WO2018141127A1 - 一种免疫缺陷大鼠模型的构建方法 - Google Patents

一种免疫缺陷大鼠模型的构建方法 Download PDF

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WO2018141127A1
WO2018141127A1 PCT/CN2017/078292 CN2017078292W WO2018141127A1 WO 2018141127 A1 WO2018141127 A1 WO 2018141127A1 CN 2017078292 W CN2017078292 W CN 2017078292W WO 2018141127 A1 WO2018141127 A1 WO 2018141127A1
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rat
generation
immunodeficient
il2rγ
gene
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French (fr)
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徐洋
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南方医科大学
广东圣赛生物科技有限公司
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Definitions

  • the embodiments of the invention relate to the field of rat model construction, and in particular to a method for constructing an immunodeficient rat model.
  • Animal models are mainly used in experimental physiology, experimental pathology, and experimental therapeutics (including screening of new drugs).
  • the development of human diseases is very complicated.
  • the human body is used as an experimental object to deeply explore the mechanism of disease occurrence, and the development of medicine is slow.
  • the experience of clinical accumulation is not only limited in time and space, but also many experiments are morally And methods are also limited.
  • With the indirect study of animal models it is possible to consciously change the factors that are impossible or difficult to exclude under natural conditions, in order to more accurately observe the experimental results of the model and compare them with human diseases, which is more convenient and more convenient. Effectively study the law of occurrence and development of human diseases and study prevention and control measures.
  • Immunodeficient animal models can be used for drug development, transplantation studies, and therapeutic mechanisms for human disease. At present, most commonly used immunodeficient animal models are mice, and the homology between rats and humans is closer, so the use of rat models has obvious advantages over the study of human-related diseases in mouse models.
  • To measure the degree of immune deficiency in the body mainly to measure the proportion of T, B lymphocytes and NK cells in the body. Japanese scholar Tomoji et al. (Mashimo T, Takizawa A, et al: Generation and characterization of severe combined immunodeficiency rats.
  • T cells in peripheral blood, bone marrow and spleen were significantly reduced, B cells and NK cells disappeared in peripheral blood and bone marrow, and only some remained in the spleen; Prkdc knockout rats CD4 - CD8 + , CD4 + CD8 - and CD4 + CD8 + T cells all disappeared, B cells also disappeared, and the number of NK cells increased. Rats with both genes knocked out, T, B lymphocytes and NK cells all disappeared.
  • the immunodeficient rat model of the prior art has at least the following problem: human CD34+ hematopoietic stem cells are transplanted on a rat model of simultaneous knockout of Prkdc and IL2R ⁇ , but no human immune system cells are produced.
  • the existing immunodeficient animal model can not meet the needs of drug testing and xenogeneic cell transplantation research, etc., which is an urgent problem to be solved by those skilled in the art.
  • the present invention provides a new immunodeficient rat model, which fills the gap of the immunodeficient rat model and can meet the test requirements.
  • a method of constructing an immunodeficient rat model comprising the steps of:
  • Injection A and injection B were injected into the cytoplasm of different rat fertilized eggs by microinjection, and then the rat fertilized eggs were transplanted into different pseudo-pregnant females, and the second was cultivated.
  • Injection C is injected into the pronucleus of rat fertilized egg by microinjection, and then the rat fertilized egg is transplanted into the pseudopregnant female, and the second generation rat C is obtained.
  • the second generation rat A, the second generation rat B and the second generation rat C were crossed to breed the F1 generation; the F1 generation was further hybridized, and the IL2R ⁇ and Prkdc genes were knocked out and transferred to humans.
  • the knockout rat Prkdc gene in step (1) adopts two gRNA sequences, respectively: first exon gRNA sequence 1: TTCCGGCACTATGGCGGACC; First exon gRNA sequence 2: GCCAGTTACCAGCTGATCCG.
  • the knockout rat IL2R ⁇ gene in step (1) adopts two gRNA sequences, respectively: The exon gRNA sequence 1: CAGCCGACCAACCTCACTAT; the fourth exon gRNA sequence 2: GAGTGAATCTCAGGTAGAAC.
  • knocking out the rat IL2R ⁇ gene also employs two additional gRNA sequences, respectively: a second exon gRNA sequence 1: CAGCCGACCAACCTCACTAT; fourth exon gRNA sequence 3: GAGCAACCGAGATCGAAGCT.
  • the in vitro transcription of the CAS9 protein mRNA is performed using a T7 transcription kit, a T3 transcription kit, or an SP6 transcription kit.
  • the in vitro transcription of the CAS9 protein mRNA is performed using a T7 transcription kit, a T3 transcription kit, or an SP6 transcription kit.
  • the pseudopregnant female in step (3) is raised under SPF level conditions.
  • the rat fertilized egg in step (3) is taken from a pregnant female mouse raised under SPF level conditions.
  • an immunodeficient rat model which knocks out the IL2R ⁇ and Prkdc genes and is transfected into the human SIRP ⁇ gene.
  • the immunodeficient rat model is constructed by any one of the methods of the first aspect.
  • a human SIRP ⁇ (hSIRPa) gene is overexpressed in Prkdc and IL2R ⁇ knockout rats, and a rat with immune function loss and overexpression of the human SIRP ⁇ gene is constructed, thereby obtaining a complete immune function to allografts.
  • Rat model this rat model does not attack immune cells other than T lymphocytes, B lymphocytes and NK cells when used for xenotransplantation.
  • Humanized xenografts are met to meet the needs of drug trials and xenogeneic cell transplantation studies.
  • the immunodeficient rat model obtained by this method not only fills the gap of the immunodeficient rat model, but also meets the test index, which can make human cells efficiently implant and the background of the strain is pure.
  • FIG. 1 is a schematic view showing the position of a gRNA used for knocking out the Prkdc gene in rat DNA in one embodiment of the method for constructing an immunodeficient rat model provided by the present invention.
  • FIG. 2 is a schematic diagram showing the position of a gRNA used for knocking out the IL2R ⁇ gene in rat DNA in one embodiment of the method for constructing an immunodeficient rat model provided by the present invention.
