CN106119284A - A kind of product for building immunodeficient animals model and application thereof - Google Patents
A kind of product for building immunodeficient animals model and application thereof Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/22—Ribonucleases RNAses, DNAses
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/10—Mammal
- A01K2227/105—Murine
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
- A01K2267/035—Animal model for multifactorial diseases
- A01K2267/0387—Animal model for diseases of the immune system
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
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Abstract
The invention discloses a kind of product for building immunodeficient animals model and application thereof.Product for building immunodeficient animals model provided by the present invention, for product first or product second or product third；Described product first is by for knocking out the material of Rag2 gene of the animal that sets out and forming for knocking out the material of the Il2rg gene of the animal that sets out；Described product second is set out the material of Rag2 gene expression of animal and forming for the set out material of Il2rg gene expression of animal of suppression by for suppression；The material that described product third is reduced by the Rag2 protein active for the animal that promotes to set out or the material lost and the Il2rg protein active for the animal that promotes to set out reduce or loses forms.It is demonstrated experimentally that utilize product provided by the present invention can build immunodeficient animals model, there is important using value.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of product for building immunodeficient animals model and
Application.
Background technology
Experimental animal model plays irreplaceable effect in biomedical research.The development of current experiment animal has
Two main trend, one is the animal model utilizing technique for gene engineering research and development genetic modification, and two is development humanized animal's model
(humanized animal model), i.e. carries the animal of the gene of the mankind, cell, tissue or organ.Humanized animal
Being produced as of model makes up human body and deposits at aspects such as gene, physiology, pathology, infectious, drug reactiones with laboratory animal
Difference provide feasible solution.The basis of tissue mosaic type humanization (disease) modelling is to obtain highly to exempt from
The animal model of epidemic disease defect, is the most just avoided that the body immunologic rejection to xenograft.There is multiple commercialization at present
The mice of hyperimmunization defect, promote humanization hemopoietic/immune system mice, humanization liver mice, human tumor move
Planting model and other human tissue, the fast development of cell transplantation mouse model, but test for some, model has it to limit to
(small, physiological and biochemical property is big with mankind's difference), such as blood specimen collection amount, precise and tiny operation technique difficulty, drug metabolism and poison
Property response feature etc..Rat model can overcome the defect of mouse model the most in many aspects, and the strain of rat, genome
The existing suitable accumulation of the research such as information, physiological feature, in the existing many application of the aspects such as disease model, medicine generation, thus research and development
Hyperimmunization defect instrument rat, is used for preparing humanization rat model, tumor model and other heteroplastic transplantation model and has weight
Want using value.
At present, the immunodeficient rats model of commercialization only has nude rat, and its thymus produces congenital because of genetic mutation
Defect, causes T Lymphocyte depletion.Although nude rat cellular immune function lacks, but still retain innate immunity function and portion
Dividing humoral immune function, immunodeficiency degree is relatively low, xenotransplantation still suffers from immunologic rejection effect, it is impossible to meet requirements at the higher level
Humanized model demand.2012, Japan Tomoji Mashimo etc., knock out Prkdc gene and the Il2rg of F344 rat
Gene, detection finds its disappearance T, B and functional NK cell, but this rat model easily occurs senilism, isozygotys and double only strikes Mus build
Having the 60% of wild-type rats, power of breeding is low, and application is restricted.Therefore, it is thus achieved that the normal hyperimmunization of reproduction ability lacks
Sunken rat model has significant application value.
CRISPR-Cas9 is that the short palindrome in interval based on antibacterial or archeobacteria rule cluster repeats CRISPR (clustered
Regularly interspaced short palindromic repeats) acquired immune system that mediates is derived
Gene editing technology.First this technology designs one section of sgRNA gene, and this RNA molecule can pass through base pair complementarity identification
Target dna sequence, the double-stranded DNA instructing the cutting of Cas9 nuclease to identify, induce homologous recombination (HDR, homologous
Directed repair) or nonhomologous end link (NHEJ, non-homologous end-joining), and then realize mesh
DNA edit.Before gene editing technology based on the exploitation of antibacterial II type immunologic mechanism has great application on genetic improvement
Scape.One of basic demand of this technology is exactly to there is strand identification RNA (sgRNA, single guiding in recipient cell
RNA), this molecule is responsible for the gene editing site of identification specificity, and then mediation combines Cas9 albumen enforcement DNA enzymatic and cuts activity,
Introduce DNA double strand breaks in the site of design, repair approach by NHEJ or HDR of intracellular DNA and introduce sudden change.Cause
This, the cutting efficiency of the Cas9 albumen of sgRNA mediation is the important component part of this technology.
Summary of the invention
The technical problem to be solved is how to obtain reproduction ability normal hyperimmunization defect rat model.
For solving above-mentioned technical problem, present invention firstly provides a kind of product for building immunodeficient animals model
Product.
Product for building immunodeficient animals model provided by the present invention, can be product first or product second or product
Third.Described product first can be by the material of the Rag2 gene of animal for knocking out with for knocking out the Il2rg base of the animal that sets out
The material composition of cause.Described product second by the material of the Rag2 gene expression of animal for suppressing and can be set out for suppression
The material composition of the Il2rg gene expression of animal.Described product third can be by the Rag2 protein active fall of animal for promoting
Low or that lose material and the Il2rg protein active being used for promoting to set out animal reduce or the material composition of forfeiture.
No. ID of described Rag2 albumen can be NP_001093998.1.
No. ID of described Il2rg albumen can be NP_543165.1.
The Gene ID of described Rag2 gene can be 295953.
The Gene ID of described Il2rg gene can be 140924.
In the said goods, described in the animal that sets out can be rat.Described rat concretely F344 rat.
In the said goods, described " for knocking out the material of the Rag2 gene of the animal that sets out " can include sgRNA-a；Described
SgRNAa can be sgRNA1, sgRNA2, sgRNA3 or sgRNA4.
The described rat genomic dna target sequence that described sgRNA1 identifies can be 5 '-
CCAGTAGGGCAGGATCTCTTAGG-3’.The described rat genomic dna target sequence that described sgRNA2 identifies can be 5 '-
TTTTCTTTGGCCAAAAAGGCTGG-3’.The described rat genomic dna target sequence that described sgRNA3 identifies can be 5 '-
CCTAAGAGATCCTGCCCTACTGG-3’.The described rat genomic dna target sequence that described sgRNA4 identifies can be 5 '-
ATCTCTTAGGCCAGCCTTTTTGG-3’。
In the said goods, described " for knocking out the material of the Il2rg gene of the animal that sets out " can include sgRNA-b；Described
SgRNAb can be sgRNA5, sgRNA6, sgRNA7 or sgRNA8.
The described rat genomic dna target sequence that described sgRNA5 identifies can be 5 '-
TAGTGCATAGTGAGGTTGGTCGG-3’.The described rat genomic dna target sequence that described sgRNA6 identifies can be 5 '-
GGTAGTACAGGAAGAAGAGAAGG-3’.The described rat genomic dna target sequence that described sgRNA7 identifies can be 5 '-
AGAAGGGGGAGGGGTAGTACAGG-3’.The described rat genomic dna target sequence that described sgRNA8 identifies can be 5 '-
CCAACCTCACTATGCACTATAGG-3’。
In the said goods, the sequence of described sgRNA3 specifically can be as shown in the sequence 2 in sequence table.
In the said goods, the sequence of described sgRNA6 specifically can be as shown in the sequence 3 in sequence table.
Described " for knocking out the material of the Rag2 gene of the animal that sets out " may also include the RNA of coding Cas9 albumen.Described
The sequence of the RNA of coding Cas9 albumen specifically can be as shown in the sequence 1 in sequence table.
Described " for knocking out the material of the Il2rg gene of the animal that sets out " may also include the RNA of coding Cas9 albumen.Described
The sequence of the RNA of coding Cas9 albumen specifically can be as shown in the sequence 1 in sequence table.
In the said goods, described " for knocking out the material of the Rag2 gene of the animal that sets out " may also include ss-DNA；Described
Ss-DNA includes section A, section B, section C, section D and section E successively.
Described section A is upstream homology arm, a length of 30～50bp, can by animal Rag2 gene described in
Some nucleotide composition of the upstream of the target sequence of sgRNA3.Described section B can be the target sequence cog region first of described sgRNA3.
Described section C can be N number of termination codon, and N is natural number and is more than or equal to 1.Described section D can be the target sequence of described sgRNA3
Row cog region second.Described section B and described section C forms the target sequence cog region of described sgRNA3.Described section E is that downstream is same
Source arm, a length of 30～50bp, can by animal Rag2 gene described in some cores in downstream of target sequence of sgRNA3
Thuja acid forms.
The nucleotide sequence of described section A specifically can be such as the 1st to 38 institute from 5 ' ends of the sequence 4 in sequence table
Show.
The nucleotide sequence of described section B specifically can be such as the 39th to 56 institute from 5 ' ends of the sequence 4 in sequence table
Show.
The nucleotide sequence of described section C specifically can be such as the 57th to 65 institute from 5 ' ends of the sequence 4 in sequence table
Show.
The nucleotide sequence of described section D specifically can be such as the 66th to 70 institute from 5 ' ends of the sequence 4 in sequence table
Show.
The nucleotide sequence of described section E specifically can be such as the 71st to 108 institute from 5 ' ends of the sequence 4 in sequence table
Show.
