CN109868285A - A kind of construction method of immunodeficient rats animal model and application - Google Patents

Abstract

The present invention relates to a kind of construction method of immunodeficient rats animal model and applications, respectively construct rat Rag1 Exon 2, the 3rd exon of Rag2 gene, Il2rg exon 1 sgRNA expression vector, it is expressed by being transcribed in vitro, obtain while having knocked out the rat of Rag1 gene and Rag2 gene, the rat that Il2rg gene is knocked is obtained, hybridization obtains the rat that Rag1, Rag2 and Il2rg gene are knocked.The rat SD-RG of the method for the present invention building has the phenotype of immune deficiency, facilitates the transplanting for carrying out kinds of tumors, and support its fast-growth, can be widely applied to the multiple fields such as humanized animal's model, stem cell and tumor research.

Description

A kind of construction method of immunodeficient rats animal model and application

Technical field

The invention belongs to animal genetic engineering and genetic modification field, more particularly, to a kind of based on gene Knockout The construction method of immunodeficient rats animal model and application.

Background technique

Source of people immune system has been rebuild in humanized animal's model, can preferably simulate human tumor microenvironment, Tumour is increasingly taken seriously in body research.Currently, humanized mouse model has been applied in the sieve of new anticancer drug The exploration various aspects of choosing and treatment method (such as cell therapy and Antybody therapy).Recombination active gene is knocked out in mouse (Recombination Activation Genes, Rag1 or Rag2) can result in the immune globulin of T cell receptor and B cell The obstacle of the genetic recombination in white encoding gene region, cause the severe immune deficiency phenotype Il2rg gene of mouse encode it is a kind of across Memebrane protein is the common subunit of several interleukin-2-receptor compounds, plays an important role in immune cell differentiation and function.It knocks out Il2rg gene can remove the intracorporal NK cell of mouse, therefore have important work in terms of establishing immunodeficient animals model With.From tail vein injection foetal liver cells, or transplanting hematopoiesis while by transplanting human fetal thymus, lymph node, spleen Stem cell (hematopoietic stem cell, HSC), can establish humanized mouse model.Known Rag and Il2rg gene The transplanting efficiency that Strains of Mouse improves human tissue and cell is knocked out, success has differentiated making for different subtype in Mice Body Haemocyte, including T, bone-marrow-derived lymphocyte, the macrophage of medullary system, Dendritic Cells and blood platelet, red blood cell etc..

Although humanized mouse model has obtained wide application in research tumour medicine, its limitation is also Significantly.Mouse it is small, blood volume is few；And limited by ethics, the size of tumour cannot surpass in PDX model Cross 1/10th of the weight of animals.If using the large mammal being more nearly with the mankind even non-human primate, Research cost will be unable to effectively control again, therefore also unrealistic.The figure (weight) of rat is 10 times of mouse, so being capable of providing More samples, and the physiological structure of rat than mouse closer to the mankind.Therefore, rat model is in correlative study than small Mouse is with greater advantage.

Summary of the invention

An object of the present invention is to provide construction method and the application of a kind of immunodeficient rats animal model.The present invention Outside by CRISPR/Cas9 technology building rat Rag1 Exon 2, the 3rd exon of Rag2 gene, Il2rg gene the 1st The expression vector for showing the sgRNA of son is expressed by being transcribed in vitro, and is obtained respectively while having been knocked out Rag1 gene and Rag2 gene Then the rat that rat and Il2rg gene are knocked then is obtained Rag1, Rag2 and Il2rg gene by hybridization and is struck The rat removed.

The invention proposes a kind of construction methods of immunodeficient rats animal model, comprising the following steps:

(1) expression vector, the Rag2 gene of the sgRNA of selectively targeted rat Rag1 Exon 2 are constructed respectively The expression vector of the expression vector of the sgRNA of 3rd exon, the sgRNA of Il2rg exon 1；

(2) it is obtained outside sgRNA, Rag2 gene the 3rd of rat Rag1 Exon 2 by the way that expression respectively is transcribed in vitro Show sgRNA the and Cas9 mRNA of sgRNA, Il2rg exon 1 of son；

(3) by the sgRNA and Cas9mRNA of the 3rd exon of sgRNA, Rag2 gene of rat Rag1 Exon 2 Respectively after purification, mixing, is injected into rat fertilized egg cell, is implanted into rat fallopian tubal, obtains while having knocked out Rag1 The rat of gene and Rag2 gene；

Respectively after purification by sgRNA the and Cas9 mRNA of rat Il2rg exon 1, mixing is injected into rat It in fertilized egg cell, is implanted into rat fallopian tubal, obtains the rat that Il2rg gene is knocked；

(4) by obtaining step (3) rat of knockout Rag1 and Rag2 gene and Il2rg gene are knocked while Rat is hybridized, and is obtained the rat that Rag1, Rag2 and Il2rg gene are knocked and is named as immunodeficient rats animal mould Type SD-RG.