  • Figure 3 is a flow cytometric map of the expression of rSIRPa in human peripheral blood and the expression of rSIRPa in human peripheral blood of normal wild rats (WT) and rats overexpressing human SIRPa (hSIRPa) in a specific embodiment of the present invention.
  • Figure 4 is a view of NK cells, B lymphocytes and T lymphocytes in peripheral blood of normal wild rats (WT), in a specific embodiment, Prkdc single knock (Prkdc +/- ) and double knock (Prkdc -/- ) rats.
  • WT normal wild rats
  • Prkdc single knock Prkdc +/-
  • Prkdc -/- double knock
  • Figure 5 is a view of NK cells, B lymphocytes and T lymphocytes in peripheral blood of normal wild rats (WT), a specific embodiment of IL2R ⁇ single knock (IL2R ⁇ +/- ) and double knock (IL2R ⁇ -/- ) rats. Flow cytometry map.
  • Figure 6 is a normal wild rat (WT), SG (IL2R ⁇ -/- , Prkdc -/- ) rat and NSG (hSIRPa, IL2R ⁇ -/- , Prkdc -/- ) rat model in a specific embodiment of the present invention.
  • WT normal wild rat
  • SG IL2R ⁇ -/- , Prkdc -/-
  • NSG hSIRPa, IL2R ⁇ -/- , Prkdc -/-
  • Fig. 7 shows the results of detection of expression of human immune cells in peripheral blood and bone marrow after 5 weeks of injection of artificial blood stem cells into the immunodeficient rat model constructed by one embodiment of the present invention.
  • Figure 8 shows the tumor formation of tumor cells and embryonic stem cells after subcutaneous transplantation in SG (IL2R ⁇ -/- , Prkdc -/- ) rats and NSG (hSIRPa, IL2R ⁇ -/- , Prkdc -/- ) rat models.
  • the inventors have found that the immunodeficient rat model of the prior art has at least the following problem: human CD34+ hematopoietic stem cells are transplanted on a rat model of simultaneous knockdown of Prkdc and IL2R ⁇ , but no human immune system cells are produced.
  • the inventors have analyzed that the main possible cause is the graft failure caused by the allogeneic rejection of macrophages and monocytes in the rat model.
  • a method for constructing an immunodeficient rat model comprising the steps of: (1) separately transfecting a gRNA encoding a Prkdc and an IL2R ⁇ gene of rat DNA in vitro, Then mixed with in vitro transcribed CAS9 protein mRNA, frozen in RNase-free ultrapure water, to obtain the corresponding injection A and injection B;
  • Injection A and injection B were injected into the cytoplasm of different rat fertilized eggs by microinjection, and then the rat fertilized eggs were transplanted into different pseudo-pregnant females, and the second was cultivated.
  • Injection C is injected into the pronucleus of rat fertilized egg by microinjection, and then the rat fertilized egg is transplanted into the pseudopregnant female, and the second generation rat C is obtained.
  • the second generation rat A, the second generation rat B and the second generation rat C were crossed to breed the F1 generation; the F1 generation was further hybridized, and the IL2R ⁇ and Prkdc genes were knocked out and transferred to humans.
  • the above microinjection method refers to direct injection of a foreign gene fragment into a prokaryotic embryo or a cultured cell by using a glass microinjection needle having a very thin tip (0.1 to 0.5 ⁇ m), and then may occur by the host genome sequence.
  • the phenomenon of recombination, deletion, replication or translocation causes the foreign gene to be embedded in the chromosome of the host.
  • the above human SIRPa genomic DNA, Signal Regulatory Protein Alpha genomic DNA can be directly purchased commercially.
  • the human SIRPa genomic DNA used in the above protocol was purchased from life technologies (BAC, RP11-993C19).
  • the artificially purchased human SIRPa genomic DNA is first amplified and purified, because the microinjection of the fertilized egg needs to be sufficiently pure, otherwise the fertilized egg may not be developed, and no positive result is obtained.
  • SIRPa is mainly expressed on the surface of monocytes and macrophages, and binds to the CD47 ligand to mediate a negative regulatory signal, and is a "don't eat me” gene.
  • the inventors performed in vitro transcription of the Prkdc and IL2R ⁇ gene knockout gRNAs of rat DNA by creative labor and a large number of experiments, respectively, and mixed with the in vitro transcribed CAS9 protein mRNA in proportion, and then each passed microinjection method.
  • the human SIRPa genomic DNA was injected into the pronucleus of another rat fertilized egg by microinjection, and the SIRPa genomic DNA was randomly inserted into the genome during the division of the fertilized egg, thereby cultivating the second generation rat C expressing the human SIRPa. .
  • the second generation rat A, the second generation rat B and the second generation rat C were crossed to breed the F1 generation, and then the F1 generation was hybridized to establish a stable homozygous Prkdc and IL2R ⁇ gene knockout and human An immunodeficient rat strain stably expressing the SIRPa (hSIRPa) gene, thereby constructing a rat model of loss of immune function and overexpression of the human SIRP ⁇ gene.
  • the rat model obtained by the above method has no immune effect on allogeneic transplantation, so that the rat model does not attack human cells except for immune cells other than T lymphocytes, B lymphocytes and NK cells for xenografting. Sexual xenografts to meet the needs of drug trials and xenogeneic cell transplantation studies.
  • the immunodeficient rat model is a good humanized rat vector.
  • the transplanted human ES cells and tumor cells in rats showed good xenograft ability and transplanted human CD34 + .
  • a humanized rat model of the hematopoietic system can be established. It can also be applied to research fields such as stem cell transplantation, tumor biology, humanized immune system reconstruction, human antibody manufacturing, and HIV research.
  • the immunodeficient rat model constructed by the above technical scheme not only fills the gap of the immunodeficient rat model, but also meets the test index, and the strain background is pure.
  • the design is simple, the operation is convenient, and the efficiency is higher.
  • gRNA also known as guide RNA, is one of the core components of the CRISPR/Cas9 technology.
  • the knockout rat Prkdc gene in the step (1) uses two gRNA sequences: a first exon gRNA sequence 1: TTCCGGCACTATGGCGGACC; a first exon gRNA sequence 2: GCCAGTTACCAGCTGATCCG.
