CN107760720A - The structure of Reconstruction in Sever Combined Immunodeciency animal model and application - Google Patents
The structure of Reconstruction in Sever Combined Immunodeciency animal model and application Download PDFInfo
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- CN107760720A CN107760720A CN201610667884.5A CN201610667884A CN107760720A CN 107760720 A CN107760720 A CN 107760720A CN 201610667884 A CN201610667884 A CN 201610667884A CN 107760720 A CN107760720 A CN 107760720A
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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
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/89—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microinjection
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New breeds of animals
- A01K67/027—New breeds of vertebrates
- A01K67/0275—Genetically modified vertebrates, e.g. transgenic
- A01K67/0276—Knockout animals
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
- C07K14/7155—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for interleukins [IL]
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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
- 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
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/07—Animals genetically altered by homologous recombination
- A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; 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; CARE OF BIRDS, FISHES, INSECTS; 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
Abstract
The present invention relates to a kind of structure and related application of Reconstruction in Sever Combined Immunodeciency animal model, the construction method by CRISPR/Cas9 technology targeting knock out encoding animals T, B cell Rag2 genes and participate in the Il2rg genes of Codocyte factor acceptor, realize the structure of the higher animal model of immune deficiency degree.
Description
Technical field
The present invention relates to a kind of construction method of Reconstruction in Sever Combined Immunodeciency animal model and application.
Background technology
The animal model for establishing immune deficiency is commonly used to the screening of tumor, the examination such as treatment method of cancer
Test.There is great effect to the cure mechanism of research tumour medicine and Other diseases using the animal model of immune deficiency,
So as to realize the treatment and prevention control to disease, so as to improve cure rate.
Researcher has found mouse T cell dysfunction after Foxn1 gene mutations within 1966, it is impossible to produces cell-specific and exempts from
Epidemic disease is reacted, and thus opens the beginning of spontaneous immunodeficient animals research.The Beige mouse of B cell defect, T cell therewith
And B cell joint defect SCID mice is also found and is used widely in the multiple fields of biomedical research in succession.To the greatest extent
The immunodeficient mouse formed after pipe T cell and/or B cell dysfunction achieves many great in biomedical research
Achievement in research, but the innate immune response reaction of its mediation such as the NK cells of remaining, macrophage, neutrophil leucocyte in vivo is still
Can so immunological rejection be produced to graft, be survived in vivo at it so as to influence tumour.In recent years, researcher has carried out many
Effort, it is intended to find the stronger laboratory mice of higher, the immune pardon of immune deficiency degree to substitute nude mice or SCID mice.
BALB/c mouse is that the research fields such as current immunology, microbiology are conventional because its biological characteristics is clear and definite
Inbred mouse.Traditional immunodeficient mouse strain BALB/c-nu/ can be cultivated into after nude channel genes BALB/c mouses
Nu, such mouse T cell defect, thus the cellular immune function degradation [6] of T cell mediation.But such mouse B cell and
Other immunocytes are normal, and these cell-mediated immune responses can often influence animal model replication effect.Such as human tumor mould
When type replicates, some Human Tumor Models are difficult to establish in BALB/c-nu/nu nude mices, in addition to tumour cell oncogenicity,
BALB/c-nu/nu nude mice immunocyte states are also to influence one of key factor that tumor model replicates.In order to overcome above disadvantage
End, it is necessary to work out that immune deficiency degree is higher, immune pardon is stronger and genetic background is from the dynamic of BALB/c mouse
Thing is used for the area researches such as immunology, microbiology, oncology.
Based on this purpose, a kind of higher animal model of immune deficiency degree has been prepared in present invention research, is advantageous to each
The structure of kind disease model, and further it is beneficial to the screening of medicine.
The content of the invention
In order to solve to establish the technological deficiency that the immunodeficient mouse strain cycle is long, strain is impure in the prior art, and
Break through the technology blockage of the U.S., Japan to China.Present invention research is prepared for that a kind of immune deficiency degree is higher, immune pardon
The construction method of stronger animal model and application, so as to realize the preparation of various disease models, further it is beneficial to control
Treatment or the screening of prophylactic agent.
One aspect of the present invention is related to a kind of construction method of the animal model of Reconstruction in Sever Combined Immunodeciency, and this method is related to pair
The targeting knock out of animal T, the Rag2 genes of B cell and participation Codocyte factor acceptor Il2rg genes.
Method described in any one of first aspect present invention, wherein described targeting knock out is by CRISPR/Cas9 skills
Art carries out the targeting knock out of animal T, the Rag2 genes of B cell and participation Codocyte factor acceptor Il2rg genes.