Described ss-DNA specifically can single strand dna as shown in the sequence 4 in sequence table.
Described ss-DNA has section A (upstream homology arm) and section E (downstream homology arm), therefore can pass through homology
Restructuring promotes the inactivation of Rag2 gene.
In the said goods, described " for knocking out the material of the Rag2 gene of the animal that sets out " can be described containing coding
The recombiant plasmid of the DNA of sgRNA3, recombiant plasmid containing the DNA encoding described sgRNA1, containing encoding described sgRNA2's
The recombiant plasmid of DNA or the recombiant plasmid containing the DNA encoding described sgRNA4.
Recombiant plasmid concretely recombiant plasmid pX330-sgRNA3 containing the DNA encoding described sgRNA3.Restructuring matter
Grain pX330-sgRNA3 is to be obtained by insertion DNA molecular 3 in the restricted enzyme BbsI recognition sequence of carrier pX330
, the nucleotides sequence of DNA molecular 3 is classified as 5 '-CCTAAGAGATCCTGCCCTAC-3 '.
Recombiant plasmid concretely recombiant plasmid pX330-sgRNA1 containing the DNA encoding described sgRNA1.Restructuring matter
Grain pX330-sgRNA1 is to be obtained by insertion DNA molecular 1 in the restricted enzyme BbsI recognition sequence of carrier pX330
, the nucleotides sequence of DNA molecular 1 is classified as 5 '-CCAGTAGGGCAGGATCTCTT-3 '.
Recombiant plasmid concretely recombiant plasmid pX330-sgRNA2 containing the DNA encoding described sgRNA2.Restructuring matter
Grain pX330-sgRNA2 is to be obtained by insertion DNA molecular 2 in the restricted enzyme BbsI recognition sequence of carrier pX330
, the nucleotides sequence of DNA molecular 2 is classified as 5 '-TTTTCTTTGGCCAAAAAGGC-3 '.
Recombiant plasmid concretely recombiant plasmid pX330-sgRNA4 containing the DNA encoding described sgRNA4.Restructuring matter
Grain pX330-sgRNA4 is to be obtained by insertion DNA molecular 4 in the restricted enzyme BbsI recognition sequence of carrier pX330
, the nucleotides sequence of DNA molecular 4 is classified as 5 '-ATCTCTTAGGCCAGCCTTTT-3 '.
In the said goods, described " for knocking out the material of the Il2rg gene of the animal that sets out " can be described containing coding
The recombiant plasmid of the DNA of sgRNA6, recombiant plasmid containing the DNA encoding described sgRNA5, containing encoding described sgRNA7's
The recombiant plasmid of DNA or the recombiant plasmid containing the DNA encoding described sgRNA8.
Recombiant plasmid concretely recombiant plasmid pX330-sgRNA6 containing the DNA encoding described sgRNA6.Restructuring matter
Grain pX330-sgRNA6 is to be obtained by insertion DNA molecular 6 in the restricted enzyme BbsI recognition sequence of carrier pX330
, the nucleotides sequence of DNA molecular 6 is classified as 5 '-GGTAGTACAGGAAGAAGAGA-3 '.
Recombiant plasmid concretely recombiant plasmid pX330-sgRNA5 containing the DNA encoding described sgRNA5.Restructuring matter
Grain pX330-sgRNA5 is to be obtained by insertion DNA molecular 5 in the restricted enzyme BbsI recognition sequence of carrier pX330
, the nucleotides sequence of DNA molecular 5 is classified as 5 '-TAGTGCATAGTGAGGTTGGT-3 '.
Recombiant plasmid concretely recombiant plasmid pX330-sgRNA7 containing the DNA encoding described sgRNA7.Restructuring matter
Grain pX330-sgRNA7 is to be obtained by insertion DNA molecular 7 in the restricted enzyme BbsI recognition sequence of carrier pX330
, the nucleotides sequence of DNA molecular 7 is classified as 5 '-AGAAGGGGGAGGGGTAGTAC-3 '.
Recombiant plasmid concretely recombiant plasmid pX330-sgRNA8 containing the DNA encoding described sgRNA8.Restructuring matter
Grain pX330-sgRNA8 is to be obtained by insertion DNA molecular 8 in the restricted enzyme BbsI recognition sequence of carrier pX330
, the nucleotides sequence of DNA molecular 8 is classified as 5 '-CCAACCTCACTATGCACTAT-3 '.
In the said goods, described " for knocking out the material of the Rag2 gene of the animal that sets out " can be also containing Cas9 albumen
The plasmid of encoding gene.
In the said goods, described " for knocking out the material of the Il2rg gene of the animal that sets out " can be also containing Cas9 albumen
The plasmid of encoding gene.
The aminoacid sequence of described Cas9 albumen is as shown in sequence 5 in sequence table.
The plasmid concretely carrier pX330 of the described encoding gene containing Cas9 albumen.Described carrier pX330 can be
Addgene Products catalog number (Cat.No.) is the product of 42230.
The application in preparing immunodeficient animals model of any of the above-described described product falls within protection scope of the present invention.
For solving above-mentioned technical problem, present invention also offers a kind of method building immunodeficient animals model.
The method of structure immunodeficient animals model provided by the present invention, it may include following steps:
(1) knock out the Rag2 gene of the animal that sets out, obtain Rag2 Gene Knock-Out Animal Model, by described Rag2 Gene Knock-Out Animal Model
With the described animals that sets out, obtain offspring animal；
(2) knock out the Il2rg gene of the animal that sets out, obtain Il2rg Gene Knock-Out Animal Model, by described Il2rg gene knockout
Animal and the described animals that sets out, obtain offspring animal；
(3) step (1) obtains offspring animal and the offspring animal copulation that step (2) obtains, it is thus achieved that described Rag2 and
The animal of the dual-gene homozygous knockout of Il2rg.
In said method, described in the animal that sets out can be rat.Described rat concretely F344 rat.
The Gene ID of described Rag2 gene can be 295953.
The Gene ID of described Il2rg gene can be 140924.
In said method, the method for described " knocking out the Rag2 gene of the animal that sets out ", it may include following steps:
(1) DNA fragmentation of coding Cas9 albumen is carried out in vitro transcription, obtain mRNA sequence, named Cas9mRNA；Will
The DNA fragmentation of the encoding gene containing described sgRNAa carries out in vitro transcription, obtains RNA sequence, named Rag2-sgRNA；Institute
Stating the target DNA that Rag2-sgRNA identifies in the animal that sets out is Rag2 gene；
(2) injecting step (1) obtains in the germ cell of the described animal that sets out described Cas9mRNA and described Rag2-
SgRNA, it is thus achieved that Rag2 Gene Knock-Out Animal Model；
The method of described " knocking out the Il2rg gene of the animal that sets out ", comprises the steps:
1. the DNA fragmentation of coding Cas9 albumen is carried out in vitro transcription, obtain mRNA sequence, named Cas9mRNA；Will
The DNA fragmentation of the encoding gene containing described sgRNAb carries out in vitro transcription, obtains RNA sequence, named Il2rg-sgRNA；
The target DNA that described Il2rg-sgRNA identifies in the described animal that sets out is Il2rg gene；
2. the described Cas9mRNA that obtains of injecting step (1) and described Il2rg-in the described germ cell set out and touch
SgRNA, it is thus achieved that Il2rg Gene Knock-Out Animal Model.
In said method, described step (2) is particularly as follows: by 40～80pg step (1) described Cas9mRNA and 20～40pg
Described Rag2-sgRNA be expelled to described in set out animal germ cell in；
In said method, described step is 2. particularly as follows: by 40～80pg step the most described Cas9mRNA and 20～40pg institutes
State Il2rg-sgRNA be expelled to described in set out animal germ cell in.
In said method, the method for described " knocking out the Rag2 gene of the animal that sets out ", also include to the described animal that sets out
Germ cell is injected ss-DNA；Described ss-DNA specifically can single strand dna as shown in the sequence 4 in sequence table." to described
Set out and the germ cell of animal injected ss-DNA " concretely: go out to start described in being expelled to by the described ss-DNA of 10～20pg
In the germ cell of thing.
Above, the immunity of the animal of described immunodeficient animals or the dual-gene homozygous knockout of described Rag2 and Il2rg lacks
Fall into concretely: compared with F344 rat, the peripheral blood T cells of immunodeficient rats, B cell, NK cell, IgG, IgA and
The content of IgM is the most relatively low, even fails to detect.Described T cell concretely CD3+CD45RA-T cell.Described B cell is concrete
Can be CD3-CD45RA+B cell.Described NK cell concretely CD3-CD161a+NK cell.
It is demonstrated experimentally that utilize the product for building immunodeficient animals model provided by the present invention to obtain breed energy
Power normal hyperimmunization defect rat model.The immunodeficiency of described hyperimmunization defect rat model is concretely: with
F344 rat is compared, the peripheral blood T cells of immunodeficient rats, B cell, NK cell, IgG, IgA and IgM content the most relatively
Low, even fail to detect.Therefore, utilize product provided by the present invention to build immunodeficient animals model, have important
Using value.
Accompanying drawing explanation
Fig. 1 is that different sgRNA is to rat Rag2 gene and the cutting efficiency testing result of rat Il2rg gene.