  • Fig. 1 shows the approximate position of gRNA guidance used in the first exon of rat DNA to knock out the Prkdc gene.
  • the Prkdc gene sequence of wild rats is:
  • CRISPR/Cas9 binds to the DNA of the targeting sequence under the guidance of gRNA.
  • the Cas9 protein is cleaved at the third base of the PAM (protospacer adjacent motif) sequence, and is cleaved.
  • the DNA will undergo end-ligation repair, and it is possible to insert some bases in this process.
  • two Prktd knockout sequences are obtained after knockout by CRISPR/Cas9 technology.
  • the first Prkdc knockout sequence is deleted from the 20th to the 114th with a total of 95 bases, a mutation of 1 base, and the mutation is the 115th, which is abrupt from A to G.
  • the sequence is as follows:
  • the second Prkdc knockout sequence lacks the 16th to 115th total of 100 bases, the mutation is 1 base, the mutation is the 116th, and the mutation is from T to C.
  • the sequence is as follows:
  • the rat IL2R ⁇ gene is knocked out in the step (1) using two gRNA sequences: a second exon gRNA sequence 1: CAGCCGACCAACCTCACTAT; a fourth exon gRNA sequence 2: GAGTGAATCTCAGGTAGAAC.
  • the rat IL2R ⁇ gene can be knocked out, and two other gRNA sequences can be used, namely: a second exon gRNA sequence 1: CAGCCGACCAACCTCACTAT; a fourth exon gRNA sequence 3: GAGCAACCGAGATCGAAGCT.
  • Figure 2 shows the approximate positions of three different gRNA guides that can be used to knock out the IL2R ⁇ gene in rat DNA.
  • the IL2rg gene sequence 1 of wild rats is:
  • the second exon gRNA sequence 1 and the fourth exon gRNA sequence 2 were guide RNAs, and the first IL2R ⁇ knockout sequence was obtained, and the 17th to the 678th total of 662 bases were deleted.
  • the sequence is as follows:
  • the IL2rg gene sequence 2 of wild rats is:
  • the second exon gRNA sequence 1 and the fourth exon gRNA sequence 3 are guide RNAs, and the second IL2R ⁇ knockout sequence is obtained, and the 17th to the 767th total of 751 bases are deleted, and After 17 bases, insert 17-27 to a total of 8 bases.
  • the sequence is as follows:
  • the in vitro transcription of the CAS9 protein mRNA is carried out by using a T7 transcription kit, a T3 transcription kit or an SP6 transcription kit.
  • the selection of the transcription kit is determined by the promoter that drives the gRNA and Cas9 proteins.
  • step (3) and step (4) are raised under SPF-grade conditions.
  • the SPF (Specefic pathogen Free) level refers to the absence of a specific pathogen level.
  • immunodeficient rats are constructed.
  • the CRISPR/Cas9 knockout may produce homozygous immunodeficient rats in the first generation, so the preferred pseudopregnant females need to be under SPF conditions.
  • the step numbers (1), (2), (3), (4), (5), and the like are only for convenience of description, and are not used to limit the order of each step in the construction method, and each step in the method. As long as it is logically reasonable, the order of the steps can vary.
  • the above step (1) may be performed independently of the step (2); for example, the step (2) may be performed before the step (1); for example, the step (3) and the step (4) may be simultaneously independent. There is no specific order limit for the two.
  • Knockout rat Prkdc gene uses two gRNA sequences: first exon gRNA sequence 1: TTCCGGCACTATGGCGGACC; first exon gRNA sequence 2: GCCAGTTACCAGCTGATCCG.
  • Knockout rat IL2R ⁇ gene uses two gRNA sequences: second exon gRNA sequence 1: CAGCCGACCAACCTCACTAT; fourth exon gRNA sequence 2: GAGTGAATCTCAGGTAGAAC.
  • Injection A and injection B were separately injected into the cytoplasm of different fertilized eggs obtained in step (3) by microinjection, and the fertilized eggs were transplanted into different SPF-level conditions.
  • the second generation rat A and the second generation rat B were cultured.
  • Knockout rat Prkdc gene uses two gRNA sequences: first exon gRNA sequence 1: TTCCGGCACTATGGCGGACC; first exon gRNA sequence 2: GCCAGTTACCAGCTGATCCG.
  • Knockout rat IL2R ⁇ gene uses two gRNA sequences: second exon gRNA sequence 1: CAGCCGACCAACCTCACTAT; fourth exon gRNA sequence 2: GAGTGAATCTCAGGTAGAAC.
  • Knockout of the rat IL2R ⁇ gene also uses two additional gRNA sequences: second exon gRNA sequence 1: CAGCCGACCAACCTCACTAT; fourth exon gRNA sequence 3: GAGCAACCGAGATCGAAGCT.
  • Injecting solution A and injection B were respectively injected into the cytoplasm of different fertilized eggs obtained in step (3) by microinjection, and transplanting rat fertilized eggs into pseudopregnancy under SPF level conditions.
  • the female the second generation rat A and the second generation rat B were cultured.
  • Figure 3 is a flow cytometric map of hSIRP ⁇ and rSIRPa expression in peripheral blood of normal wild rats (WT) and rats overexpressing human SIRPa (hSIRPa) in the present invention (i.e., second generation rat C).
  • Figure 4 is a flow cytometric measurement of NK cells, B lymphocytes and T lymphocytes in peripheral blood of normal wild rats (WT), Prkdc single knock (Prkdc +/- ) and double knock (Prkdc -/- ) rats. Map.
  • Figure 5 is a flow cytometric measurement of NK cells, B lymphocytes and T lymphocytes in peripheral blood of normal wild rats (WT), IL2R ⁇ single knock (IL2R ⁇ +/- ) and double knock (IL2R ⁇ -/- ) rats. Map.
  • Figure 6 shows NK cells in peripheral blood of normal wild rat (WT), SG (IL2R ⁇ -/- , Prkdc -/- ) rats and NSG (hSIRPa, IL2R ⁇ -/- , Prkdc -/- ) rat models, Flow cytometric map of B lymphocytes and T lymphocytes, and flow cytometric map of hSIRPa and rSIRPa expression.