Method described in any one of first aspect present invention, wherein described targeting knock out be related to for Il2rg genes and
Rag2 genes design and synthesize sgRNA.
Method described in any one of first aspect present invention, wherein distinguishing for the sgRNA of Il2rg genes and Rag2 genes
It is to be directed to IL2rg exon 1s and Rag2 Exon 2s.
Method described in any one of first aspect present invention, wherein the structure of the sgRNA is specifically as shown in table 1.
Method described in any one of first aspect present invention, wherein described animal is mouse.
Second aspect of the present invention is related to sgRNA for Il2rg and Rag2 genes and moved preparing Reconstruction in Sever Combined Immunodeciency
Application in thing model.
Application described in any one of second aspect of the present invention, wherein described for Il2rg genes and the sgRNA of Rag2 genes
It is to be directed to Il2rg exon 1s and Rag2 Exon 2s respectively.
Application described in any one of second aspect of the present invention, wherein the structure of the sgRNA is specifically as shown in table 1.
Application described in any one of second aspect of the present invention, wherein described animal is mouse.
Third aspect present invention is related to a kind of method for building Reconstruction in Sever Combined Immunodeciency Mice, comprises the following steps：
(1) oligonucleotide synthesis and plasmid construction；
(2) in-vitro transcription；
(3) mRNA microinjections and fertilized oocyte；
(4) foundation of knock out mice strain and the identification of genotype and phenotype.
Oligonucleotide synthesis in method described in any one of third aspect present invention, wherein step (1) is to be directed to Il2rg
Exon 1 and Rag2 Exon 2s design and synthesize sgRNA.
Method described in any one of third aspect present invention, wherein specific for the sgRNA structures of Il2rg and Rag2 genes
As shown in table 1.
Method described in any one of third aspect present invention, through annealing after the sgRNA synthesis in step (1), it is connected into pX330
Carrier, build the pX330 plasmids containing sgRNA.
Method described in any one of third aspect present invention, plasmid is obtained in step (1) after sequencing shows that structure is correct,
Clone sgRNA and Cas9 simultaneously carries out in-vitro transcription under the effect of T7 promoters, and transcription product is single band after electroresis appraisal.
Method described in any one of third aspect present invention, step (3) is related to obtains fertilized egg cell at fallopian tubal position,
The 20 μ g/ μ L Rag2-sgRNA transcribed and 10 μ g/ μ L Il2rg-sgRNA and 5 μ g/ μ L Cas9mRNA are taken to mix respectively,
It is expelled to Eppendorf NK2 microinjection instruments in fertilized egg cell's kytoplasm.
Method described in any one of third aspect present invention, step (3) are further related to fertilized egg cell in 37 DEG C of 5%CO2
Continue 2~3h of culture in incubator, take growth conditions good, it is defeated to ICR false pregnancy mouse to be developed to 2 cell stage fertilized oocytes
Oviduct position.
Method described in any one of third aspect present invention, step (4) are related to the F0 of mutation is small with wild type for mouse
Mouse mates, and obtains F1 generation hybrid mice, and F1 generation hybrid mice is further mutually handed over to the homozygote for obtaining dual-gene mutation
Mouse.
Method described in any one of third aspect present invention, the genotype detection in step (4) refer to take out raw 2 weeks or so
F0 for mouse tail tissue, mouse gene group DNA is extracted with kit after cracking, PCR method clone F0 for mouse Rag2 and
Il2rg genes.PCR reaction conditions are：94 DEG C of pre-degenerations 3min, 94 DEG C of denaturation 30s, 60 DEG C of annealing 40s, 65 DEG C of extension 40s, altogether
Carry out 35 circulations.Circulation terminate after with 65 DEG C extension 6min.Take 10 μ L PCR primers to carry out electrophoresis, surveyed after size is correct
Sequence.
Method described in any one of third aspect present invention, the Phenotypic examination in step (4) is to take 50 μ by cutting tail method
L F0 are eluted with FACS buffer solution after erythrocyte cracked liquid cracking for mouse peripheral blood, add 1:The FITC-CD3 of 1000 dilutions,
PE-NKp46 and APC-B220 antibody at room temperature lucifuge dyes 30min.Upper machine testing mouse peripheral blood is white after FACS buffer solution elution
CD3, B220 and NKp46 positive cell situation in cell.