Fig. 2 is the sequencing result that the F344 rat Rag2 gene knockout head of numbered R9 builds Mus.
Fig. 3 is Rag2 gene and the qualification result of Il2rg gene mutation in immunodeficient rats.
Fig. 4 is the peripheral blood T cells of flow cytometry analysis immunodeficient rats, B cell and the frequency of NK cell.
Fig. 5 is the content of IgG, IgA and IgM in the peripheral blood detecting immunodeficient rats.
Detailed description of the invention
Being further described in detail the present invention below in conjunction with detailed description of the invention, the embodiment be given is only for explaining
The bright present invention rather than in order to limit the scope of the present invention.
Experimental technique in following embodiment, if no special instructions, is conventional method.
Material used in following embodiment, reagent etc., if no special instructions, the most commercially obtain.
Common products purification kit is TIANGEN Biotech's product, and catalog number is DP204.
Ligation kit is takara Products, and catalog number is D6022.Blood tissues cellular genome extracts test kit
TIANGEN Biotech's product, catalog number is DP304.T7endonuclease is NEB Products,
Catalog number is M0302L.Carrier pBlunt is Beijing Quanshijin Biotechnology Co., Ltd's product, and catalog number is
CB111-02.IOTest Anti-Rat CD3-FITC, CD45RA-PC7, CD161a-APC and IOTest Anti-Rat CD3-
FITC is the product of Beckman Coulter company.Rat IgG、IgA and IgM ELISA quantitation kits
Product for Bethyl Laboratories company.Carrier pX330 is Addgene Products, and catalog number is 42230；
Nucleotide sequence containing coding Cas9 albumen and eukaryotic expression regulating and controlling sequence on carrier pX330, it is possible to express Cas9 albumen.Limit
Property restriction endonuclease BbsI, EcoRI-HF and AgeI-HF processed are NEB Products, and catalog number is respectively R0539L, R3101L
And R3552S.10 × T4ligation buffer and T4polynucleotide kinase is NEB Products, product mesh
Record number is respectively B0202S and M0201S.Ligation kit is takara Products, and catalog number is D6022.F344
The pregnant Mus of strain is Beijing Vital River Experimental Animals Technology Co., Ltd.'s product, and catalog number is 112.PBS is
HyClone Products, catalog number is SH30256.01.Calf serum is Gibco Products, and catalog number is
REF16000-044.DMEM in high glucose culture medium is HyClone Products, and catalog number is SH30022.01B.Plasmid is little to be carried
Test kit is TIANGEN Biotech's product, and catalog number is DP103.nucleofectorTMFor amaxa
Products.NEB buffer2 is NEB Products, and catalog number is M0302L.mMESSAGE mMACHINE
T7ULTRA Kit and MEGAclearTMKit is Ambion Products, and catalog number is respectively AM1345 and AM1908.
It is Axygen Products that agarose gel reclaims test kit, and catalog number is AP-GS-250.MEGAshortscriptTM
T7Kit is Ambion Products, and catalog number is AM1354.Female F344 rat and normal male F344 rat are north
Capital laboratory animal Technology Co., Ltd. of dimension tonneau China product, catalog number is 112.Injection serum gonadotrophin and injection floss promote
Property element is Ningbo Sansheng Pharmaceutical Co., Ltd.'s product, and batch number is respectively S150406 and B141105.P3Primary
CellX Kit L is Lonza Products, and catalog number is V4XP-3024.
The solute of TE buffer and concentration thereof are 10mM Tris-HCl and 1mM EDTA, and solvent is distilled water, and pH value is
8.0。
Sodium acetate solution: by 40.8g NaAc 3H2O is dissolved in deionized water, with glacial acetic acid regulation pH value to 5.2,
Then it is settled to 100mL with deionized water.
Embodiment 1, the selection of sgRNA sequence
One, design identifies the sgRNA sequence of rat Rag2 gene and identifies the sgRNA sequence of rat Il2rg gene
According to Rag2 gene (full name is recombination activating gene 2, and Gene ID is 295953)
Nucleotide sequence design number is the sgRNA sequence of 1～4, and according to Il2rg gene, (full name is interleukin 2receptor
Subunit gamma, Gene ID is 140924) the sgRNA sequence that nucleotide sequence design number is 5～8, each sgRNA
The rat genomic dna target sequence identified is shown in Table 1.
Table 1
| Numbering | Sequence (5 '-3 ') |
| 1 | CCAGTAGGGCAGGATCTCTTAGG |
| 2 | TTTTCTTTGGCCAAAAAGGCTGG |
| 3 | CCTAAGAGATCCTGCCCTACTGG |
| 4 | ATCTCTTAGGCCAGCCTTTTTGG |
| 5 | TAGTGCATAGTGAGGTTGGTCGG |
| 6 | GGTAGTACAGGAAGAAGAGAAGG |
| 7 | AGAAGGGGGAGGGGTAGTACAGG |
| 8 | CCAACCTCACTATGCACTATAGG |
Two, the structure of carrier
1, the structure of recombiant plasmid pX330-sgRNA1
(1) with restricted enzyme BbsI enzyme action carrier pX330, reclaim about 8.5kb's with common products purification kit
Carrier framework.
(2) synthetic primer 1-F:5 '-ACCG -3 ' (single underscore is enzyme action position
Point sticky end, square frame is initiation transcription site, and wave is sgRNA sequence) and primer 1-R:5 '-AAAC -3 ' (single underscore is restriction enzyme site sticky end, and wave is sgRNA sequence).Use TE
Primer 1-F and primer 1-R is diluted to 100 μMs by buffer respectively, obtains primer 1-F diluent and primer 1-R diluent；Then
Carry out annealing reaction, form DNA molecular, be 100ng/ μ L with deionized water dilution DNA molecule to its concentration.
Annealing system: primer 1-F diluent 1 μ L, primer 1-R diluent 1 μ L, 10 × T4ligation buffer 1 μ L,
T4polynucleotide kinase1 μ L, deionized water 6 μ L, cumulative volume 10 μ L.
Cycle of annealing: 37 DEG C of 30min, 95 DEG C of 5min, 90 DEG C of 1min, 85 DEG C of 1min, 80 DEG C of 1min, 75 DEG C of 1min, 70 DEG C
1min, 65 DEG C of 1min, 60 DEG C of 1min, 55 DEG C of 1min, 50 DEG C of 1min, 45 DEG C of 1min, 40 DEG C of 1min, 35 DEG C of 1min, 30 DEG C of 1min,
25 DEG C of 1min, 4 DEG C of preservations.
(3) after completing step (1) and (2), according to the operating procedure of ligation kit by carrier framework and DNA molecular 1
Connect, obtain recombiant plasmid pX330-sgRNA1.
According to sequencing result, recombiant plasmid pX330-sgRNA1 is carried out structure and is described as follows: to the restriction of carrier pX330
Property restriction endonuclease BbsI recognition sequence in insert DNA molecular 1, the nucleotides sequence of DNA molecular 1 is classified as 5 '-
CCAGTAGGGCAGGATCTCTT-3’。
2, the structure of recombiant plasmid pX330-sgRNA2
According to above-mentioned steps 1, primer 1-F is replaced with primer 2-F:5 '-ACCG -
3 ' (single underscore is restriction enzyme site sticky end, and square frame is initiation transcription site, and wave is sgRNA sequence), primer 1-R replaces
Be changed to primer 2-R:5 '-AAAC -3 ' (single underscore is restriction enzyme site sticky end, wave
Line is sgRNA sequence), other step is the most constant, obtains recombiant plasmid pX330-sgRNA2.
According to sequencing result, recombiant plasmid pX330-sgRNA2 is carried out structure and is described as follows: to the restriction of carrier pX330
Property restriction endonuclease BbsI recognition sequence in insert DNA molecular 2, the nucleotides sequence of DNA molecular 2 is classified as 5 '-
TTTTCTTTGGCCAAAAAGGC-3’。
3, the structure of recombiant plasmid pX330-sgRNA3
According to above-mentioned steps 1, primer 1-F is replaced with primer 3-F:5 '-ACCG -
3 ' (single underscore is restriction enzyme site sticky end, and square frame is initiation transcription site, and wave is sgRNA sequence), primer 1-R replaces
Be changed to primer 3-R:5 '-AAAC -3 ' (single underscore is restriction enzyme site sticky end, wave
Line is sgRNA sequence), other step is the most constant, obtains recombiant plasmid pX330-sgRNA3.
According to sequencing result, recombiant plasmid pX330-sgRNA3 is carried out structure and is described as follows: to the restriction of carrier pX330
Property restriction endonuclease BbsI recognition sequence in insert DNA molecular 3, the nucleotides sequence of DNA molecular 3 is classified as 5 '-
CCTAAGAGATCCTGCCCTAC-3’。
4, the structure of recombiant plasmid pX330-sgRNA4
According to above-mentioned steps 1, primer 1-F is replaced with primer 4-F:5 '-ACCG -
3 ' (single underscore is restriction enzyme site sticky end, and square frame is initiation transcription site, and wave is sgRNA sequence), primer 1-R replaces
Be changed to primer 4-R:5 '-AAAC -3 ' (single underscore is restriction enzyme site sticky end, wave
Line is sgRNA sequence), other step is the most constant, obtains recombiant plasmid pX330-sgRNA4.