  • Figure 2 shows two possible knockouts of Prkdc single knock (Prkdc +/- ) and double knock (Prkdc -/- ) rats, second generation rat A.
  • Figure 5 shows two possible knockouts of IL2R ⁇ single knock (IL2R ⁇ +/- ) and double knock (IL2R ⁇ -/- ) rats, ie second generation rat B.
  • the NSG rat of Figure 6 is an immunodeficient rat model constructed in accordance with a preferred embodiment of the invention.
  • Human hematopoietic stem cell CD34 tail vein injection was performed in NSG rats, and 1*10 6 human CD34+ cells were transplanted. After 5 weeks, human immune cells were detected in peripheral blood and bone marrow, and in the case of untransplanted human thymus, mainly human B cells (hCD19 positive), accounting for more than 65%, and T cells ( hCD3 positive) and monocytes/macrophages (hCD14 positive) accounted for a small proportion. This fully indicates that NSG rats can be used to establish humanized rats.
  • Fig. 7 shows the case where human hematopoietic stem cell CD34 was injected intravenously into the immunodeficient rat model constructed by one embodiment of the present invention to develop adult peripheral blood cells after 5 weeks.
  • Lung cancer tumor cells H460 and human embryonic stem cells H9 were subcutaneously injected with 1*10 5 cells in SG and NSG rat models, respectively.
  • Figures 8A-8C show the tumor formation (size, volume, growth) of H460 lung cancer cells subcutaneously in SG and NSG rats.
  • Figures 8D-8F show the tumor formation (size, volume, growth) of human embryonic stem cell H9 subcutaneously in SG and NSG rats.

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Abstract

一种免疫缺陷大鼠模型的构建方法，包括以下步骤：将针对大鼠DNA的Prkdc和IL2Rγ基因敲除的gRNA分别进行体外转录，再分别与体外转录的CAS9蛋白mRNA混合，冻存于无RNA酶的超纯水中，得到相应的注射液A及B；将人SIRPa基因组DNA扩增，纯化，冻存于无RNA酶的超纯水中，得注射液C；采用显微注射法将注射液A和B分别注射进不同的大鼠受精卵的胞质中，将注射液C注射进另一大鼠受精卵的原核中，将三种受精卵移植入不同假孕雌鼠体内，培育分别得到第二代大鼠A，B和C；将第二代大鼠A、B和C进行杂交，繁殖F1代；再将F1代进行杂交，得到敲除了IL2Rγ和Prkdc基因并转入了人SIRPa基因的免疫缺陷大鼠模型。该大鼠模型可以让人类细胞高效植入，符合试验指标，品系背景纯正。

Description

一种免疫缺陷大鼠模型的构建方法
本申请要求于2017年02月06日提交中国专利局、申请号为201710065515.3、发明名称为“一种免疫缺陷大鼠模型的构建方法”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明实施例涉及大鼠模型构建领域，具体涉及一种免疫缺陷大鼠模型的构建方法。
背景技术
动物模型主要用于实验生理学、实验病理学和实验治疗学(包括新药筛选)研究。人类疾病的发展十分复杂，以人本身作为实验对象来深入探讨疾病发生机制，推动医药学的发展来之缓慢，临床积累的经验不仅在时间和空间上都存在局限性，而且许多实验在道义上和方法上也受到限制。而借助于动物模型的间接研究，可以有意识地改变那些在自然条件下不可能或不易排除的因素，以便更准确地观察模型的实验结果并与人类疾病进行比较研究，有助于更方便、更有效地研究人类疾病的发生发展规律，研究防治措施。