Wherein, the nucleotide sequence of the rat Rag1 Exon 2 is as shown in SEQ ID NO.4, it may be assumed that 5 '- gtagcttc gccaaaatgg ctgtcccctt gccatctacc ctgagactca gttctgcacc tgatgaaatt cagcacccgc acatcaaatt ttccgagtgg aaatttaagc tgtttagggt gagatccttt-3’。

Wherein, the nucleotide sequence of the 3rd exon of rat Rag2 gene is as shown in SEQ ID NO.5, it may be assumed that 5 '- ataaaaga cctactcaca atcaaaaaat gtccctgcag atggttacag tgggtcataa catagcctta attcaaccag gcttctcact gatgaatttt gatggtcaag tttttttctt tggccaaaaa ggctggccta agagatcctg ccctactgga-3’。

Wherein, the nucleotide sequence of the rat Il2rg exon 1 is as shown in SEQ ID NO.6, it may be assumed that 5 '- atgttg aaaccattat tgccatctag atccttctta ctccttcagc tgcttctgct gagggtaggg tggagctcca aggtcctcat gtccagtggg aatgaagaca ccaaatctg-3’。

Preferably, in the step (1), using CRISPR/Cas9 technology, for rat Rag1 Exon 2, greatly The 3rd exon of mouse Rag2 gene, rat Il2rg exon 1 separately design targeting DNA sequence dna, construct rat respectively The expression vector, big of the expression vector of the sgRNA of Rag1 Exon 2, the sgRNA of the 3rd exon of rat Rag2 gene The expression vector of the sgRNA of mouse Il2rg exon 1；Targeting DNA sequence dna, that is, the sgRNA is respectively SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 sequence.

In the step (2), by transcribing Cas9 mRNA respectively obtained, rat Rag1 Exon 2 SgRNA, the sgRNA of the 3rd exon of rat Rag2 gene, rat Il2rg exon 1 sgRNA adsorbed respectively Cas9 mRNA, each sgRNA after purification is utilized respectively spectrophotometric determination concentration by column purification.

In the present invention, it is preferable that the final concentration of 20-100ng/ μ l of Cas9 mRNA after purification, rat after purification SgRNA, the rat Il2rg exon 1 of the sgRNA of Rag1 Exon 2, the 3rd exon of rat Rag2 gene The final concentration of sgRNA be respectively 10-50ng/ μ l.

The present invention, which innovates, to be proposed to carry out absorption column purification after transcription step, to avoid DNA and protein contamination, is significantly mentioned The high efficiency of subsequent fertilized eggs injection.And preferably conform to Cas9 mRNA, sgRNA final concentration requirement after purification.

In the step (3), when the sgRNA is mixed with Cas9 mRNA, the mass ratio of the two is 1:2.That is, when mixing Mass ratio of the sum of the sgRNA of the 3rd exon of sgRNA and Rag2 gene of rat Rag1 Exon 2 with Cas9mRNA For 1:2.The mass ratio of sgRNA and the Cas9 mRNA of rat Il2rg exon 1 are 1:2 when mixing.

The invention also provides a kind of expression vectors, respectively containing outside the selectively targeted rat Rag1 gene the 2nd Aobvious son, the 3rd exon of Rag2 gene, Il2rg exon 1 sgRNA DNA sequence dna carrier.Preferably, the table Contain pX330 up to carrier, the targeting DNA that the expression vector contains carrier pX330 and connect with the carrier pX330 is described Targeting DNA sequence dna is respectively SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 sequence.