The advantageous effects of the present invention：Traditional gene knockout mainly using homologous recombination principle by insertion mutation and
Gene targeting loses target gene function, but these method fabrication cycles are grown, can only be in some specialized laboratory's ability
Complete.The present invention is specifically bound using the sgRNA and DNA of homology, played by sgRNA by CRISPR/Cas9 technologies
Targeting, guiding endonuclease Cas9 is cut at target spot, so as to reach the purpose of genetic modification.The technology of the present invention
Rag2 and II2rg gene sequencings in scheme show, the two genes close to thereon, downstream molecules, and overlap,
Large fragment deletion mutation is such as carried out to it will influence whether upstream and downstream developed by molecule.Accordingly, the present invention with Il2rg genes the 1st outside
Aobvious son and Rag2 Exon 2s are that target spot carries out point mutation, and this mutation can not only cause Rag2 and II2rg gene functions
Inactivation, and to thereon, downstream gene expression has no significant effect.Compared with traditional gene knockout method, technology of the invention
Scheme has the characteristics of quick, reliable and knockout efficiency high.The present invention has knocked out two different genes by a step, after knockout
T cell, B cell and NK cells substantially completely lack, and obstacle also occurs for cell factor receptor body function, and inoculation human breast carcinoma is thin
Growth of tumour cell is good after born of the same parents system, shows the biological characteristics similar to traditional immunization deficient animals.It is thus obtained to exempt from
Higher, the immune pardon of epidemic disease defect level is stronger and genetic background can be widely used for being immunized from the animal of BALB/c mouse
The area researches such as, microbiology, oncology.
Brief description of the drawings
Fig. 1 sgRNA sequences Design schematic diagrames.
The genotype identification of Fig. 2 knock out mice, wherein Fig. 2A identify that Fig. 2 B are target gene for the PCR of mutant mice
Sequencing result.
The phenotypic evaluation of Fig. 3 knock out mice, wherein Fig. 3 A are FCM analysis T cell, B cell and NK cell numbers
Amount；Fig. 3 B are that knock out mice is inoculated with tumour growth situation after SKBR-2HL cells.
Embodiment
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.It is unreceipted specific in embodiment
Condition person, the condition suggested according to normal condition or manufacturer are carried out.Agents useful for same or the unreceipted production firm person of instrument, it is
The conventional products of acquisition purchased in market can be passed through.
The oligonucleotide synthesis of embodiment 1 and plasmid construction
Rag2 the and Il2rg gene orders reported according to Genbank, respectively for the 2nd extron of Rag2 genes and
The 1st extron sgRNA of the design with targeting of Il2rg genes, sgRNA sequences are shown in Table 1.Through annealing after sgRNA synthesis,
PX330 carriers are connected into, build the pX330 plasmids containing sgRNA.Plasmid is small to be carried and shows that plasmid construction is correct after sequencing.
After sgRNA and Cas9 clones, in-vitro transcription is carried out under the effect of T7 promoters, transcription product is single bar after electroresis appraisal
Band, and OD260/280>1.9, OD260/230>2.3.
The sgRNA target spots of table 1 and oligonucleotide sequence
| SgRNA titles | SgRNA oligonucleotide sequences |
| Rag2-up | 5’-caccggcctaagagatcctgtcctac-3’ |
| Rag2-down | 5’-aaacgtaggacaggatctcttaggcc-3’ |
| Il2rg-up | 5’-caccggagcagctgaaggactaaga-3’ |
| Il2rg-down | 5’-aaactcttagtccttcagctgctcc-3’ |
The in-vitro transcription of embodiment 2, mRNA microinjections and fertilized oocyte
Using pX330-Rag2-sgRNA and pX330-Il2rg-sgRNA plasmids as template, PCR methods clone promoter containing T7
Rag2-sgRNA and Il2rg-sgRNA.Same procedure clones Cas9 genes, reclaims after purification, DNA, which is dissolved in DEPC water, to be preserved.
200ng Rag2-sgRNA, Il2rg-sgRNA and Cas9 product after purification is taken to carry out in-vitro transcription, quantitative rear -80 DEG C of guarantors respectively
Deposit.
8 week old BALB/c mouses male is mated with female mice using the previous day, and the female mice solution for seeing bolt is taken after 24h
Cut open, fertilized egg cell is obtained at fallopian tubal position.The 20 μ g/ μ L Rag2-sgRNA transcribed and 10 μ g/ μ L are taken respectively
Il2rg-sgRNA and 5 μ g/ μ L Cas9mRNA mixing, fertilized egg cell born of the same parents are expelled to Eppendorf NK2 microinjection instruments
In matter.Fertilized egg cell continues 2~3h of culture in 37 DEG C of 5%CO2 incubators, takes growth shape body good, is developed to 2 cell stages
Fertilized oocyte is to ICR false pregnancy mouse fallopian tubals position.
