttgaaa aggaccacag atttgttata tacatgctga 240
aaagggagta atacttactt gtggatgaag tagtccccag caaggcttcc attccttcag 300
tgtaggaaaa acctcctgca ctggctgtgt gaatctgttc atgggaacta ccacaaatgt 360
atgtcaggtg gaagagagga aagggtgcag acttaaagtg gaatgtttaa ttggagtttc 420
aggtctgaga aattggccat tagaaaggag aggcagaaaa taaacatctc caagagtaca 480
tggctcacat tgtcatgact ccgacaccca aaaaaaggtg gttaattttt caatgtcctg 540
aggctccatc cagttaccat gcaaaattaa acatcacaag ttacacaaga tttgtgggtg 600
ctcatcacag tgaccatgtt tttttttccg ccacagtgag gtcaaccggg aatctctgga 660
agcgatcctt ccacagctga agtgccattt cacctggaat ttattcaggg aaggaagtat 720
gtccagtcat atggaagaca gggtgtgcaa ccaggtcgaa catttaaact ctgaggagaa 780
ggcaacaatg tatgacttat tggcctacat aaagcaccta gatggcgaaa gcaaggccgc 840
cctggagtgc ttagggcaag ctgaagattt aaggaagtca gagcacaatg atcaatcaga 900
aattcgtcga ctggtcacct ggggaaacta cgcctggatc tactatcaca tgggccgtct 960
ctcagaagct caggcttacg ttgacaaggt gagacaagtt tgccaaaagt ttgcgaatcc 1020
ttacagcatg gaatgcccag aacttgaatg tgaggaagga tggacacgcc taaagtgtgg 1080
aagaaatgaa cgagcaaaaa tgtgctttga aaaggctcta gaagagaagc caaaggaccc 1140
agagtgctcc tctgggatgg ccatcgccat gttccgccta gaagaaaaac ctgagaagca 1200
gttctccgtg gatgctctga agcaggccat ggagttgaat cctcagaacc agtacctgaa 1260
agttctcctg gccctgaaac tgctgaggat gggagaagaa gctgaagggg agcgattgat 1320
taaagatgct ttggggaaag ctcctaatca aacggatgtc ctccaaaagg cagctcagtt 1380
ttacaagaaa aagggtaacc tagacagagc tattgagtta cttggaaaag cactgcgatc 1440
cacagtgaac aacagtcctc tctactcttt ggtcatgtgc cgttacaggg aaatactgga 1500
gcagctacag aataaaggag atgctgacag cagtgagaga agacagagga tggcagaact 1560
gagacgatta acgatggagt tcatgcagaa gactcttcag aggaggcgaa gtcctttgaa 1620
ctcctactca gatctcatcg atttcccgga agtagagaga tgctatcaga tggtcatcag 1680
taaggagagc cccgatgttg aggaagaaga cctctatgag cgctattgca acctccagga 1740
gtaccacagg aagtctgaag acctcgcagc cctggagtgt ttgttgcaat ttcccagaaa 1800
tgaaaggtca atcgagaagg aagaggttaa agagcaaaca tagcaagcag atcttaacct 1860
ccagtagcaa attgtggtgg attcttggca gttgcaggga taaaggagtg gctgaatggt 1920
tttggggttt gggaggcaac gcactttggg gcacaggcag gcttttcctg gcaccatgaa 1980
cctgaggaca accggaagtg tgtcagagtg cagacagaca gtctctcagc tctgtactgt 2040
gagacagatg tgctgtggag tgctgcttat ggggagaatg tgctgaaaaa agcatgagcc 2100
ttcctgccaa ggattgctga caaactgctc ttgattgttt ctttaaggaa ctgctttctc 2160
tccctgactc ctctgctcat cctagccata caattttcca gtcagcaaac ctcattacta 2220
atcatgtagg gaatggagct taaagcagac agagcacctt ttgatcacat tttttttctc 2280
tgcaataaat gacaactccc aacaaaattc actcttgttt cttcattcca tttcactgta 2340
gttgcttaag cctgaaggat taacctttgc ctaaggctag aatattcttt gcacatcatt 2400
tttgtattct actgtggttt tagggctgtg tgtgtgtgag tgtgtgtgtg tgtgtgtgtg 2460
tgtgtgtgtg tgtgtgtgtt tgtgtatccc taagaatgat tctaaagtta acatatatgc 2520
caaatagaaa ttgtagaata aatgtaaaag tttcaatcaa aatacagtct tttgtgtggt 2580
ggtaagagac agcaaaataa tacagtctta ttaagtctca gtgtttcttc ttttatcatt 2640