The invention also provides single strain oligonucleotide sequence, the sequence is respectively as follows:

A pair of positive and negative single-stranded SEQ ID NO.7 and SEQ ID NO.8, it is annealed to form SEQ ID NO.1；

A pair of positive and negative single-stranded SEQ ID NO.9 and SEQ ID NO.10, it is annealed to form SEQ ID NO.2；

A pair of positive and negative single-stranded SEQ ID NO.11 and SEQ ID NO.12, it is annealed to form SEQ ID NO.3.

Wherein, SEQ ID NO.7 and SEQ the ID NO.8 is directed to the sgRNA target spot of Rag1 Exon 2.SEQ ID NO.9 and SEQ ID NO.10 is directed to the sgRNA target spot of the 3rd exon of Rag2 gene.SEQ ID NO.11 and SEQ ID NO.12 is directed to the sgRNA target spot of Il2rg exon 1.

Wherein, aobvious outside selectively targeted Rag1 Exon 2, the 3rd exon of Rag2 gene and Il2rg gene the 1st The expression vector of the sgRNA of son, is made of carrier pX330 and the targeting DNA connecting with carrier pX330, and targeting DNA sequence dna is SgRNA is SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 sequence.

It is described to contain sgRNA and Cas9 mRNA into the cell the invention also provides a kind of non-human mammalian cell. Wherein, the sgRNA is respectively sgRNA, such as SEQ ID of the rat Rag1 Exon 2 as shown in SEQ ID NO.1 The sgRNA of the 3rd exon of rat Rag2 gene and the rat Il2rg gene as shown in SEQ ID NO.3 shown in NO.2 The sgRNA of 1 exon.

The invention also provides be respectively used to knock out the CRISPR-Cas9 expression system of rat Rag1, Rag2, Il2rg gene System, respectively containing outside selectively targeted rat Rag1 Exon 2, the 3rd exon of Rag2 gene and Il2rg gene the 1st Show the expression vector of the sgRNA of son.PX330 carrier can express Cas9 mRNA.The expression vector includes pX330 carrier.

The present invention also proposes the immunodeficient rats animal model constructed by the method for the present invention, expression vector, inhuman Class mammalian cell is screening or is preparing the application in immunoregulation medicament or screening anti-tumor medicine.

The invention also provides outside rat Rag1 Exon 2, the 3rd exon of Rag2 gene, Il2rg gene the 1st Aobvious son is preparing the purposes in immunodeficient rats animal model.The nucleotide sequence of the rat Rag1 Exon 2 As shown in SEQ ID NO.4；The nucleotide sequence of the 3rd exon of rat Rag2 gene is as shown in SEQ ID NO.5；It is described The nucleotide sequence of rat Il2rg exon 1 is as shown in SEQ ID NO.6.

The invention also provides rat Rag1 Exon 2, the 3rd exon of rat Rag2 gene, Il2rg genes The sgRNA of 1 exon is knocking out rat Rag1 Exon 2, the 3rd exon of rat Rag2 gene, rat Il2rg gene Application in 1st exon.The nucleotide sequence of the rat Rag1 Exon 2 is as shown in SEQ ID NO.4；It is described The nucleotide sequence of the 3rd exon of rat Rag2 gene is as shown in SEQ ID NO.5；The nucleotide of the rat Il2rg gene Sequence is as shown in SEQ ID NO.6.

The CRISPR-Cas9 that the invention also provides a kind of for knocking out rat Rag1, Rag2 and Il2rg gene expresses system System, the expression system contain the sgRNA of selectively targeted rat Rag1 Exon 2, rat Rag2 gene the 3rd respectively The expression vector of the sgRNA of exon, the sgRNA of rat Il2rg exon 1.The sgRNA is respectively SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 sequence.

The invention also provides a kind of immunodeficient rats animal model SD-RG constructed by the method for the present invention, features Can be verified by following Testing index: (1) DNA of SD-RG rat has corresponding mutant nucleotide sequence；(2) SD-RG rat immunity group Knit middle shortage T, B and NK cell；(3) growth rate of the subcutaneous transplantation tumour in SD-RG rat is more than other animals such as mouse Model.

Immunodeficient rats animal model and its construction method proposed by the present invention and application, pass through CRISPR/Cas9 skill Art obtains the rat (SD-RG immunodeficient rats animal model) of Rag1, Rag2, Il2rg gene knockout, with prior art tradition Knockout technology (allophenic mice) compares, the present invention it is more efficient, the time cycle is shorter, and rat have mouse do not have it is excellent Gesture.The SD-RG rat severe immune deficiency that the present invention constructs lacks T, B and NK cell, the growth of transplantation tumor in immuning tissue Rate provides a kind of reliable animal mould significantly beyond other animal models such as mouse, for immune and tumour related fields research Type.The present invention can not only promote the basic research of immune system related fields, but also can grind for the new drug of the diseases such as tumour Hair provides high-caliber animal model.