According to sequencing result, recombiant plasmid pX330-sgRNA4 is carried out structure and is described as follows: to the restriction of carrier pX330
Property restriction endonuclease BbsI recognition sequence in insert DNA molecular 4, the nucleotides sequence of DNA molecular 4 is classified as 5 '-
ATCTCTTAGGCCAGCCTTTT-3’。
5, the structure of recombiant plasmid pX330-sgRNA5
According to above-mentioned steps 1, primer 1-F is replaced with primer 5-F:5 '-ACCG -
3 ' (single underscore is restriction enzyme site sticky end, and square frame is initiation transcription site, and wave is sgRNA sequence), primer 1-R replaces
Be changed to primer 5-R:5 '-AAAC -3 ' (single underscore is restriction enzyme site sticky end, wave
Line is sgRNA sequence), other step is the most constant, obtains recombiant plasmid pX330-sgRNA5.
According to sequencing result, recombiant plasmid pX330-sgRNA5 is carried out structure and is described as follows: to the restriction of carrier pX330
Property restriction endonuclease BbsI recognition sequence in insert DNA molecular 5, the nucleotides sequence of DNA molecular 5 is classified as 5 '-
TAGTGCATAGTGAGGTTGGT-3’。
6, the structure of recombiant plasmid pX330-sgRNA6
According to above-mentioned steps 1, primer 1-F is replaced with primer 6-F:5 '-ACCG -
3 ' (single underscore is restriction enzyme site sticky end, and square frame is initiation transcription site, and wave is sgRNA sequence), primer 1-R replaces
Be changed to primer 6-R:5 '-AAAC -3 ' (single underscore is restriction enzyme site sticky end, wave
Line is sgRNA sequence), other step is the most constant, obtains recombiant plasmid pX330-sgRNA6.
According to sequencing result, recombiant plasmid pX330-sgRNA6 is carried out structure and is described as follows: to the restriction of carrier pX330
Property restriction endonuclease BbsI recognition sequence in insert DNA molecular 6, the nucleotides sequence of DNA molecular 6 is classified as 5 '-
GGTAGTACAGGAAGAAGAGA-3’。
7, the structure of recombiant plasmid pX330-sgRNA7
According to above-mentioned steps 1, primer 1-F is replaced with primer 7-F:5 '-ACCG -
3 ' (single underscore is restriction enzyme site sticky end, and square frame is initiation transcription site, and wave is sgRNA sequence), primer 1-R replaces
Be changed to primer 7-R:5 '-AAAC 3 ' (single underscore is restriction enzyme site sticky end, wave
Line is sgRNA sequence), other step is the most constant, obtains recombiant plasmid pX330-sgRNA7.
According to sequencing result, recombiant plasmid pX330-sgRNA7 is carried out structure and is described as follows: to the restriction of carrier pX330
Property restriction endonuclease BbsI recognition sequence in insert DNA molecular 7, the nucleotides sequence of DNA molecular 7 is classified as 5 '-
AGAAGGGGGAGGGGTAGTAC-3’。
8, the structure of recombiant plasmid pX330-sgRNA8
According to above-mentioned steps 1, primer 1-F is replaced with primer 8-F:5 '-ACCG -
3 ' (single underscore is restriction enzyme site sticky end, and square frame is initiation transcription site, and wave is sgRNA sequence), primer 1-R replaces
Be changed to primer 8-R:5 '-AAAC -3 ' (single underscore is restriction enzyme site sticky end, wave
Line is sgRNA sequence), other step is the most constant, obtains recombiant plasmid pX330-sgRNA8.
According to sequencing result, recombiant plasmid pX330-sgRNA8 is carried out structure and is described as follows: to the restriction of carrier pX330
Property restriction endonuclease BbsI recognition sequence in insert DNA molecular 8, the nucleotides sequence of DNA molecular 8 is classified as 5 '-
CCAACCTCACTATGCACTAT-3’。
Three, detection cutting efficiency
1, the separation of rat embryo fibroblast
Use continuous steps digestion method separation rat embryo fibroblast, specifically comprise the following steps that
1. taking the pregnant Mus of F344 strain of conceived 14～15d, disconnected neck is put to death, and puts into 75% (percent by volume) alcohol water blend
Middle immersion 1min；Then this F344 strain pregnant Mus outside of belly is upwards placed in dissecting pan, with cotton ball soaked in alcohol moistening, sterilization abdominal part, depends on
Secondary cutting off skin layer, Musclar layer, open abdominal cavity, cut off the connective tissue and fat being connected on uterus, that takes out containing Mus embryo is complete
Whole uterus is placed in aseptic culture dish (a diameter of 6cm).
2. complete step 1. after, by the intact uteri iris scissors containing Mus embryo and ophthalmic tweezers strip off Uterus wall, by Mus embryo with
Embryo outside organization, Placenta Hominis etc. separate, and are transferred to by Mus embryo in another culture dish filling PBS (a diameter of 6cm)；So
Removing the head of deratization embryo, extremity and internal organs with ophthalmic tweezers afterwards, torso portion is transferred in another culture dish (a diameter of 6cm),
And wash three times with PBS.
3. complete step 2. after, the torso portion elbow iris scissors of Mus embryo is cut into 1mm3The piece of tissue of size, uses PBS
Buffer solution 1～3 times, be then placed in centrifuge tube at the bottom of the cone filling PBS by piece of tissue, after room temperature stands 2～3min,
Abandon supernatant.
4. complete step 3. after, in centrifuge tube at the bottom of described cone add Digestive system first (Digestive system first is by containing 0.25% (volume
Percentage ratio) the EDTA solution of pancreatin and PBS be that 1:2 mixing forms according to volume ratio；Wherein EDTA solution is that Gibco is public
Department's product, catalog number is REF25200-056) soak, blow with dropper even, digest 10min under room temperature, be subsequently adding (volume
For long-pending more than 2 times of Digestive system nail body) DMEM in high glucose culture medium containing 10% (percent by volume) calf serum, quiet after mixing
Put, obtain rat embryo fibroblast suspension and piece of tissue precipitation first.
5. complete step 4. after, add appropriate Digestive system second (Digestive system second be by containing 0.25% (body in piece of tissue precipitation first
Long-pending percentage ratio) the EDTA solution of pancreatin and PBS be that 1:1 mixing forms according to volume ratio；Wherein EDTA solution is Gibco
Products, catalog number is REF25200-056), blow with dropper even, digest 5～10min under room temperature, be subsequently adding (body
Amass more than 2 times for Digestive system second volume) containing the DMEM in high glucose culture medium of 10% (percent by volume) calf serum, quiet after mixing
Put, obtain rat embryo fibroblast suspension and piece of tissue precipitation second.
According to step step process piece of tissue precipitation second 5., hang until collecting enough rat embryo fibroblasts
Liquid.
6. complete step 5. after, take rat embryo fibroblast suspension, 1000rpm is centrifuged 10min, and the precipitation that obtains is used
DMEM in high glucose culture medium (hereinafter referred to as rat embryo fibroblast culture medium) containing 10% (percent by volume) calf serum
Resuspended, then it is seeded in the culture dish of a diameter of 10cm, adds rat embryo fibroblast culture medium to 8mL, after shaking up
It is placed in 37 DEG C of incubators cultivation, the rat embryo fibroblast culture medium renewed every 24h during cultivation, until rat embryonic
At the bottom of tire fibroblast is paved with bottle, can carry out frozen.
2, consideration convey
(1) extraction of recombiant plasmid
1. the little extraction reagent kit of plasmid is used to extract recombiant plasmid (recombiant plasmid pX330-sgRNA1, recombiant plasmid pX330-
SgRNA2, recombiant plasmid pX330-sgRNA3, recombiant plasmid pX330-sgRNA4, recombiant plasmid pX330-sgRNA5, restructuring matter
Grain pX330-sgRNA6, recombiant plasmid pX330-sgRNA7 or recombiant plasmid pX330-sgRNA8).
2. complete step 1. after, take recombiant plasmid, add 2 times of volume dehydrated alcohol and 0.1 times of volume sodium acetate solution (vinegar
The preparation method of acid sodium solution is: by 40.8g NaAc 3H2O is dissolved in deionized water, with glacial acetic acid regulation pH value extremely
5.2, be then settled to 100mL with deionized water), after mixing ,-20 DEG C stand 30min, 4 DEG C, 12000rpm be centrifuged 10min,
To precipitation.
3. complete step 2. after, take precipitation, with 70% (percent by volume) ethanol water wash 2 times, after drying, with 20
μ L TE buffer solution.
(2) take the rat embryo fibroblast that step 1 separates, become unicellular with trypsinization.
(3) after completing step (1) and (2), according to preparation consideration convey system (Nucleofector solution shown in table 2
P4 and Supplement is P3Primary CellAssembly in X Kit L), then use
nucleofectorTMCarry out consideration convey, obtain system after consideration convey.
(4), after completing step (3), system after consideration convey is seeded to fill the cultivation of rat embryo fibroblast culture medium
Ware, cultivates 3d, obtains turning the rat embryo fibroblast of recombiant plasmid for 37 DEG C.
Table 2 consideration convey system
| Component | Volume |
| Nucleofector solution P4 | 16.4μL |
| Supplement | 3.6μL |
| Rat embryo fibroblast | 1×105Individual |
| Recombiant plasmid | 2μg |
3, the extraction of genomic DNA and the recovery of pcr amplification product
(1) extract the operating procedure of test kit according to blood tissues cellular genome, what extraction step 2 obtained respectively turns weight
The genomic DNA of the rat embryo fibroblast of group plasmid.