Embodiment 3:The foundation of knock out mice strain and genotype identification
Raw 2 weeks or so F0 is taken out for mouse tail tissue, mouse gene group DNA, PCR method are extracted with kit after cracking
F0 is cloned for mouse Rag2 and Il2rg gene.PCR reaction conditions are：94 DEG C of pre-degeneration 3min, 94 DEG C of denaturation 30s, 60 DEG C are annealed
40s, 65 DEG C of extension 40s, carries out 35 circulations altogether.Circulation terminate after with 65 DEG C extension 6min.10 μ L PCR primers are taken to carry out electricity
Swimming, is sequenced after size is correct.The F0 of mutation mates for mouse with wild-type mice, obtains F1 generation hybrid mice, F1 generation
Hybrid mice mutually hands over the homozygote mouse that can obtain dual-gene mutation to collect 740 pieces of BALB/c mouse embryo number altogether, wherein
609 pieces are fertilized egg cell, there is 249 pieces of survivals after microinjection, and preparation F0 builds mouse for head after being transplanted to ICR acceptor mouse.Sequencing
After take F0 to mate with wild type BALB/c mouse for mutant mice to obtain F1 generation mouse, the F1 generation mouse of mutation obtains after mutually handing over
The F2 of the dual-gene mutation of Rag2 and Il2rg is for homozygote mouse.F0, F1 and F2 are born for mouse after about 2w, clip Mouse Tail-tip
3mm~5mm organizes to be used for extracting genome DNA.Take 1 μ L DNA to enter performing PCR reaction for template, sent out after 1% gel electrophoresis identification
Existing Rag2 genetic fragments size is 821bp, and Il2rg clip sizes are 686bp, is consistent (Fig. 2A) with expected size.Sequencing result
Show that F0, F1 and F2 there are two saltant types for mouse IL2rg genes, be 10bp and 11bp deletion mutation respectively；And Rag2 bases
Because of an only saltant type, the deletion mutation (Fig. 2 B) for being 8bp.
The knock out mice phenotypic evaluation of embodiment 3
50 μ L F0 are taken to be eluted for mouse peripheral blood, erythrocyte cracked liquid after cracking with FACS buffer solution by cutting tail method,
Add 1:FITC-CD3, PE-NKp46 and APC-B220 antibody at room temperature lucifuge dyeing 30min of 1000 dilutions.FACS buffer solution is washed
The positive cell situation of CD3, B220 and NKp46 in machine testing mouse peripheral blood leucocyte are gone up after de-.
Compared with normal wild type mouse, knock out mice CD3+T cells, B220+B cells and NKp46+NK cell numbers
Amount declines obvious (Fig. 3 A), shows that CRISPR/Cas9 technologies enter after edlin to have influence on the two bases to Rag2 and Il2rg genes
Because of the biological function of coding, T cell, B cell and NK cell quantities is caused to reduce.Further pass through the tumour transplatation of duplicator source
Model verifies knock out mice biological characteristics, and human breast cancer cell SKBR-2HL is inoculated with 3 knock out mice respectively, 1
In mouse tumor growth, and as time went on, gross tumor volume constantly increases (Fig. 3 B) visual tumors cell after week.With reference to streaming
Testing result, it is seen that not only T cell, B cell and NK cells lack Rag2 and Il2rg knock out mice substantially, and it is exempted from
Epidemic disease functional defect, graft can breed in vivo at it.
Claims (10)
1. a kind of construction method of the animal model of Reconstruction in Sever Combined Immunodeciency, this method are related to the Rag2 to animal T, B cell
The targeting knock out of the Il2rg genes of gene and participation Codocyte factor acceptor.
2. the method described in claim 1, wherein described targeting knock out is to carry out animal T, B by CRISPR/Cas9 technologies
The targeting knock out of the Rag2 genes of cell and the Il2rg genes of participation Codocyte factor acceptor.
3. the method described in claim 2, designed simultaneously for Il2rg genes and Rag2 genes wherein described targeting knock out is related to
Synthesize sgRNA.
4. the method described in claim 3, wherein the sgRNA for Il2rg genes and Rag2 genes is directed to respectively
Il2rg exon 1s and Rag2 Exon 2s.
5. the method described in claim 4, wherein the structure of the sgRNA is specifically as shown in table 1.
6. purposes of the animal model that the method described in claim any one of 1-5 prepares in disease model is prepared.
7. it is directed to applications of the sgRNA of Il2rg and Rag2 genes in Reconstruction in Sever Combined Immunodeciency animal model is prepared.
8. the application described in claim 7, wherein the sgRNA for Il2rg genes and Rag2 genes is directed to respectively
IL2rg exon 1s and Rag2 Exon 2s.
9. the application described in claim 8, wherein the structure of the sgRNA is specifically as shown in table 1.
10. method or application described in any one of foregoing claim, wherein described animal is mouse.
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