In the present invention, Rag1 and Rag2 are in same chromosome, while after knocking out, then hybridize with Il2rg, in this way can be simultaneously The immunodeficiency type rat of different genotype is obtained, by the method for the invention, available dual-gene (Rag1, Rag2) is knocked out Rat, the rat that also available single-gene (Il2rg) knocks out, also available three gene (Rag1, Rag2, Il2rg) knocks out Rat.And simplify the process of identification.

The present invention takes the step of first knocking out heterozygosis again respectively, that is, first knocks out two bases of the rat on item chromosome Because of Rag1, Rag2, the individual gene Il2rg of another rat is knocked out, then again hybridizes two rats, to obtain three The rat that gene (Rag1, Rag2, Il2rg) knocks out.Inventive process avoids problems of the existing technology, e.g., existing skill It can be caused because of irregular permutation and combination between several genes caused by knocking out not exclusively when once knocking out multiple genes in art The operational difficulties screened again after striking obtain the low efficiency of purpose object.

The method of the present invention proposes while knocking out two genes Rag1, Rag2 of rat, thoroughly causes T cell and B is thin The immune deficiency of born of the same parents, so that available more preferably immunodeficient animals model by the method for the invention, avoids existing skill It is presented not on the animal phenotype that can occur in missing Rag1 in art but with Rag2 or missing Rag2 but in the case of there is Rag1 The problem of thorough immune deficiency.

In the present invention, gRNA and Cas9 mRNA after purification, and carries out fertilized eggs injection in mass ratio for 1:2, improves and strikes Except efficiency.

The SD-RG rat that the present invention constructs is able to carry out tumour cell and tissue transplantation, and the growth rate of tumour is remote Much higher than the existing model such as mouse.This greatly shortens experimental period, improves conventional efficient.And SD-RG rat figure is big, than Mouse surgical experiments are easy, are more suitable for orthotopic transplantation.

In addition, the figure and metabolic characteristic of mouse are unsuitable for pathology and toxicological experiment, and the SD-RG that the present invention constructs is big Mouse can directly carry out pathology and toxicological experiment while transplanting, improve the utilization efficiency of tumour PDX model.

Detailed description of the invention

Fig. 1 shows the schematic diagrames that Il2rg knock out mice of the present invention is established using CRISPR/Cas9 technology.

Fig. 2, which is illustrated in the SD-RG rat immunity tissue of the invention constructed, lacks T, B and NK cell；Wherein, A indicates SD- The bis- positive T cells of CD4+CD8+, the mono- positive T cell of CD3+CD4+, the mono- positive T cell of CD3+CD8+ in RG thymus gland significantly drop It is low；B indicates that pro/pre B cell and non-mature B cell in SD-RG marrow significantly reduce；C is indicated in SD-RG peripheral blood Total T, B, NK cell all significantly reduces；D indicates that total T, B, NK cell in SD-RG spleen significantly reduces.

Fig. 3 illustrates that growth rate of the subcutaneous transplantation tumour in the SD-RG rat that the method for the present invention constructs is more than mouse Deng other animal models；Wherein, A indicate SD-RG rat of the present invention, control group (the bis- knockouts of wild type (WT), Rag1Rag2, The mono- knockout rat, immune-deficient mice NSG of Il2rg)；It is wild that B indicates that the tumor growth rate in SD-RG rat is significantly higher than The bis- knockouts of type (WT), Rag1Rag2, the mono- knockout rat of Il2rg and immune-deficient mice NSG；C indicates to dye by HE aobvious Show the group characteristic for remaining initial cell tumor formation of the transplantation tumor loyalty of SD-RG rat, while antibody dyeing display transplanting Tumour contains the distinctive pan-CK marker of NCI-H460 cell line.

Specific embodiment

Following embodiment and attached drawing are not intended to limit the scope of the invention for illustrating the present invention.Unless otherwise specified, The conventional means that technological means used in embodiment is well known to those skilled in the art, raw materials used is commercial goods. Do not make the experimental methods of molecular biology illustrated in following embodiment, referring to " Molecular Cloning:A Laboratory guide " (third edition) J. specific method described in one book of Pehanorm Brooker carries out, or carries out according to kit and product description.