(2) after completing step (1), to turn the genome of the rat embryo fibroblast of recombiant plasmid pX330-sgRNA1
DNA, turn the genomic DNA of the rat embryo fibroblast of recombiant plasmid pX330-sgRNA2, turn recombiant plasmid pX330-
The genomic DNA of the rat embryo fibroblast of sgRNA3 or turn the rat embryo of recombiant plasmid pX330-sgRNA4 and become fiber
The genomic DNA of cell is template, with F1:5 '-TTGATGGTCAAGTTTATTCAATTACAC-3 ' and R1:5 '-
ACCAACACTTTTTCCTCGGCTA-3 ' is that primer carries out PCR amplification, obtains the pcr amplification product first of about 832bp.
(3) after completing step (1), the rat embryo fibroblast turning recombiant plasmid pX330-sgRNA5 extracted with step (1)
The genomic DNA tieing up cell, the genomic DNA turning the rat embryo fibroblast of recombiant plasmid pX330-sgRNA6, turn weight
Organize the genomic DNA of the rat embryo fibroblast of plasmid pX330-sgRNA7 or turn the big of recombiant plasmid pX330-sgRNA8
The genomic DNA of rat embryo fibroblast cell is template, with F2:5 '-GGCTGTCTGTCTCACTACTGCTT-3 ' and R2:5 '-
GAACAATCTATAAACTCCAGTGCCT-3 ' is that primer carries out PCR amplification, obtains the pcr amplification product second of about 702bp.
4, T7E1 abrupt climatic change
(1) preparation of sample
1. reclaim pcr amplification product first with common products purification kit, be recycled product first；Purify by common products
Test kit reclaims pcr amplification product second, is recycled product second.
2. complete step 1. after, prepare annealing reaction system: annealing reaction system include reclaim product first or reclaim product
Second 500ng, NEB buffer2 2 μ L, use ddH2O mends to 20 μ L.
3. complete step 2. after, carry out annealing reaction.Reaction condition is: first 98 DEG C 10min, then slow cooling (fall
Temperature speed < 1 DEG C/10s) to 25 DEG C, last 25 DEG C of 5min.
Complete step 3. after annealing reaction system be the sample of preparation.
(2) T7endonuclease process
Take sample 20 μ L prepared by step (1), add 0.5 μ L T7endonuclease, obtain system for handling；Then 37
1h is reacted under the conditions of DEG C.
(3) electrophoretic analysis and result judge
The agarose gel electrophoresis analysis that system for handling concentration is 1.5% of step (2) will be completed, then carry out as follows
Result judges:
If pcr amplification product first can be two sections by T7endonuclease enzyme action and size respectively may be about 500bp and
, then there is the sudden change of Rag2 gene in 300bp；If pcr amplification product first by the size after T7endonuclease enzyme action without bright
Aobvious change, then do not cause the sudden change of Rag2 gene；
If pcr amplification product second can be two sections by T7endonuclease enzyme action and size respectively may be about 450bp and
, then there is the sudden change of Il2rg gene in 250bp；If pcr amplification product second by the size after T7endonuclease enzyme action without bright
Aobvious change, then do not cause the sudden change of Il2rg gene.
T7E1 abrupt climatic change experimental result is shown in that (A is that T7E1 enzyme action detects Rag2 gene to Fig. 1: 1 is recombiant plasmid pX330-
SgRNA1,2 is recombiant plasmid pX330-sgRNA2, and 3 is recombiant plasmid pX330-sgRNA3, and 4 is recombiant plasmid pX330-
SgRNA4, M are DNA Marker；B is that T7E1 enzyme action detects Il2rg gene: 1 is recombiant plasmid pX330-sgRNA5, and 2 is restructuring
Plasmid pX330-sgRNA6,3 is recombiant plasmid pX330-sgRNA7, and 4 is recombiant plasmid pX330-sgRNA8, and M is DNA
Marker).Result shows, recombiant plasmid pX330-sgRNA1～recombiant plasmid pX330-sgRNA4 all can cause Rag2 gene
Sudden change, and the cutting efficiency of recombiant plasmid pX330-sgRNA3 is the highest；Recombiant plasmid pX330-sgRNA5～recombiant plasmid
PX330-sgRNA8 all can cause the sudden change of Il2rg gene, and the cutting efficiency of recombiant plasmid pX330-sgRNA6 is the highest.
Take the pcr amplification product first that in step 3, (2) obtain as template using recombiant plasmid pX330-sgRNA3, with carrier
PBlunt connects, and obtains recombiant plasmid first.Checking order recombiant plasmid first, result shows, recombiant plasmid pX330-sgRNA3
Really on rat embryo fibroblast, cause the sudden change of Rag2 gene.
Take the pcr amplification product second that in step 3, (3) obtain as template using recombiant plasmid pX330-sgRNA6, with carrier
PBlunt connects, and obtains recombiant plasmid second.Checking order recombiant plasmid second, result shows, recombiant plasmid pX330-sgRNA6
Really on rat embryo fibroblast, cause the sudden change of Il2rg gene.
Embodiment 2, the acquisition of immunodeficient rats
One, the in vitro transcription of Cas9mRNA, Rag2sgRNA and Il2rg sgRNA and purification
1, the in vitro transcription of Cas9mRNA and purification
(1) with restriction enzyme A geI-HF enzyme action carrier pX330, about 8.5kb is reclaimed with common products purification kit
Carrier framework.
(2) synthetic primer T7-F:5 '-CCGG -3 ' (single underscore is enzyme action position
Point sticky end, wave is T7 promoter sequence) and primer T7-R:5 '-CCGG -3 ' (single
Underscore is restriction enzyme site sticky end, and wave is T7 promoter sequence).With TE buffer respectively by primer T7-F and primer
T7-R is diluted to 100 μMs, obtains primer 1-F diluent and primer 1-R diluent；Then carry out annealing reaction, form DNA and divide
Son, is 100ng/ μ L with deionized water dilution DNA molecule to its concentration.
Annealing system: primer T7-F diluent 1 μ L, primer T7-R diluent 1 μ L, 10 × T4ligation buffer 1 μ
L, T4polynucleotide kinase1 μ L, deionized water 6 μ L, cumulative volume 10 μ L.
Cycle of annealing: 37 DEG C of 30min, 95 DEG C of 5min, 90 DEG C of 1min, 85 DEG C of 1min, 80 DEG C of 1min, 75 DEG C of 1min, 70 DEG C
1min, 65 DEG C of 1min, 60 DEG C of 1min, 55 DEG C of 1min, 50 DEG C of 1min, 45 DEG C of 1min, 40 DEG C of 1min, 35 DEG C of 1min, 30 DEG C of 1min,
25 DEG C of 1min, 4 DEG C of preservations.
(3) after completing step (1) and (2), according to the operating procedure of ligation kit by carrier framework and DNA molecular T7
Connect, obtain recombiant plasmid pX330-T7.
According to sequencing result, recombiant plasmid pX330-T7 is carried out structure and is described as follows: to carrier pX330 restricted in
Cut in enzyme AgeI recognition sequence insertion DNA molecular T7, the nucleotides sequence of DNA molecular T7 is classified as 5 '-
taatacgactcactatagg-3’。
(4) after completing step (3), with restricted enzyme EcoRI enzyme action carrier pX330-T7, then by concentration it is successively
The E.C. 3.4.21.64 aqueous solution of 100 μ g/mL and the SDS aqueous solution of 0.5% (mass percent) process (purpose is for removing RNase),
To linearizing carrier pX330-T7.
(5) after completing step (4), take linearizing carrier pX330-T7, add equal-volume phenol/chloroform and be stripped,
12000rpm is centrifuged 10min, obtains supernatant；Take described supernatant, add 2 times of volume dehydrated alcohol and 0.1 times of volume acetic acid
Sodium solution, is placed in-20 DEG C of 1h, and 12000rpm is centrifuged 10min, the precipitation obtained 70% (percent by volume) ethanol aqueous wash
Wash 2 times, add 20 μ L TE buffer after drying, obtain the solution of linearizing carrier pX330-T7.
(6), after completing step (5), according to the operating procedure of mMESSAGE mMACHINE T7 ULTRA Kit, body is carried out
Transcribe outward.Comprise the following steps that (wherein Nuclease-free water, T7 2 × NTP/ARCA, 10 × T7 Reaction
Buffer、T7 Enzyme Mix、TURBO DNase、mMESSAGET7 Ultra reaction、5×E-PAP
Buffer, ATP Solution, E-PAP and Lithium chloride is mMESSAGE mMACHINE T7ULTRA Kit
In assembly):
1. transcribe system shown in preparation table 3, be subsequently placed in 37 DEG C of reaction 2h, obtain transcribing rear system.
System transcribed by table 3
| Component | Volume |
| Nuclease-free water | Mend to 20 μ L |
| T7 2×NTP/ARCA | 10μL |
| 10×T7 Reaction Buffer | 2μL |
| Linearizing carrier pX330-T7 | 2μg |
| T7 Enzyme Mix | 2μL |
2. rear system adds 1 μ L TURBO DNase to transcribing, 37 DEG C of reaction 20min.