tggggaacct tataagacct ctctacccct ttctgaagaa acaatggcag ttttgctagg 2700
agaggccaag aaatcaggtg gggaaacatg gtggggaatc ctagtggtac atgatgggaa 2760
gtgcaggagg atggacatga tcaatatttg ttatacacat ggatgaaaat ttcccagtct 2820
tttcaaggtc cttcctctgc taagatgatt ggaaattgtg aatgctcatc agagtatcaa 2880
aacacaggtg ttttagcatc tatgtct 2907
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<213>Artificial sequence
<400> 2
gggtcatggg tatagaaccg g 21
<210> 3
<211> 23
<212> DNA
<213>Artificial sequence
<400> 3
ctaggagagg ccaagaaatc agg 23
<210> 4
<211> 394
<212> DNA
<213>Mouse（Mus musculus）
<400> 4
gtcctcttct ttctgaggtg cacctgccac cttgtaaatt agaacaggga taaagaatga 60
caacctctga aattatacta tgtttctgtc ctgaatcata agcagctttt gcagatgatt 120
aaagtgcaaa attgcaggga attcaagatt cttgaatcaa taaatctaag gcaaggctaa 180
gaagggtcat gggtataggg agaggcgggg aaacatggtg gggaatccta gtggtacatg 240
atgggaagtg caggaggatg gacatgatca atatttgtta tacacatgga tgaaaatttc 300
ccagtctttt caaggtcctt cctctgctaa gatgattgga aattgtgaat gctcatcaga 360
gtatcaaaac acaggtgttt tagcatctat gtct 394
<210> 5
<211> 380
<212> DNA
<213>Mouse（Mus musculus）
<400> 5
gtcctcttct ttctgaggtg cacctgccac cttgtaaatt agaacaggga taaagaatga 60
caacctctga aattatacta tgtttctgtc ctgaatcata agcagctttt gcagatgatt 120
aaagtgcaaa attgcaggga attcaagatt cttgaatcaa taaatctaag gcaaggctaa 180
gaagggtcag gtggggaaac atggtgggga atcctagtgg tacatgatgg gaagtgcagg 240
aggatggaca tgatcaatat ttgttataca catggatgaa aatttcccag tcttttcaag 300
gtccttcctc tgctaagatg attggaaatt gtgaatgctc atcagagtat caaaacacag 360
gtgttttagc atctatgtct 380
<210> 6
<211> 394
<212> DNA
<213>Mouse（Mus musculus）
<400> 6
gtcctcttct ttctgaggtg cacctgccac cttgtaaatt agaacaggga taaagaatga 60
caacctctga aattatacta tgtttctgtc ctgaatcata agcagctttt gcagatgatt 120
aaagtgcaaa attgcaggga attcaagatt cttgaatcaa taaatctaag gcaaggctaa 180
gaagggtcat gggtataggg agaggcgggg aaacatggtg gggaatccta gtggtacatg 240
atgggaagtg caggaggatg gacatgatca atatttgtta tacacatgga tgaaaatttc 300
ccagtctttt caaggtcctt cctctgctaa gatgattgga aattgtgaat gctcatcaga 360
gtatcaaaac acaggtgttt tagcatctat gtct 394
<210> 7
<211> 380
<212> DNA
<213>Mouse（Mus musculus）
<400> 7
gtcctcttct ttctgaggtg cacctgccac cttgtaaatt agaacaggga taaagaatga 60
caacctctga aattatacta tgtttctgtc ctgaatcata agcagctttt gcagatgatt 120
aaagtgcaaa attgcaggga attcaagatt cttgaatcaa taaatctaag gcaaggctaa 180
gaagggtcag gtggggaaac atggtgggga atcctagtgg tacatgatgg gaagtgcagg 240
aggatggaca tgatcaatat ttgttataca catggatgaa aatttcccag tcttttcaag 300
gtccttcctc tgctaagatg attggaaatt gtgaatgctc atcagagtat caaaacacag 360
gtgttttagc atctatgtct 380

Claims (10)

  1. A kind of 1. method for establishing Ifit3-eKO1 gene knock-out mice models, it is characterised in that described method is based on CRISPR/Cas9 gene Knockouts, described method comprise the following steps：