Building of the embodiment 1 for the CRISPR-Cas9 expression system of Rag1, Rag2 and Il2rg gene

Rag1, Rag2 and Il2rg knock out mice are established using CRISPR/Cas9 technology.

As shown in Figure 1, the A in Fig. 1 is indicated: being directed to Rag1 Exon 2, the 3rd exon of Rag2 gene and Il2rg Exon 1 separately designs targeting sequence.B in Fig. 1 is indicated: the base deletion of generation and the mutation of insertion.

1, it according to rat Rag1, Rag2 and Il2rg gene order, carries out the design of sgRNA and obtains the sequence of sgRNA Information.

Wherein, the sgRNA of selectively targeted Rag1 Exon 2, DNA sequence dna is as shown in SEQ ID NO.1 (GAGTCTCAGGGTAGATGGCA).Wherein, (SEQ ID NO.4 is Rag1 gene to SEQ ID NO.1 targeting SEQ ID NO.4 A part).It is respectively synthesized positive and negative single-stranded SEQ ID NO.7 and SEQ ID NO.8, then annealing forms SEQ ID NO.1.

Wherein, the sgRNA of selectively targeted the 3rd exon of Rag2 gene, DNA sequence dna is as shown in SEQ ID NO.2 (AAGAGATCCTGCCCTACTGG).Wherein (SEQ ID NO.5 is Rag2 gene to SEQ ID NO.2 targeting SEQ ID NO.5 A part).It is respectively synthesized positive and negative single-stranded SEQ ID NO.9 and SEQ ID NO.10, then annealing forms SEQ ID NO.2.

Wherein, the sgRNA of selectively targeted Il2rg exon 1, DNA sequence dna is as shown in SEQ ID NO.3 (AAGGATCTAGATGGCAATAA).Wherein (SEQ ID NO.6 is Il2rg gene to SEQ ID NO.3 targeting SEQ ID NO.6 A part).It is respectively synthesized positive and negative single-stranded SEQ ID NO.11 and SEQ ID NO.12, then annealing forms SEQ ID NO.3.

2, the building of sgRNA vector plasmid:

(1) separately design and synthesize identification Rag1 Exon 2, the 3rd exon of Rag2 gene and Il2rg gene the The sgRNA cog region DNA sequence dna of 1 exon, i.e. SEQ ID NO.7 and SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10, SEQ ID NO.11 and SEQ ID NO.12, it is specific such as table 2；

(2) each DNA is first synthesizing single-stranded, and gradient cooling is annealed after the single-stranded sgRNA sequence after synthesis carries out phosphorylation, shape At double-stranded DNA；The specific steps are the oligoDNA that will be synthesized (SEQ ID NO.7, SEQ ID NO.9 and eq ID NO.11 conduct Normal chain oligoDNA, SEQ ID NO.8, SEQ ID NO.10 and eq ID NO.12 are as anti-chain sequence, by positive and negative chain OligoDNA is added after being mixed with 10 × T4 DNA ligase buffer and T4 polynucleotide kinase with the volume ratio of 2:2:1 The water polishing system of 3 times of volumes, then 37 DEG C of incubation 30min, then 95 DEG C, 5min denaturation, later with 5 DEG C of rates per minute 25 DEG C of completion reactions are cooled to generate phosphorylation cohesive end；BbsI digestion carrier pX330 generates cohesive end simultaneously；Annealing Reaction system is as shown in table 1.

1 annealing reaction system of table

2 sgRNA cog region DNA sequence dna of table

(3) with T4DNA ligase by SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 double-stranded DNA respectively with PX330 is attached, and obtains eukaryon CRISPR-Cas9 expression system carrier.Coupled reaction system such as table 3.Connect the temperature of reaction Degree is 16 degrees Celsius, and the connection reaction time is 1h-3h.

3 coupled reaction system of table

The pX330 sheared refers to the pX330 after carrying out linearization process using BbsI restriction enzyme.