免疫缺陷动物模型可用于药物开发、移植研究以及人类疾病的治疗机理研究。目前，常用的免疫缺陷动物模型多为小鼠，而大鼠与人类的同源性更接近，所以使用大鼠模型比小鼠模型研究人类相关疾病具有明显的优势。衡量机体免疫缺陷的程度，主要测量机体内T、B淋巴细胞和NK细胞所占的比例。日本学者Tomoji等人(Mashimo T,Takizawa A,et al:Generation and characterization of severe combined immunodeficiency rats.Cell reports 2012,2:685-694.、Mashimo T,Takizawa A,et al:Generation of knockout rats with X-linked severe combined immunodeficiency(X-SCID)using zinc-finger nucleases.PloS one 2010,5:e8870.)用Zinc-Finger技术得到IL2Rγ和Prkdc基因敲除的大鼠，并证实IL2Rγ纯合敲除大鼠外周血、骨髓和脾脏中T细胞明显减少，B细胞和NK细胞在外周血和骨髓中消失，仅在脾脏中有一些残留；Prkdc 基因敲除的大鼠CD4-CD8+、CD4+CD8-和CD4+CD8+T细胞全部消失，B细胞也明显消失，而NK细胞数量增加；两个基因同时敲除的大鼠，T、B淋巴细胞和NK细胞全部消失。但是，发明人发现现有技术中的免疫缺陷大鼠模型至少存在如下的问题：在Prkdc和IL2Rγ同时敲除的大鼠模型上移植人的CD34+造血干细胞，却并没有产生人的免疫系统细胞。
因此，现有的免疫缺陷动物模型还不能满足药物试验和异种细胞移植研究等的需求，这是本领域技术人员亟待解决的问题。
发明内容
为解决上述技术问题，本发明实施例提供了一种新的免疫缺陷大鼠模型，填补了免疫缺陷大鼠模型品系空缺，能够符合试验需求。
第一方面，提供了一种免疫缺陷大鼠模型的构建方法，包括以下步骤：
(1)将针对大鼠DNA的Prkdc和IL2Rγ基因敲除的gRNA分别进行体外转录，再分别与体外转录的CAS9蛋白mRNA混合，冻存于无RNA酶的超纯水中，得到相应的注射液A及注射液B；
(2)将人SIRPa基因组DNA扩增，纯化，冻存于无RNA酶的超纯水中，得到注射液C；
(3)采用显微注射法将注射液A和注射液B分别注射进不同的大鼠受精卵的胞质中，然后将大鼠受精卵分别移植入不同假孕雌鼠体内，培育得到第二代大鼠A和第二代大鼠B；
(4)采用显微注射法将注射液C注射进大鼠受精卵的原核中，然后将大鼠受精卵移植入假孕雌鼠体内，培育得到第二代大鼠C；
(5)将第二代大鼠A、第二代大鼠B和第二代大鼠C进行杂交，繁殖F1代；再将F1代进行杂交，得到敲除了IL2Rγ和Prkdc基因并转入了人SIRPa基因的免疫缺陷大鼠模型。
结合第一方面，在第一方面的一种可能的实现方式中，步骤(1)中的敲除大鼠Prkdc基因采用两条gRNA序列，分别为：第一外显子gRNA序列1：TTCCGGCACTATGGCGGACC；第一外显子gRNA序列2： GCCAGTTACCAGCTGATCCG。
结合第一方面或上述某些可能的实施方式，在第一方面的一种可能的实现方式中，步骤(1)中的敲除大鼠IL2Rγ基因采用两条gRNA序列，分别为：第二外显子gRNA序列1：CAGCCGACCAACCTCACTAT；第四外显子gRNA序列2：GAGTGAATCTCAGGTAGAAC。
结合第一方面或上述某些可能的实施方式，在第一方面的一种可能的实现方式中，敲除大鼠IL2Rγ基因还采用另外两条gRNA序列，分别为：第二外显子gRNA序列1：CAGCCGACCAACCTCACTAT；第四外显子gRNA序列3：GAGCAACCGAGATCGAAGCT。
结合第一方面或上述某些可能的实施方式，在第一方面的一种可能的实现方式中，体外转录CAS9蛋白mRNA采用T7转录试剂盒、T3转录试剂盒或SP6转录试剂盒。
结合第一方面或上述某些可能的实施方式，在第一方面的一种可能的实现方式中，体外转录CAS9蛋白mRNA采用T7转录试剂盒、T3转录试剂盒或SP6转录试剂盒。
结合第一方面或上述某些可能的实施方式，在第一方面的一种可能的实现方式中，步骤(3)中的假孕雌鼠在SPF级别条件下饲养。
结合第一方面或上述某些可能的实施方式，在第一方面的一种可能的实现方式中，步骤(3)中的大鼠受精卵取自在SPF级别条件下饲养的受孕雌鼠。
第二方面，提供了一种免疫缺陷大鼠模型，所述大鼠模型敲除了IL2Rγ和Prkdc基因并转入了人SIRPa基因。
结合第二方面，在第二方面的一种可能的实现方式中，所述免疫缺陷大鼠模型由第一方面的任一种方法构建而成。
上述技术方案中，在Prkdc和IL2Rγ敲除的大鼠中过表达人的SIRPa(hSIRPa)基因，构建免疫功能丧失和过表达人的SIRPa基因的大鼠，从而得到了完全对异体移植无免疫作用的大鼠模型，使得这种大鼠模型在用于进行异种移植时除T淋巴细胞、B淋巴细胞和NK细胞以外的免疫细胞也不攻 击人源性的异种移植物，满足药物试验和异种细胞移植研究等的需求。该方法得到的免疫缺陷大鼠模型不但填补了免疫缺陷大鼠模型品系空缺，符合试验指标，可以让人类细胞高效植入，而且品系背景纯正。
附图说明
图1为本发明提供的免疫缺陷大鼠模型构建方法的其中一个具体实施方式中敲除Prkdc基因所使用的gRNA在大鼠DNA中的位置示意图。
图2为本发明提供的免疫缺陷大鼠模型构建方法的其中一个具体实施方式中敲除IL2Rγ基因所使用的gRNA在大鼠DNA中的位置示意图。
图3为正常野生大鼠(WT)和本发明一个具体实施方式中过表达人SIRPa(hSIRPa)大鼠的外周血中人的hSIRPa和大鼠的rSIRPa表达情况的流式细胞测量图谱。
图4为正常野生大鼠(WT)、一个具体实施方式中Prkdc单敲(Prkdc+/-)和双敲(Prkdc-/-)大鼠的外周血中NK细胞、B淋巴细胞和T淋巴细胞的流式细胞测量图谱。
图5为正常野生大鼠(WT)、一个具体实施方式中IL2Rγ单敲(IL2Rγ+/-)和双敲(IL2Rγ-/-)大鼠的外周血中NK细胞、B淋巴细胞和T淋巴细胞的流式细胞测量图谱。
