CN113106101A - 一种nod遗传背景双基因缺陷小鼠模型的制备方法及应用 - Google Patents
一种nod遗传背景双基因缺陷小鼠模型的制备方法及应用 Download PDFInfo
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Abstract
本发明提供一种NOD遗传背景双基因免疫缺陷小鼠模型的制备方法，利用基因编辑技术敲除NOD小鼠的RAG1基因的第2号外显子全部编码区DNA片段以及JAK3基因上包括18‑21号外显子序列在内的DNA片段；敲除的后的RAG1基因序列如SEQ ID NO:9所示，敲除的后的JAK3基因序列如SEQ ID NO:10所示。NOD遗传背景小鼠中利用CRISPR/Cas9***同时敲除RAG1与JAK3基因在国内外尚属首次，这也是首例RAG1和JAK3双敲基因的纯合子小鼠免疫缺陷小鼠模型，具有很高的原创性和非常重要的基础研究和实际应用价值。
Description
技术领域
本发明属于基因工程技术领域，具体涉及一种NOD遗传背景中RAG1基因与JAK3基因同时缺失的小鼠模型的制备方法及应用。
背景技术
RAG1基因(recombination activating gene 1,重组活化基因1)位于小鼠2号染色体，包含2个外显子，编码区全部位于2号外显子，该基因编码由1040个氨基酸残基组成的Rag1蛋白。淋巴细胞独有的体细胞DNA重排现象也称为V(D)J重组，是免疫反应中B细胞产生抗体和T细胞受体多样性的分子基础。Rag1与其家族蛋白Rag2组成异二聚体，参与免疫球蛋白V(D)J重组过程中的DNA切口的引入(Ru,H.,P.Zhang,and H.Wu,Structural gymnasticsof RAG-mediated DNA cleavage in V(D)J recombination.Curr Opin Struct Biol,2018.53:178-186；Schatz,D.G.,M.A.Oettinger,and D.Baltimore,The V(D)Jrecombination activating gene,RAG-1.Cell,1989.59(6):1035-48.)，可以发生V(D)J重组的基因座包括B淋巴细胞的免疫球蛋白重链、κ和λ轻链，以及T淋巴细胞的T细胞受体α、β、γ和δ链。该基因缺失小鼠，由于V(D)J重组过程受阻而不能产生成熟的T细胞。由于没有T细胞的辅助，B细胞的成熟也会受影响(Mombaerts,P.,et al.,RAG-1-deficient mice haveno mature B and T lymphocytes.Cell,1992.68(5):869-77)。但Rag1功能缺失并不会影响天然免疫反应***，比如NK细胞的成熟，在Rag1缺失小鼠的非淋巴组织中甚至能观察到NK细胞数量有所增加(Grundy,M.A.and C.L.Sentman,Immunodeficient mice haveelevated numbers of NK cells in non-lymphoid tissues.Exp Cell Res,2006.312(19):p.3920-6)。
JAK3基因位于小鼠8号染色体，包含23个外显子，其编码蛋白属于Janus激酶家族成员的受体酪氨酸激酶。其中Jak3蛋白主要表达在免疫细胞中，是细胞因子IL-2/4/7/9/15/21gamma受体亚单位的下游信号分子，介导受体/JAK3/STAT信号通路。由于骨髓中NK前体细胞成熟需要IL2/4/15/21的刺激，胸腺中T细胞的成熟需要IL2/4/7/15/21参与。JAK3基因功能受损直接影响这些细胞因子的信号传导。因此，JAK3基因突变个体可出现T、B细胞和NK细胞的缺失(Thomis,D.C.,et al.,Defects in B lymphocyte maturation and Tlymphocyte activation in mice lacking Jak3.Science,1995.270(5237):794-7；Robinette,M.L.,et al.,Jak3 deficiency blocks innate lymphoid celldevelopment.Mucosal Immunol,2018.11(1):50-60.)。