Embodiment 2 is transcribed in vitro

The in-vitro transcription mediated with T7 promoter is carried out using T7-Cas9 PCR and T7-sgRNA PCR product, i.e., with T7 Promoter realizes the transcription from DNA to mRNA using RNA polymerase, specifically as the promoter being transcribed in vitro in vitro Method are as follows: T7-Cas9 PCR is transcribed in vitro with mMESSAGE mMACHINE T7 ULTRA kit (Life Technologies) T7-sgRNA PCR product is transcribed in vitro with MEGAshortscript T7 kit (Life Technologies) in product.It will turn The mRNA that record generates is purified, method particularly includes: Cas9 mRNA is pure with MEGAclear kit (Life Technologies) Change, sgRNAs is purified with ethanol precipitation methods, dissolves mRNA with pure water, and mRNA after purification is dense using spectrophotometric determination Degree.

T7-Cas9 PCR primer be shown in Table 4, T7-sgRNA PCR primer be shown in Table 5, gRNA-Rag1-F, gRNA-Rag2-F, GRNA-Il2rg-F is respectively the forward primer for being directed to SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, reverse primer For gRNA-R.PCR reaction system is shown in Table 6, PCR reaction condition and is shown in Table 7.

4 T7-Cas9 PCR primer of table

5 T7-sgRNA PCR primer of table

6 PCR reaction system of table

7 PCR reaction condition of table

Embodiment 3 prepares gene knockout using the CRISPR-Cas9 system mRNA for Rag1, Rag2 and Il2rg gene Rat

1, procaryotic injection and embryo transfer

The protokaryon phase fertilized eggs for taking rat, it is pre- mixed by what is obtained in previous embodiment 2 using microinjection instrument Cas9mRNA/sgRNA mixture (final concentration of 10ng/ μ l of final concentration of 20ng/ the μ l, sgRNA of Cas9mRNA), is injected to rat Then fertilized egg cell's matter migrates in the fallopian tubal of receptor female rat, producer gene target practice rat, aforementioned Cas9mRNA/sgRNA The injection volume of mixture is 0.5-1ul.

2, genotype identification

After the production of replace-conceive female rat, long to 2 week old clip 1cm or so rat-tail to newborn mouse, Proteinase K, phenol is imitative after 55 DEG C of digestion Rat-tail genomic DNA is extracted in extracting.Using rat genomic dna as template, separately design for Rag1 Exon 2, The primer of Rag2 gene the 3rd exon and Il2rg exon 1, is expanded, and the PCR product of acquisition is sequenced, Primer sequence such as the following table 8.

Wherein, Rag1-L and Rag1-R corresponds to SEQ ID NO.4；Rag1-L and Rag1-R corresponds to SEQ ID NO.5； Il2rg-F and Il2rg-R corresponds to SEQ ID NO.6.

The identification primer of 8 gene targeted mice of table

It then may be to practice shooting successfully if bimodal situation occurs in sequencing result target practice location proximate.Select bimodal sample PCR again, TA is cloned into carrier T after the recycling of product glue, and picking positive colony is sequenced again after conversion.

If base insertion or base deletion nearby occur for sequencing result Rag1, Rag2 and Il2rg gene target site, cause to read Frameshift mutations then can determine whether as Rag1, Rag2 and Il2rg gene knockout.

In Figure 1B, for SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 knockout rat in spy Base deletion and/or insertion has occurred in fixed site.Wherein, there is the missing of 1564 bases (bp), Rag2 base in Rag1 gene Because there is the missing of 86 bases (bp), and then there is the insertion of 1 base (bp) in Il2rg gene.These mutation lead to correlation Frameshift mutation when gene expression, to cause the expression deletion of related gene, i.e. gene knockout.

Embodiment 4 verifies the immunodeficient rats animal model that the present invention obtains using fluidic cell

The red rear corresponding antibody of T, B, NK cell is split in the immuning tissue for taking the SD-RG rat obtained in above-described embodiment 3 It is incubated for (being the antibody of AntiCD3 McAb, CD4, CD8, CD45 and CD161 respectively), passes through flow cytometry analysis.With wild type (WT), The bis- knockouts of Rag1Rag2 and the mono- knockout rat of Il2rg are compared, the CD4+CD8 such as Fig. 2A, in immunodeficient rats SD-RG thymus gland + bis- positive T cells (CD4+CD8+DP), the mono- positive T cell of CD3+CD4+ (CD3+CD4+SP) and the mono- positive T cell of CD3+CD8+ (CD3+CD8+SP) it significantly reduces；Pro/pre B cell (pro/pre B cells) He Feicheng such as Fig. 2 B, in SD-RG marrow Ripe B cell (immature B cells) significantly reduces；Such as Fig. 2 C, total T, B, NK cell in SD-RG peripheral blood all significantly drops It is low；Such as Fig. 2 D, total T, B, NK cell in SD-RG spleen is all significantly reduced.These all illustrate SD-RG rat its with most serious Immunodeficient pheno.