图6为正常野生大鼠(WT)、本发明一个具体实施方式中SG(IL2Rγ-/-，Prkdc-/-)大鼠和NSG(hSIRPa，IL2Rγ-/-，Prkdc-/-)大鼠模型的外周血中NK细胞、B淋巴细胞和T淋巴细胞的流式细胞测量图谱，以及hSIRPa和rSIRPa表达情况的流式细胞测量图谱。
图7为人造血干细胞尾静脉注射本发明其中一个实施方式所构建出的免疫缺陷大鼠模型5周后人的免疫细胞在外周血和骨髓中的表达情况检测结果。
图8为肿瘤细胞和胚胎干细胞在SG(IL2Rγ-/-，Prkdc-/-)大鼠和NSG(hSIRPa，IL2Rγ-/-，Prkdc-/-)大鼠模型皮下移植后的成瘤情况。
具体实施方式
为更清楚的对本发明技术方案予以阐述，下面将结合具体实施方式对本 发明的技术方案进行进一步阐述：
发明人发现现有技术中的免疫缺陷大鼠模型至少存在如下的问题：在Prkdc和IL2Rγ同时敲除的大鼠模型上移植人的CD34+造血干细胞，却并没有产生人的免疫系统细胞。发明人经过分析认为，主要可能原因是该大鼠模型内的巨噬细胞和单核细胞的异体排斥作用导致的移植失败。
为此，在一个具体的实施方式中，提供了一种免疫缺陷大鼠模型的构建方法，包括以下步骤：(1)将针对大鼠DNA的Prkdc和IL2Rγ基因敲除的gRNA分别进行体外转录，再分别与体外转录的CAS9蛋白mRNA混合，冻存于无RNA酶的超纯水中，得到相应的注射液A及注射液B；
(2)将人SIRPa基因组DNA扩增，纯化，冻存于无RNA酶的超纯水中，得到注射液C；
(3)采用显微注射法将注射液A和注射液B分别注射进不同的大鼠受精卵的胞质中，然后将大鼠受精卵分别移植入不同假孕雌鼠体内，培育得到第二代大鼠A和第二代大鼠B；
(4)采用显微注射法将注射液C注射进大鼠受精卵的原核中，然后将大鼠受精卵移植入假孕雌鼠体内，培育得到第二代大鼠C；
(5)将第二代大鼠A、第二代大鼠B和第二代大鼠C进行杂交，繁殖F1代；再将F1代进行杂交，得到敲除了IL2Rγ和Prkdc基因并转入了人SIRPa基因的免疫缺陷大鼠模型。
上述的显微注射法是指利用管尖极细(0.1至0.5μm)的玻璃微量注射针，将外源基因片段直接注射到原核期胚或培养的细胞中，然后藉由宿主基因组序列可能发生的重组、缺失、复制或易位等现象而使外源基因嵌入宿主的染色体内。
上述人SIRPa基因组DNA，即Signal Regulatory ProteinAlpha基因组DNA，可通过商业途径直接采购。例如，上述方案中使用的人SIRPa基因组DNA采购自life technologies(BAC,RP11-993C19)。初始购买的人SIRPa基因组DNA先进行扩增纯化，因为受精卵显微注射需要足够纯，否则容易导致受精卵发育不下去，从而得不到阳性结果。
上述技术方案中，SIRPa主要表达于单核细胞和巨噬细胞表面，与CD47配体结合后介导负性调节信号，是一个“别吃我”的基因。发明人经过创造性劳动和大量实验，将针对大鼠DNA的Prkdc和IL2Rγ基因敲除的gRNA分别进行体外转录，再分别与体外转录制备好的CAS9蛋白mRNA按比例混合，再各自通过显微注射法注射进入受孕雌鼠的不同的受精卵的胞质或原核中，在受精卵中依赖真核翻译系统将Cas9的mRNA翻译成蛋白质，即Cas9蛋白；Cas9蛋白与gRNA结合，在gRNA的介导下结合到作用靶点，Cas9蛋白对所结合靶点进行切割，破坏免疫相关基因序列，从而使免疫相关基因的功能丧失，将受精卵移植入假孕雌鼠后培育得到敲除了Prkdc基因的第二代大鼠A和敲除了IL2Rγ基因的第二代大鼠B。将人SIRPa基因组DNA通过显微注射法注射进入另一大鼠受精卵的原核中，SIRPa基因组DNA在受精卵分裂期间随机插入基因组中，从而使培育得到表达人的SIRPa的第二代大鼠C。最后通过将第二代大鼠A、第二代大鼠B和第二代大鼠C进行杂交，繁殖F1代，再将F1代进行杂交，建立稳定纯合的Prkdc和IL2Rγ基因敲除和人SIRPa(hSIRPa)基因稳定表达的免疫缺陷大鼠品系，从而构建出免疫功能丧失和过表达人的SIRPa基因的大鼠模型。
通过上述方法培育得到的大鼠模型对异体移植完全无免疫作用，使得这种大鼠模型在用于进行异种移植时除T淋巴细胞、B淋巴细胞和NK细胞以外的免疫细胞也不攻击人源性的异种移植物，从而满足药物试验和异种细胞移植研究等的需求。该免疫缺陷大鼠模型是一种良好的人源化大鼠的载体，通过大鼠体内移植人的ES细胞、肿瘤细胞，结果均呈现出很好的异种移植能力，并且在移植了人CD34+造血干细胞后，可以建立造血系统人源化大鼠模型。此外还可以应用于干细胞移植、肿瘤生物学、人源化免疫系统重建、人类抗体制造、HIV研究等研究领域。
上述技术方案构建的免疫缺陷大鼠模型不但填补了免疫缺陷大鼠模型品系空缺，符合试验指标，而且品系背景纯正。
上述敲除了Prkdc基因的第二代大鼠A和敲除了IL2Rγ基因的第二代大鼠B都是采用CRISPR/Cas9技术构建的。CRISPR/Cas9技术与之前的Zine-Finger 和TALEN技术都可以实现对基因的敲除，但因为每个技术对基因编辑位点都有一定的要求，所以最终都到大鼠DNA敲除序列(位点)不一样，此外CRISPR/Cas9技术设计简单，操作方便，效率更高。
上述的gRNA又称引导RNA，是CRISPR/Cas9技术的核心组成部分之一。
进一步地，步骤(1)中的敲除大鼠Prkdc基因采用两条gRNA序列，分别为：第一外显子gRNA序列1：TTCCGGCACTATGGCGGACC；第一外显子gRNA序列2：GCCAGTTACCAGCTGATCCG。
请结合图1，图1中示意了大鼠DNA第一外显子中要敲除Prkdc基因所使用的gRNA引导的大概位置。
野生大鼠的Prkdc基因序列为：
Figure PCTCN2017078292-appb-000001
通过CRISPR/Cas9技术对基因进行编辑，CRISPR/Cas9在gRNA的介导下结合到靶向序列的DNA，Cas9蛋白在PAM(protospacer adjacent motif)序列的第三个碱基处进行切割，断开的DNA会进行末端连接修复，此过程有可能插入一些碱基。在本方案中，通过CRISPR/Cas9技术敲除后可得到两个Prkdc敲除序列。