    Step 1: determine Ifit3-eKO1 mouse gene to be knocked out specific target sites sgRNA1, sgRNA2, and with Cas9 cores Sour enzyme in-vitro transcription is into mRNA；
    Step 2: active sgRNA and Cas9RNA microinjections are entered in mouse fertilized egg, Ifit3-eKO1 genes are obtained Knock-out mice.
  2. 2. according to the method for claim 1, it is characterised in that sgRNA1 such as SEQ ID NO:2, sgRNA 2 such as SEQ ID NO:Shown in 3.
  3. 3. according to any described method of claim 1 or 2, it is characterised in that step 2 is following steps in described method：
    (1), mouse ovulation induction and in vitro fertilization, cultivation embryonated egg；
    (2), by active sgRNA and Cas9RNA microinjections into mouse fertilized egg；
    (3), the cultivation of embryonated egg in vitro culture, implantation acceptor and target gene modification animal.
  4. 4. according to any described method of claim 1 or 2, it is characterised in that described method comprises the following steps：
    (1) Ifit3-eKO1 genes target spot to be knocked out, is determined, and by sgRNA and Cas9 nuclease mRNA in-vitro transcriptions；
    (2), mouse ovulation induction, in vitro fertilization, embryonated egg microinjection；
    (3), taking after injection the zygote transplation survived, output mouse, as F0 is for mouse in false pregnancy rat body；
    (4) F0, is extracted to expand for mouse tail DNA, PCR and send product to sequencing；
    (5), positive mice is mated with wild type opposite sex mouse and obtains F1 generation hybrid mice；
    (6), F1 generation hybrid mice is hybridized and obtains F2 for homozygote mouse, as mouse model.
  5. 5. according to any described method of claim 1 or 2, it is characterised in that described method also includes step 3, step 3 For：Identify Ifit3-eKO1 knock out mice animal models.
  6. 6. according to the method for claim 5, it is characterised in that step 3 is following steps in described method：
    Taking after injection the zygote transplation survived, output mouse, as F0 is for mouse in false pregnancy rat body；
    F0 is extracted to expand for mouse tail DNA, PCR and send product to sequencing；
    Positive mice is mated with wild type opposite sex mouse and obtains F1 generation hybrid mice；
    F1 generation hybrid mice is hybridized and obtains F2 for homozygote mouse, as mouse model.
  7. 7. according to application of any described methods of claim 1-6 in tumor research.
  8. 8. the knockout Ifit3-eKO1 genes of the mouse model obtained according to any described methods of claim 1-6 is thin Born of the same parents.
  9. 9. a kind of sgRNA based on CRISPR/Cas9 gene Knockouts structure Ifit3-eKO1 gene knock-out mice models, its It is characterised by, sgRNA1 such as SEQ ID NO:2, sgRNA2 such as SEQ ID NO:Shown in 3.
  10. 10. applications of the sgRNA according to claim 9 in deficient mice is established.
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