The advance of 5 transplantation tumor experimental verification SD-RG rat of embodiment

It can be in the tumor cell lines such as SD-RG rat skin lower injection NCI-H460 and the tumor tissues of transplant patient.With For NCI-H460 tumor cell line, as shown in Figure 3A, with wild type (WT), the bis- knockouts of Rag1Rag2, the mono- knockout rat of Il2rg And immune-deficient mice NSG is control group, hypodermic initial concentration is 2.5X10⁶.As shown in Figure 3B, SD-RG Tumor growth rate in rat is significantly higher than the bis- knockouts of wild type (WT), Rag1Rag2, the mono- knockout rat of Il2rg and is immunized Deficient mice NSG.After injection 20 days, the gross tumor volume in SD-RG rat has been more than 20cm³, bis- better than Rag1Rag2 to strike Except with the mono- knockout rat of Il2rg, and be significantly higher than NSG mouse model and wild type (WT) rat.Meanwhile as shown in Figure 3 C, HE The group characteristic for remaining initial cell tumor formation of the transplantation tumor loyalty of dyeing display SD-RG rat, while antibody dyeing is aobvious Show that transplantation tumor contains the distinctive pan-CK marker of NCI-H460 cell line.

It is only that detailed description is subject to the present invention in a manner of general explanation, specific embodiment etc. in summary, is not Limitation to interest field of the present invention.On the basis of without departing from the present invention, those skilled in the art are done on the basis of the present invention Further modify or improve, belong to claimed interest field.

SEQUENCE LISTING

<110>Jiangsu Ai Erkang biological medicine Science and Technology Ltd.

<120>a kind of construction method of immunodeficient rats animal model and application

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<170> PatentIn version 3.3

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<212> DNA

<213>artificial sequence

<400> 22

catctcctac taagtctttc tctgtacaac 30

<210> 23

<211> 30

<212> DNA

<213>artificial sequence

<400> 23

tattttccat ctatccttcc tagactgtac 30

<210> 24

<211> 29

<212> DNA

<213>artificial sequence

<400> 24

ctagcaggct ccagattgta tagaaagag 29

Claims (12)

1. a kind of construction method of immunodeficient rats animal model, which is characterized in that the described method comprises the following steps:

(1) difference construction of expression vector；The expression vector is respectively selectively targeted rat Rag1 Exon 2 Expression vector, the rat Il2rg exon 1 of the expression vector of sgRNA, the sgRNA of the 3rd exon of rat Rag2 gene SgRNA expression vector；

(2) the 3rd exon of sgRNA, Rag2 gene of rat Rag1 Exon 2 is obtained by the way that expression respectively is transcribed in vitro SgRNA, Il2rg exon 1 sgRNA and Cas9mRNA；

(3) sgRNA and Cas9mRNA of the 3rd exon of sgRNA, Rag2 gene of rat Rag1 Exon 2 are distinguished After purification, it mixes, is injected into rat fertilized egg cell, is implanted into rat fallopian tubal, obtain while having knocked out Rag1 gene With the rat of Rag2 gene；

Respectively after purification by the sgRNA and Cas9mRNA of rat Il2rg exon 1, mixing is injected into rat fertilized eggs It in cell, is implanted into rat fallopian tubal, obtains the rat for having knocked out Il2rg gene；

(4) by the rat that is described while having knocked out Rag1 and Rag2 gene that step (3) obtain and described Il2rg gene has been knocked out Rat hybridization, obtain the immunodeficient rats animal model SD-RG that knocks out of Rag1, Rag2 and Il2rg gene.

2. construction method as described in claim 1, which is characterized in that the nucleotide of the rat Rag1 Exon 2 Sequence is as shown in SEQ ID NO.4；The nucleotide sequence of the 3rd exon of rat Rag2 gene is as shown in SEQ ID NO.5； The nucleotide sequence of the rat Il2rg exon 1 is as shown in SEQ ID NO.6.