第一Prkdc敲除序列缺失第20-114个共95个碱基，突变1个碱基，突变的为第115个，由A突变为G。其序列具体如下：
Figure PCTCN2017078292-appb-000002
第二Prkdc敲除序列缺失第16-115个共100个碱基，突变1个碱基，突变的为第116个，由T突变为C。其序列具体如下：
Figure PCTCN2017078292-appb-000003
进一步地，步骤(1)中敲除大鼠IL2Rγ基因采用两条gRNA序列，分别为：第二外显子gRNA序列1：CAGCCGACCAACCTCACTAT；第四外显子gRNA序列2：GAGTGAATCTCAGGTAGAAC。
进一步地，步骤(1)中敲除大鼠IL2Rγ基因还可以采用另外两条gRNA序列，分别为：第二外显子gRNA序列1：CAGCCGACCAACCTCACTAT；第四外显子gRNA序列3：GAGCAACCGAGATCGAAGCT。
请结合图2，图2中示意了大鼠DNA中要敲除IL2Rγ基因可使用的3条不同gRNA引导的大概位置。
野生大鼠的IL2rg基因序列1为：
Figure PCTCN2017078292-appb-000004
Figure PCTCN2017078292-appb-000005
采用CRISPR/Cas9技术，第二外显子gRNA序列1和第四外显子gRNA序列2为引导RNA，获得第一IL2Rγ敲除序列，缺失第17-678个共662个碱基。其序列具体如下：
Figure PCTCN2017078292-appb-000006
野生大鼠的IL2rg基因序列2为：
Figure PCTCN2017078292-appb-000007
Figure PCTCN2017078292-appb-000008
采用CRISPR/Cas9技术，第二外显子gRNA序列1和第四外显子gRNA序列3为引导RNA，获得第二IL2Rγ敲除序列，缺失第17-767个共751个碱基，并在第16个碱基后插入第17-27个共8个碱基。其序列具体如下：
Figure PCTCN2017078292-appb-000009
进一步地，步骤(1)中，体外转录CAS9蛋白mRNA采用T7转录试剂盒、T3转录试剂盒或SP6转录试剂盒。转录试剂盒的选取由启动gRNA和Cas9蛋白的启动子决定。
进一步地，步骤(3)和步骤(4)中的假孕雌鼠在SPF级别条件下饲养。
SPF(Specefic pathogen Free)级别是指无特定病原体级别。本方案中要构建的是免疫缺陷大鼠，CRISPR/Cas9技术敲除可能在第一代就产生纯合的免疫缺陷大鼠，所以优选的假孕雌鼠需要在SPF条件下。
本发明的技术方案中步骤编号(1)、(2)、(3)、(4)、(5)等编号仅为了便于表述，不用于限定构建方法中各个步骤的顺序，方法中的各个步骤，只要逻辑上合理，各步骤的顺序可以变化。例如，上述的步骤(1)可以与步骤(2)同时独立进行；又例如，步骤(2)可以放在步骤(1)之前进行；还例如，步骤(3)和步骤(4)可以同时独立进行，二者没有特定的顺序限制。
下面通过实施例进一步说明上述具体实施方式，但并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法，按照常规方法和条件，或按照商品说明书选择。其他未做特别说明的试剂、原料和仪器设备均可通过商业途径直接购得。
实施例一
(1)将针对大鼠DNA的Prkdc和IL2Rγ基因敲除的gRNA分别进行体外转录，再分别与体外转录的CAS9蛋白mRNA混合，冻存于无RNA酶的超纯水 中，得到相应的注射液A、注射液B，备用。
敲除大鼠Prkdc基因采用两条gRNA序列，分别为：第一外显子gRNA序列1：TTCCGGCACTATGGCGGACC；第一外显子gRNA序列2：GCCAGTTACCAGCTGATCCG。
敲除大鼠IL2Rγ基因采用两条gRNA序列，分别为：第二外显子gRNA序列1：CAGCCGACCAACCTCACTAT；第四外显子gRNA序列2：GAGTGAATCTCAGGTAGAAC。
体外转录CAS9蛋白mRNA采用T7转录试剂盒。
(2)将人SIRPa DNA扩增，纯化，冻存于无RNA酶的超纯水中，得到注射液C，备用。
(3)在SPF级别条件下饲养的受孕雌鼠腹腔中获取输卵管，用浓度为0.9％的生理盐水清洗后，显微镜下观察、挑取并收集受精卵。
(4)采用显微注射法将注射液A和注射液B分别注射进步骤(3)获取的不同的受精卵的胞质中，将大鼠受精卵分别移植入SPF级别条件下饲养的不同的假孕雌鼠体内，培育得到第二代大鼠A和第二代大鼠B。
(5)将注射液C注射进步骤(3)获取的另一受精卵的原核中，将大鼠受精卵移植入另一SPF级别条件下饲养的假孕雌鼠体内，培育得到第二代大鼠C；
(6)将第二代大鼠A，第二代大鼠B，第二代大鼠C进行杂交，繁殖F1代；再将F1代进行杂交，得到敲除了IL2Rγ和Prkdc基因并转入了人SIRPa基因的免疫缺陷大鼠模型。
实施例二
(1)将针对大鼠DNA的Prkdc和IL2Rγ基因敲除的gRNA分别进行体外转录，再分别与体外转录的CAS9蛋白mRNA混合，冻存于无RNA酶的超纯水中，得到相应的注射液A和B，备用。
敲除大鼠Prkdc基因采用两条gRNA序列，分别为：第一外显子gRNA序列1：TTCCGGCACTATGGCGGACC；第一外显子gRNA序列2：GCCAGTTACCAGCTGATCCG。
敲除大鼠IL2Rγ基因采用两条gRNA序列，分别为：第二外显子gRNA序列1：CAGCCGACCAACCTCACTAT；第四外显子gRNA序列2：GAGTGAATCTCAGGTAGAAC。
敲除大鼠IL2Rγ基因还采用另外两条gRNA序列，分别为：第二外显子gRNA序列1：CAGCCGACCAACCTCACTAT；第四外显子gRNA序列3：GAGCAACCGAGATCGAAGCT。
体外转录CAS9蛋白mRNA采用T7转录试剂盒。