3. complete step 2. after, prepare the tailing system shown in table 4, then 37 DEG C reaction 30～45min.After obtaining tailing
System.
Table 4 tailing system
4. complete step 3. after, add 50 μ L Lithium chloride, be placed in-20 DEG C of 30min, 12000rpm be centrifuged
10min, the precipitation obtained 70% (percent by volume) ethanol water washs 2 times, adds 20 μ L deionized waters, obtain after drying
The in vitro transcription solution of Cas9 mRNA.
(7) after completing step (6), according to MEGAclearTMThe operating procedure of Kit, is purified, wherein Elution
Solution, Binding Solution, adsorption column, Wash Solution and RNase-free water are
MEGAclearTMAssembly in Kit.Specifically comprise the following steps that
Taking the in vitro transcription solution of Cas9 mRNA, adding Elution Solution is 100 μ L to volume, then adds successively
Enter 350 μ L Binding Solution and 250 μ L dehydrated alcohol, obtain mixed solution.Mixed solution is added adsorption column,
12000rpm is centrifuged 1min, then washes twice with 500 μ L Wash Solution, and each 12000rpm is centrifuged 1min.Dry
After, in adsorption column, adding 50 μ L Elution Solution, 12000rpm is centrifuged 1min, and obtaining concentration is 985ng/ μ L
The purification solution of Cas9 mRNA ,-80 DEG C save backup.
The sequence of the RNA in the purification solution of Cas9 mRNA is as shown in the sequence 1 in sequence table.
2, the in vitro transcription of Rag2 sgRNA and purification
(1) acquisition of Rag2 sgRNA3 template
With carrier pX330 as template, with the primer Rag2-sgRNA3-F:5 ' of synthetic-
TTAATACGACTCACTATAGCCTAAGAGATCCTGCCCTACGTTTTAGAGCTAGAAAT AGC-3 ' and primer sgRNA-R:
5 '-AAAAAAGCACCGACTCGGTGCC-3 ' are primer, carry out PCR amplification, reclaim test kit with agarose gel and return after electrophoresis
Receive pcr amplification product.
Response procedures is: 95 DEG C of degeneration 2min；95 DEG C of degeneration 30sec, 50 DEG C of annealing 30sec, 68 DEG C extend 20sec, altogether
30 circulations；68 DEG C extend 7min；4 DEG C of preservations.
(2) after completing step (1), according to MEGAshortscriptTMThe operating procedure of T7Kit, carries out in vitro transcription, its
Middle TURBO DNase, Water (Nuclease-free), T7 10 × Reaction Buffer, T7ATP Solution
(75mM), T7CTP Solution (75mM), T7GTP Solution (75mM), T7UTP Solution (75mM) and
T7Enzyme Mix is MEGAshortscriptTMAssembly in T7Kit.Specifically comprise the following steps that
1. transcribe system shown in preparation table 5, be subsequently placed in 37 DEG C of reaction 3h, obtain transcribing rear system.
System transcribed by table 5
| Component | Volume |
| Water(Nuclease-free) | Mend to 20ul |
| T7 10×Reaction Buffer | 2μL |
| T7ATP Solution(75mM) | 2μL |
| T7CTP Solution(75mM) | 2μL |
| T7GTP Solution(75mM) | 2μL |
| T7UTP Solution(75mM) | 2μL |
| T7Enzyme Mix | 2μL |
| Reclaim pcr amplification product | 1μg |
2. rear system adds 1 μ L TURBO DNase to transcribing, 37 DEG C of reaction 15min, obtain the body of Rag2-sgRNA3
Transcribe outward solution.
(5) after completing step (4), according to MEGAclearTMThe operating procedure of Kit, is purified, wherein Elution
Solution, Binding Solution, adsorption column, Wash Solution and RNase-free water are
MEGAclearTMAssembly in Kit.Specifically comprise the following steps that
Taking the in vitro transcription solution of Rag2-sgRNA3, adding Elution Solution is 100 μ L to volume, more successively
Add 350 μ L Binding Solution and 250 μ L dehydrated alcohol, obtain mixed solution.Mixed solution is added adsorption column,
12000rpm is centrifuged 1min, then washes twice with 500 μ L Wash Solution, and each 12000rpm is centrifuged 1min.Dry
After, in adsorption column, adding 50 μ L Elution Solution, 12000rpm is centrifuged 1min, and obtaining concentration is 635ng/ μ L
The purification solution of Rag2-sgRNA3 ,-80 DEG C save backup.
In the purification solution of Rag2-sgRNA3, the sequence of RNA is as shown in the sequence 2 in sequence table.
3, the in vitro transcription of Il2rg gRNA and purification
According to the method for above-mentioned steps 2, primer Rag2-sgRNA3-F is replaced with primer I l2rg-sgRNA6-F:5 '-
TTAATACGACTCACTATAGGATGGGGAGGGGGAAGAGTAGTTTTAGAGCTAGAAAT AGC-3 ', other step is the most not
Becoming, obtain the purification solution of the Il2rg-sgRNA6 that concentration is 1100ng/ μ L ,-80 DEG C save backup.
In the purification solution of Il2rg-sgRNA6, the sequence of RNA is as shown in the sequence 3 in sequence table.
Two, the synthesis of ss-DNA
Design according to the nucleotide sequence of the sgRNA sequence of numbered 3 in Rag2 gene and embodiment 1 table 1 and by raw work
Biological engineering (Shanghai) limited company synthesis ss-DNA.Ss-DNA is the single strand dna shown in sequence 4 in sequence table:
5’-TTGATGGTCAAGTTTTTTTCTTTGGCCAAAAAGGCTGG -3 ' (single underscore is left homology arm, and square frame is embodiment
From 5 ' ends the 1st to 18 of the target sequence cog region of numbered 3 in 1 table 1, double underline is 3 even termination codoies, thick lines
For from 5 ' ends the 19th to 23 of the sgRNA sequence of numbered 3 in embodiment 1 table 1, wave is right homology arm).Spend
Ss-DNA is diluted by ionized water, obtains the ss-DNA diluent that concentration is 100ng/ μ L, and-80 DEG C save backup.
Three, the acquisition of Rag2 knockout rat and qualification
1, Rag2 gene intends knocking out the acquisition of rat
(1) taking some of the female F344 rat of 5～6 week old, vaginal orifice is closed, then at the experiment 14:00 of first day,
The injection serum gonadotrophin of every female F344 rats by intraperitoneal injection 30IU.
(2) after completing step (1) 48～50h, the injection chorionic gonadotrophin of every female F344 rats by intraperitoneal injection 50IU,
Then (purpose is for providing germ cell) is mated with normal male F344 rat；Take vaginal orifice flushing and more than 2 monthly ages female simultaneously
Property some of F344 rat, Mus public with ligation mates.
(3) observe after completing the 17～19h of step (2), take out the female F344 rat of 5～6 week old has smart bolt person
As donor, take out female F344 rats more than 2 monthly ages has smart bolt person as receptor.
(4), after completing the 1～2h of step (3), disconnected neck puts to death donor, by operation take out fallopian tube be placed in containing
M2 liquid (the Sigma company of 0.05% (percent by volume) hyaluronidase (Sigma Products, catalog number is H3884)
Product, catalog number is M7167), tear ampulla of uterine tube with tweezers under the microscope, germ cell in company with granular cell is
Together flow out.After the granular cell around germ cell departs from, sucking-off germ cell is placed in M2 liquid washing, is finally placed inM-RECM liquid (Millipore Products, catalog number is MR-166-D), 5%CO2, 37 DEG C cultivate
0.5～1h.
(5), after completing step (4), full, zona pellucida is selected clear and have the germ cell of two protokaryons, then at high power lens
Under, the injection premixed liquid first thrusting injection 1～2pL in germ cell protokaryon with entry needle (contains in injection premixed liquid first
Cas9mRNA, Rag2-sgRNA3 and ss-DNA, wherein the concentration of Cas9mRNA is 40ng/ μ L, and the concentration of Rag2-sgRNA3 is
20ng/ μ L, the concentration of ss-DNA is 10ng/ μ L), then 5%CO2, 37 DEG C cultivate 0.5～1h, collect the germ cell survived.
(6) after completing step (5), take receptor, first take out ovary after anesthesia and connect fallopian tube, the zygote transplation that will survive
Enter fallopian tube, then ovary is put back to abdominal cavity, suture muscles and skin together with fallopian tube.
(7) step (6) filial mice childbirth in latter about 21 days is completed, it is thus achieved that 26 Rag2 genes are intended knocking out rat, number respectively
For R1～R26.
2, Rag2 gene intends knocking out the qualification of rat
(1) treat that 26 Rag2 genes that step 1 obtains intended knocking out Growth in Rats to 7 days, cut tail respectively and extract genome
DNA。
(2) genomic DNA extracted with step (1) respectively is as template, with 5 '-CCTAAGAGATCCTGCCCTTGATAGT-
3 ' and 5 '-GACCGTCCTCCAAAGAGAACACC-3 ' are primer, carry out PCR amplification.Response procedures is: 94 DEG C of degeneration 5min；
94 DEG C of degeneration 30sec, 62 DEG C of annealing 30sec, 72 DEG C extend 55sec, totally 35 circulations；72 DEG C extend 5min；4 DEG C of preservations.