3. construction method as described in claim 1, which is characterized in that in the step (1), using CRISPR/Cas9 technology, It is set respectively for rat Rag1 Exon 2, the 3rd exon of rat Rag2 gene, rat Il2rg exon 1 Meter targeting DNA sequence dna, expression vector, the rat Rag2 gene the 3rd for constructing the sgRNA of rat Rag1 Exon 2 are aobvious outside The expression vector of the expression vector of sgRNA of son, the sgRNA of rat Il2rg exon 1；The targeting DNA sequence dna point It Wei not SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 sequence.

4. construction method as described in claim 1, which is characterized in that the step (2) is respectively obtained by transcription Cas9mRNA, sgRNA carry out absorption column purification respectively；

Wherein, the final concentration of 20-100ng/ μ l of Cas9mRNA after purification；Rat Rag1 Exon 2 after purification SgRNA, the sgRNA of the 3rd exon of rat Rag2 gene, rat Il2rg exon 1 sgRNA final concentration difference For 10-50ng/ μ l.

5. construction method as described in claim 1, which is characterized in that in the step (3), the sgRNA and Cas9mRNA When mixing, the mass ratio of sgRNA and Cas9mRNA are 1:2.

6. a kind of expression vector, which is characterized in that the expression vector is respectively aobvious outside selectively targeted rat Rag1 gene the 2nd The expression vector of sgRNA of son, the expression vector of the sgRNA of the 3rd exon of rat Rag2 gene, outside rat Il2rg gene the 1st The expression vector of the sgRNA of son is shown, the expression vector respectively includes carrier pX330 and connect with the carrier pX330 DNA is targeted, the targeting DNA sequence dna is respectively SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 sequence.

7. a kind of single strain oligonucleotide sequence, which is characterized in that the sequence is respectively as follows:

The annealed positive and negative single-stranded SEQ ID NO.7 and SEQ ID NO.8 of a pair for forming SEQ ID NO.1；

The annealed positive and negative single-stranded SEQ ID NO.9 and SEQ ID NO.10 of a pair for forming SEQ ID NO.2；

The annealed positive and negative single-stranded SEQ ID NO.11 and SEQ ID NO.12 of a pair for forming SEQ ID NO.3.

8. a kind of non-human mammalian cell, which is characterized in that the cell contains sgRNA and Cas9mRNA, wherein described SgRNA includes the sgRNA of rat Rag1 Exon 2 as shown in SEQ ID NO.1, as shown in SEQ ID NO.2 The sgRNA of the 3rd exon of the rat Rag2 gene and sgRNA of the rat Il2rg gene as shown in SEQ ID NO.3.

9. a kind of for knocking out the CRISPR-Cas9 expression system of rat Rag1, Rag2 and Il2rg gene, which is characterized in that institute It states expression system and contains selectively targeted rat Rag1 Exon 2, the 3rd exon of rat Rag2 gene, rat respectively The expression vector of the sgRNA of Il2rg exon 1.

10. the immunodeficient rats animal model that construction method as described in claim 1 obtains, table as claimed in claim 6 Immunoregulation medicament or antineoplastic are being screened or prepared up to carrier, non-human mammalian cell as claimed in claim 8 Application in object screening.

11. rat Rag1 Exon 2, the 3rd exon of rat Rag2 gene and/or rat Il2rg exon 1 Preparing the purposes in immunodeficient rats animal model, which is characterized in that the nucleosides of the rat Rag1 Exon 2 Acid sequence is as shown in SEQ ID NO.4；The nucleotide sequence such as SEQ ID NO.5 institute of the 3rd exon of rat Rag2 gene Show；The nucleotide sequence of the rat Il2rg exon 1 is as shown in SEQ ID NO.6.

12. the sgRNA of rat Rag1 Exon 2, the 3rd exon of rat Rag2 gene, Il2rg exon 1 In knockout rat Rag1 Exon 2, the 3rd exon of rat Rag2 gene, mouse Il2rg exon 1 Using, which is characterized in that the nucleotide sequence of the rat Rag1 Exon 2 is as shown in SEQ ID NO.4；It is described big The nucleotide sequence of the 3rd exon of mouse Rag2 gene is as shown in SEQ ID NO.5；The rat Il2rg exon 1 Nucleotide sequence is as shown in SEQ ID NO.6.