(2)将人SIRPa DNA扩增，纯化，冻存于无RNA酶的超纯水中，得到注射液C，备用。
(3)在SPF级别条件下饲养的受孕雌鼠腹腔中获取输卵管，用浓度为0.9％的生理盐水清洗后，显微镜下观察、挑取并收集受精卵。
(4)采用显微注射法将注射液A和注射液B分别注射进步骤(3)获取的不同的受精卵的胞质中，将大鼠受精卵分别移植入SPF级别条件下饲养的假孕雌鼠体内，培育得到第二代大鼠A和第二代大鼠B。
(5)将注射液C注射进步骤(3)获取的另一受精卵的原核中，将大鼠受精卵移植入另一SPF级别条件下饲养的假孕雌鼠体内，培育得到第二代大鼠C；
(6)将第二代大鼠A，第二代大鼠B，第二代大鼠C进行杂交，繁殖F1代；再将F1代进行杂交，得到敲除了IL2Rγ和Prkdc基因并转入了人SIRPa基因的免疫缺陷大鼠模型。
图3为正常野生大鼠(WT)和本发明实施例中过表达人SIRPa(hSIRPa)大鼠(即第二代大鼠C)的外周血中hSIRPa和rSIRPa表达情况的流式细胞测量图谱。
图4为正常野生大鼠(WT)、Prkdc单敲(Prkdc+/-)和双敲(Prkdc-/-)大鼠的外周血中NK细胞、B淋巴细胞和T淋巴细胞的流式细胞测量图谱。
图5为正常野生大鼠(WT)、IL2Rγ单敲(IL2Rγ+/-)和双敲(IL2Rγ-/-)大鼠的外周血中NK细胞、B淋巴细胞和T淋巴细胞的流式细胞测量图谱。
图6为正常野生大鼠(WT)、SG(IL2Rγ-/-，Prkdc-/-)大鼠和NSG(hSIRPa， IL2Rγ-/-，Prkdc-/-)大鼠模型的外周血中NK细胞、B淋巴细胞和T淋巴细胞的流式细胞测量图谱，以及hSIRPa和rSIRPa表达情况的流式细胞测量图谱。
图4中的Prkdc单敲(Prkdc+/-)和双敲(Prkdc-/-)大鼠即第二代大鼠A可能的两种敲除情况。图5中IL2Rγ单敲(IL2Rγ+/-)和双敲(IL2Rγ-/-)大鼠即第二代大鼠B可能的两种敲除情况。图6中的NSG大鼠即本发明的一个优选的实施例所构建的免疫缺陷大鼠模型。
比较例一
采用NSG大鼠进行人的造血干细胞CD34尾静脉注射移植，移植1*106个人的CD34+细胞。5周后，外周血和骨髓中均检测到了人的免疫细胞，并且在未移植人的胸腺情况下，主要是以人的B细胞(hCD19阳性)为主，占65％以上，而T细胞(hCD3阳性)和单核细胞/巨噬细胞(hCD14阳性)占很少比例。这充分表明NSG大鼠可以用于建立人源化大鼠。
图7为人的造血干细胞CD34尾静脉注射移植本发明其中一个实施方式所构建出的免疫缺陷大鼠模型5周后发育成人外周血细胞的情况。
比较例二
用肺癌肿瘤细胞H460和人胚胎干细胞H9分别在SG和NSG大鼠模型皮下注射1*105个细胞。
图8A-8C为H460肺癌细胞在SG和NSG大鼠皮下的成瘤情况(大小、体积、生长情况)。
图8D-8F为人胚胎干细胞H9在SG和NSG大鼠皮下的成瘤情况(大小、体积、生长情况)。
最后应说明的是：以上各实施例仅用以帮助理解本发明的技术方案及核心思想，而非对其限制；尽管参照前述各实施例对本发明进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分或者全部技术特征进行等同替换，而这些修改或者替换也落入本发明权利要求的保护范围内。

Claims (8)

  1. 一种免疫缺陷大鼠模型的构建方法，其特征在于，包括以下步骤：
    (1)将针对大鼠DNA的Prkdc和IL2Rγ基因敲除的gRNA分别进行体外转录，再分别与体外转录的CAS9蛋白mRNA混合，冻存于无RNA酶的超纯水中，得到相应的注射液A及注射液B；
    (2)将人SIRPa基因组DNA扩增，纯化，冻存于无RNA酶的超纯水中，得到注射液C；
    (3)采用显微注射法将注射液A和注射液B分别注射进不同的大鼠受精卵的胞质中，然后将大鼠受精卵分别移植入不同假孕雌鼠体内，培育得到第二代大鼠A和第二代大鼠B；
    (4)采用显微注射法将注射液C注射进大鼠受精卵的原核中，然后将大鼠受精卵移植入假孕雌鼠体内，培育得到第二代大鼠C；
    (5)将第二代大鼠A、第二代大鼠B和第二代大鼠C进行杂交，繁殖F1代；再将F1代进行杂交，得到敲除了IL2Rγ和Prkdc基因并转入了人SIRPa基因的免疫缺陷大鼠模型。
  2. 根据权利要求1所述的免疫缺陷大鼠模型的构建方法，其特征在于，步骤(1)中的敲除大鼠Prkdc基因采用两条gRNA序列，分别为：第一外显子gRNA序列1：TTCCGGCACTATGGCGGACC；第一外显子gRNA序列2：GCCAGTTACCAGCTGATCCG。
  3. 根据权利要求1所述的免疫缺陷大鼠模型的构建方法，其特征在于，步骤(1)中的敲除大鼠IL2Rγ基因采用两条gRNA序列，分别为：第二外显子gRNA序列1：CAGCCGACCAACCTCACTAT；第四外显子gRNA序列2：GAGTGAATCTCAGGTAGAAC。
  4. 根据权利要求3所述的免疫缺陷大鼠模型的构建方法，其特征在于，敲除大鼠IL2Rγ基因还采用另外两条gRNA序列，分别为：第二外显子gRNA序列1：CAGCCGACCAACCTCACTAT；第四外显子gRNA序列3：GAGCAACCGAGATCGAAGCT。
  5. 根据权利要求1所述的免疫缺陷大鼠模型的构建方法，其特征在于，步骤(1)中，体外转录CAS9蛋白mRNA采用T7转录试剂盒、T3转录试剂盒或SP6转录试剂盒。
  6. 根据权利要求1所述的免疫缺陷大鼠模型的构建方法，其特征在于，步骤(3)和步骤(4)中的假孕雌鼠在SPF级别条件下饲养。
  7. 根据权利要求1所述的免疫缺陷大鼠模型的构建方法，其特征在于，步骤(3)中的大鼠受精卵取自在SPF级别条件下饲养的受孕雌鼠。
  8. 一种免疫缺陷大鼠模型，其特征在于，所述大鼠模型敲除了IL2Rγ和Prkdc基因并转入了人SIRPa基因。
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