(3) pcr amplification product that step (2) obtains is connected with carrier pBlunt, obtains recombiant plasmid.Then to restructuring
Plasmid checks order.
Test result indicate that (Fig. 2 and Biao 6, Fig. 2 are the order-checking that the F344 rat Rag2 gene knockout head of numbered R9 builds Mus
Result), the Rag2 gene of numbered R1, R2, R3, R9, R11, R25 and R26 intends knocking out rat, and to be accredited as Rag2 gene knockout big
Mus (i.e. F344 rat Rag2 gene knockout head builds Mus), wherein R9 is the Rag2 clpp gene knocking in TGATAGTAA termination codon
Except rat.
The qualification result of table 6 Rag2 knockout rat
| Numbering | Sex | Embryo's background | Qualification result | Sequence (5 '-3 ') |
| R1 | ♀ | F344 | Insert aim sequence | CCTAAGAGATCCTGCCCT{TGATAGTAA}ACTGGAGTCTTT |
| R2 | ♀ | F344 | Insert aim sequence | CCTAAGAGATCCTGCCCT{TGATAGTAA}ACTGGAGTCTTT |
| R3 | ♀ | F344 | Insert aim sequence | CCTAAGAGATCCTGCCCT{TGATAGTAA}ACTGGAGTCTTT |
| R9 | ♂ | F344 | Insert aim sequence | CCTAAGAGATCCTGCCCT{TGATAGTAA}ACTGGAGTCTTT |
| R11 | ♀ | F344 | Insert aim sequence | CCTAAGAGATCCTGCCCT{TGATAGTAA}ACTGGAGTCTTT |
| R25 | ♂ | F344 | Insert aim sequence | CCTAAGAGATCCTGCCCT{TGATAGTAA}ACTGGAGTCTTT |
| R26 | ♂ | F344 | Insert aim sequence | CCTAAGAGATCCTGCCCT{TGATAGTAA}ACTGGAGTCTTT |
Note: be the concrete sequence (i.e. aim sequence) inserted in { }.
Four, the acquisition of Il2rg knockout rat and qualification
1, Il2rg gene intends knocking out the acquisition of rat
(1) with (1) in step 3.
(2) with (2) in step 3.
(3) with (3) in step 3.
(4) with (4) in step 3.
(5), after completing step (4), full, zona pellucida is selected clear and have the germ cell of two protokaryons, then at high power lens
Under, the injection premixed liquid second thrusting injection 1～2pL in germ cell protokaryon with entry needle (contains in injection premixed liquid second
Cas9mRNA and Il2rg-sgRNA6, wherein the concentration of Cas9mRNA is 40ng/ μ L, and the concentration of Il2rg-sgRNA6 is 20ng/ μ
L), then 5%CO2, 37 DEG C cultivate 1h, collect the germ cell survived.
(6) with (6) in step 3.
(7) step (6) filial mice childbirth in latter about 21 days is completed, it is thus achieved that 12 Il2rg genes are intended knocking out rat, number respectively
For L1～L12.
2, Il2rg gene intends knocking out the qualification of rat
(1) treat that 12 Il2rg genes that step 1 obtains intended knocking out Growth in Rats to 7 days, cut tail respectively and extract genome
DNA。
(2) genomic DNA extracted with step (1) respectively is as template, with 5 '-GGCTGTCTGTCTCACTACTGCTT-3 '
It is primer with 5 '-GAACAATCTATAAACTCCAGTGCCT-3 ', carries out PCR amplification.Response procedures is: 94 DEG C of degeneration 5min；
94 DEG C of degeneration 30sec, 62 DEG C of annealing 30sec, 72 DEG C extend 55sec, totally 35 circulations；72 DEG C extend 5min；4 DEG C of preservations.
(3) pcr amplification product that step (2) obtains is connected with carrier pBlunt, obtains recombiant plasmid.Then to restructuring
Plasmid checks order.
Test result indicate that (table 7), the Il2rg gene of numbered L1, L2, L3, L4, L7, L9, L10 and L12 is intended knocking out greatly
Mus is accredited as Il2rg knockout rat.
The qualification result of table 7 Il2rg knockout rat
| Numbering | Sex | Embryo's background | Testing result | Sequence (5 '-3 ') |
| L1 | ♀ | F344 | Δ4 | CACTATGCACTATAGGTAT[GAGA]AGGGGGAGGGGTAG |
| L2 | ♀ | F344 | Δ10 | CCTCACTATGCACTAT[AGGTATGAGA]AGGGGGAGGGGTA |
| L3 | ♀ | F344 | Δ10 | CCTCACTATGCACTAT[AGGTATGAGA]AGGGGGAGGGGTA |
| L4 | ♂ | F344 | Δ10 | CCTCACTATGCACTAT[AGGTATGAGA]AGGGGGAGGGGTA |
| L7 | ♀ | F344 | Δ10 | CCTCACTATGCACTAT[AGGTATGAGA]AGGGGGAGGGGTA |
| L9 | ♂ | F344 | Δ4 | CACTATGCACTATAGGTAT[GAGA]AGGGGGAGGGGTAG |
| L10 | ♂ | F344 | Δ10 | CCTCACTATGCACTAT[AGGTATGAGA]AGGGGGAGGGGTA |
| L12 | ♂ | F344 | Δ3 | ACTATAGGTATG[AGA]AGGGGGAGGGGTAGTACAGGA |
Note: Δ 10 is for deleting 10bp, and Δ 4 is for deleting 4bp, and Δ 3, for deleting 3bp, is the concrete nucleoside deleted in bracket
Acid sequence.
Five, the acquisition of immunodeficient rats and qualification
1, the acquisition of immunodeficient rats
(1) R9 step 3 obtained and female F344 rat copulation, expanding propagation F1 generation, obtain heterozygosis Mus.
(2) L4 step 4 obtained and female F344 rat copulation, expanding propagation F1 generation, obtain heterozygosis Mus.
(3) the mutual copulation of heterozygosis Mus that heterozygosis Mus step (1) obtained and step (2) obtain, obtains Rag2 and Il2rg
F344 rat (the named F344-Rag2 of dual-gene homozygous knockoutem1Il2rgem2/ Vst, is called for short F344RG rat, i.e. immunity and lacks
Fall into rat), the F344 rat of the dual-gene homozygous knockout of Rag2 and Il2rg, female F344 rat and normal male F344 rat.
The F344 rat of the dual-gene homozygous knockout of above-mentioned Rag2 and Il2rg refers on two homologous chromosomes of this rat
Rag2 gene and Il2rg gene all there occurs sudden change.The F344 rat of the dual-gene homozygous knockout of above-mentioned Rag2 and Il2rg refers to
It is that the Rag2 gene of a homologous chromosome in two homologous chromosomes of this rat there occurs sudden change, the dyeing of another homology
The Rag2 gene of body is not undergone mutation, and the Il2rg gene of a homologous chromosome in two homologous chromosomes of this rat
There occurs sudden change, the Il2rg gene of another homologous chromosome is not undergone mutation.
2, the qualification of immunodeficient rats
(1) Rag2 gene
1. treat that female F344 rat that step 1 obtains, normal male F344 rat, dual-gene the isozygotying of Rag2 and Il2rg are struck
The F344 Growth in Rats of the F344 rat removed or the dual-gene homozygous knockout of Rag2 or Il2rg, to 7 days, is cut tail respectively and is extracted genome
DNA。
1. the genomic DNA extracted with step the most respectively is as template, with Rag2-test-F1, Rag2-test-R, Rag2-
AS-MF and Rag2-AS-WR is that primer carries out PCR amplification.Rag2-test-F1, Rag2-test-R, Rag2-AS-MF and Rag2-
The nucleotide sequence of AS-WR refers to table 10.
Response procedures is: 94 DEG C of degeneration 5min；94 DEG C of degeneration 30sec, 62 DEG C of annealing 30sec, 72 DEG C extend 55sec, altogether
35 circulations；72 DEG C extend 5min；4 DEG C of preservations.
3. pcr amplification product step 2. obtained carries out agarose gel electrophoresis, then according to mesh in pcr amplification product
The size of band and table 8 shown in criterion judge genotype: genotype is that wild type (+/+) refers to the two of this rat
Rag2 gene on bar homologous chromosome is not all undergone mutation；Genotype is two that heterozygote (+/-) refers to this rat
The Rag2 gene of a homologous chromosome in homologous chromosome there occurs sudden change, another homologous chromosome Rag2 gene not
Undergoing mutation, genotype is that the Rag2 gene that homozygote (-/-) refers on two homologous chromosomes of this rat all there occurs
Sudden change.
Table 8 Rag2 knockout rat genotype criterion
(2) Il2rg gene
1. treat that female F344 rat that step 1 obtains, normal male F344 rat, dual-gene the isozygotying of Rag2 and Il2rg are struck
The F344 Growth in Rats of the F344 rat removed or the dual-gene homozygous knockout of Rag2 and Il2rg, to 7 days, is cut tail respectively and is extracted genome
DNA。
1. the genomic DNA extracted with step the most respectively as template, with Il2rg-F, Il2rg-R, Il2rg-WR and
Il2rg-MF is that primer carries out PCR amplification.The nucleotide sequence of Il2rg-F, Il2rg-R, Il2rg-WR and Il2rg-MF refers to
Table 10.
Response procedures is: 94 DEG C of degeneration 5min；94 DEG C of degeneration 30sec, 59.5 DEG C of annealing 30sec, 72 DEG C extend 55sec,
Totally 35 circulations；72 DEG C extend 5min；4 DEG C of preservations.
3. pcr amplification product step 2. obtained carries out agarose gel electrophoresis, then according to mesh in pcr amplification product
The size of band and table 9 shown in criterion judge genotype: genotype is that wild type (+/+) refers to the two of this rat
Il2rg gene on bar homologous chromosome is not all undergone mutation；Genotype is two that heterozygote (+/-) refers to this rat
The Il2rg gene of a homologous chromosome in homologous chromosome there occurs the Il2rg gene of sudden change, another homologous chromosome
Not undergoing mutation, genotype is that the Il2rg gene that homozygote (-/-) refers on two homologous chromosomes of this rat all occurs
Sudden change.
Table 9 Il2rg knockout rat genotype criterion
The sequence of table 10 primer
Experimental result is shown in that (A is the qualification result of Rag2 gene to Fig. 3, and B is the qualification result of Il2rg gene, and WT is wild type
(+/+), +/-is heterozygote (+/-),-/-for homozygote (-/-), M is DNA Marker).Authentication method according to above-mentioned offer
And criterion, (3) can identify from step 1 the F344 rat of the dual-gene homozygous knockout of Rag2 and Il2rg, i.e. immunity
Defect rat.
Six, the immunodeficiency degree analyzing of immunodeficient rats
(1) analysis of T cell
Take the peripheral blood of immunodeficient rats, add IOTest Anti-Rat CD3-FITC and CD45RA-PC7, pass through
CD3 in flow cytometry analysis immunodeficient rats+CD45RA-The frequency of T cell.
According to the method described above, immunodeficient rats being replaced with wild-type rats, other step is the most constant, obtains wild type
CD3 in rat+CD45RA-The frequency of T cell.
Test result indicate that, compared with wild-type rats, CD3 in the peripheral blood of immunodeficient rats+CD45RA-T cell
Frequency is the most relatively low, i.e. lacks T cell.
(2) analysis of B cell
Take the peripheral blood of immunodeficient rats, add IOTest Anti-Rat CD3-FITC and CD45RA-PC7, pass through
CD3 in flow cytometry analysis immunodeficient rats-CD45RA+The frequency of B cell.
According to the method described above, immunodeficient rats being replaced with wild-type rats, other step is the most constant, obtains wild type
CD3 in rat-CD45RA+The frequency of B cell.
Test result indicate that (in Fig. 4 left figure, F344RG rat is immunodeficient rats, and F344rat is F344 rat), with
F344 rat is compared, CD3 in the peripheral blood of immunodeficient rats-CD45RA+The frequency of B cell is the most relatively low, i.e. lacks CD3-
CD45RA+B cell.
(3) analysis of NK cell
Take the peripheral blood of immunodeficient rats, add CD161a-APC and IOTest Anti-Rat CD3-FITC, pass through
CD3 in flow cytometry analysis immunodeficient rats-CD161a+The frequency of NK cell.
According to the method described above, immunodeficient rats being replaced with wild-type rats, other step is the most constant, obtains wild type
CD3 in rat-CD161a+The frequency of NK cell.
Test result indicate that (in Fig. 4 right figure, F344RG rat is immunodeficient rats, and F344rat is F344 rat), with
F344 rat is compared, CD3 in the peripheral blood of immunodeficient rats-CD161a+The frequency of NK cell is the most relatively low, i.e. lacks CD3-
CD161a+NK cell.
(4) analysis of IgM, IgG and IgA
Take the peripheral blood of immunodeficient rats, according to Rat IgG, IgAand IgM ELISA quantitation kits
Operating procedure analyze the content of IgG, IgA and IgM in immunodeficient rats.
Experimental result is shown in Fig. 5 (FRG is immunodeficient rats, and F344 is F344 rat).Result shows, with F344 rat phase
Ratio, is substantially not detectable IgG, IgA and IgM in the peripheral blood of immunodeficient rats.
The above results shows, the immunodeficient rats that step 5 obtains meets hyperimmunization defect rat feature.
Claims (10)
1. for building the product of immunodeficient animals model, for product first or product second or product third；
Described product first is by the material of the Rag2 gene of animal for knocking out with for knocking out the Il2rg gene of the animal that sets out
Material composition；
The material of described product second Rag2 gene expression of animal by for suppression and the Il2rg of the animal that sets out for suppression
The material composition of gene expression；
Described product third is reduced by the Rag2 protein active of animal for promote or the material lost and setting out for promoting
The material composition that the Il2rg protein active of animal reduces or loses.
2. product as claimed in claim 1, it is characterised in that the animal that sets out described in: is rat.
3. product as claimed in claim 1 or 2, it is characterised in that:
Described " for knocking out the material of the Rag2 gene of the animal that sets out " includes sgRNAa；Described sgRNAa be sgRNA1,
SgRNA2, sgRNA3 or sgRNA4；The genomic DNA target sequence of described rat that described sgRNA1 identifies is 5 '-
CCAGTAGGGCAGGATCTCTTAGG-3’；The genomic DNA target sequence of described rat that described sgRNA2 identifies is 5 '-
TTTTCTTTGGCCAAAAAGGCTGG-3’；The genomic DNA target sequence of described rat that described sgRNA3 identifies is 5 '-
CCTAAGAGATCCTGCCCTACTGG-3’；The genomic DNA target sequence of described rat that described sgRNA4 identifies is 5 '-
ATCTCTTAGGCCAGCCTTTTTGG-3’；
Described " for knocking out the material of the Il2rg gene of the animal that sets out " includes sgRNAb；Described sgRNAb be sgRNA5,
SgRNA6, sgRNA7 or sgRNA8；The genomic DNA target sequence of described rat that described sgRNA5 identifies is 5 '-
TAGTGCATAGTGAGGTTGGTCGG-3’；The described rat genomic dna target sequence that described sgRNA6 identifies is 5 '-
GGTAGTACAGGAAGAAGAGAAGG-3’；The genomic DNA target sequence of described rat that described sgRNA7 identifies is 5 '-
AGAAGGGGGAGGGGTAGTACAGG-3’；The described rat genomic dna target sequence that described sgRNA8 identifies is 5 '-
CCAACCTCACTATGCACTATAGG-3’。
4. product as claimed in claim 3, it is characterised in that: the sequence of described sgRNA3 is as shown in the sequence 2 in sequence table；
The sequence of described sgRNA6 is as shown in the sequence 3 in sequence table.
5. the product as described in Claims 1-4 is arbitrary, it is characterised in that:
Described " for knocking out the material of the Rag2 gene of the animal that sets out " also includes the RNA encoding Cas9 albumen；
Described " for knocking out the material of the Il2rg gene of the animal that sets out " also includes the RNA encoding Cas9 albumen.
6. the product as described in claim 1 to 5 is arbitrary, it is characterised in that:
Described " for knocking out the material of the Rag2 gene of the animal that sets out " also includes ss-DNA；Described ss-DNA includes section successively
A, section B, section C, section D and section E；
Described section A is upstream homology arm, a length of 30～50bp, by animal Rag2 gene described in the target of sgRNA3
Some nucleotide composition of the upstream of sequence；Described section B is the target sequence cog region first of described sgRNA3；Described section C is N
Individual termination codon, N is natural number and is more than or equal to 1；Described section D is the target sequence cog region second of described sgRNA3；Described
Section B and described section C forms the target sequence cog region of described sgRNA3；Described section E is downstream homology arm, a length of 30～
50bp, some nucleotide in the downstream of the target sequence of sgRNA3 described in the Rag2 gene by the animal that sets out form.
7. the product as described in claim 1 to 6 is arbitrary, it is characterised in that:
Described " for knocking out the material of the Rag2 gene of the animal that sets out " is the restructuring matter containing the DNA encoding described sgRNA3
Grain, recombiant plasmid containing the DNA encoding described sgRNA1, containing encoding the recombiant plasmid of DNA of described sgRNA2 or containing
Encode the recombiant plasmid of the DNA of described sgRNA4；
Described " for knocking out the material of the Il2rg gene of the animal that sets out " is for containing the weight containing the DNA encoding described sgRNA6
Group plasmid, containing encode described sgRNA5 DNA recombiant plasmid, containing encode described sgRNA7 DNA recombiant plasmid or
Recombiant plasmid containing the DNA encoding described sgRNA8.
8. arbitrary described product application in preparing immunodeficient animals model in claim 1 to 7.
9. the method building immunodeficient animals model, comprises the steps:
(1) knock out the Rag2 gene of the animal that sets out, obtain Rag2 Gene Knock-Out Animal Model, by described Rag2 Gene Knock-Out Animal Model and institute
State the animals that sets out, obtain offspring animal；
(2) knock out the Il2rg gene of the animal that sets out, obtain Il2rg Gene Knock-Out Animal Model, by described Il2rg Gene Knock-Out Animal Model
With the described animals that sets out, obtain offspring animal；
(3) step (1) obtains offspring animal and the offspring animal copulation that step (2) obtains, it is thus achieved that described Rag2 and Il2rg is double
The animal that gene pure knocks out.
10. method as claimed in claim 9, it is characterised in that the animal that sets out described in: is rat.
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