NOD小鼠是日本学者对远交系Jcl：JCR鼠进行近交培育第6代时从白内障易感亚系中分离出非肥胖糖尿病品系(NOD)和非肥胖正常品系(nod)，是I型糖尿病研究和人源化模型建立中使用最为广泛的小鼠品系，目前一些经典的人源化小鼠模型如NOG和NSG等均是在NOD小鼠遗传背景中制作获得的。目前世界范围内还没有关于RAG1基因与JAK3基因同时缺失的免疫缺陷小鼠模型制作成功的报道。这是由于NOD小鼠是Ⅰ型糖尿病小鼠，单独敲除JAK3后的纯合子小鼠，生长至6-7周就会患有糖尿病，导致新生仔死亡率很高；该小鼠9-12周时，肚子非常大，根本无法怀孕。因此，RAG1基因敲除的纯合子与JAK3基因敲除的纯合子很难整合在同一只小鼠上。所以，在NOD遗传背景小鼠中制作获得RAG1基因与JAK3基因同时缺失的免疫缺陷小鼠模型，将为开启此类小鼠在异种移植中的研究提供更好的小鼠模型，也为人源化小鼠的研究开启全新的视野。
发明内容
为了解决上述技术问题，本发明提供了一种NOD遗传背景下RAG1基因与JAK3基因同时缺失的小鼠模型的制备方法，该方法可操作性强，构建成功率高。
为了实现上述目的，本发明所采用的技术方案是：
一种NOD遗传背景双基因缺陷小鼠模型的制备方法，利用基因编辑技术敲除NOD小鼠的RAG1基因的第2号外显子全部编码区DNA片段以及JAK3基因上包括18-21号外显子序列在内的DNA片段；敲除的后的RAG1基因序列如SEQ ID NO:9所示，敲除的后的JAK3基因序列如SEQ ID NO:10所示。
本发明首次在NOD遗传背景小鼠模型中利用CRISPR/Cas9***实现了对控制小鼠免疫***的关键基因RAG1与JAK3进行特异性敲除，获得一种在NOD遗传背景下重症联合免疫缺陷表型的小鼠动物模型，该模型小鼠中免疫细胞完全消失。本发明敲除了RAG1与JAK3基因的部分份片段，敲除后的RAG1基因序列如SEQ ID NO:9所示，敲除后的JAK3基因序列如SEQ ID NO:10所示。
优选地，所述基因编辑技术为CRISPR-Cas9基因编辑工具。
优选地，所述的方法，包括以下步骤：
(1)针对NOD小鼠的RAG1基因的第2号外显子设计两端sgRNA识别位点，其识别序列如下：
Ragl-5sgRNA-1：TAATAGGTACCAGGGACGTTGGG(SEQ ID NO:1)
Ragl-5sgRNA-2：CTAATAGGTACCAGGGACGTTGG(SEQ ID NO:2)
Ragl-3sgRNA-1：CTTGGAGCAGCGGTAGCTGCAGG(SEQ ID NO:3)
Ragl-3sgRNA-2：AGCGGTAGCTGCAGGGGACCAGG(SEQ ID NO:4)；
(2)针对NOD小鼠的JAK3基因的第18号至21号外显子设计两端sgRNA识别位点，其识别序列如下：
Jak3-17sgRNA-1：CACAGACTGGCGTCACTGCATGG(SEQ ID NO:5)
Jak3-17sgRNA-2：CGACAAGATGTTCTCATCTGAGG(SEQ ID NO:6)
Jak3-21sgRNA-1：TTCTAGCTCCGTCCCTCCGCAGG(SEQ ID NO:7)
Jak3-21sgRNA-2：TCTAGCTCCGTCCCTCCGCAGGG(SEQ ID NO:8)；
(3)利用体外转录技术获得相应sgRNA mRAN和Cas9 mRNA；可利用本领域的CRISPR/Cas9***试剂盒获得sgRNA mRAN和Cas9 mRNA。
(4)将sgRNA mRAN和Cas9 mRNA共同注射入NOD小鼠受精***内，然后将所述受精卵移植入受体母鼠产生RAG1与JAK3双基因缺失NOD小鼠模型。
发明人通过大量实验研究发现选择敲除的位点是基因失活的关键因素。即使技术上可以敲除该基因，但获得的小鼠不一定具有相关的生理、病理表型。本发明中试图敲除了RAG1基因上的其他位点，通过检测该小鼠的免疫细胞，发现其仍然含有与未敲除RAG1基因小鼠相同免疫细胞数。如图5所示，RAG1敲除NOD小鼠中，其T细胞，B细胞，NK细胞与正常NOD小鼠比较，没有太大区别。本发明中还试图敲除了JAK3基因上的其他位点，通过检测该小鼠的免疫细胞，发现其仍然含有与未敲除JAK3基因小鼠相同免疫细胞数。如图6所示，JAK3基因敲除的NOD小鼠中，其T细胞，B细胞，NK细胞与正常NOD小鼠比较，没有太大区别。而本发明中RAG1与JAK3双基因敲除NOD小鼠中，如图4所示，完全没有检测到T细胞和B细胞，NK细胞比正常NOD小鼠明显少。这表明，本发明sgRNA结合的靶点可以彻底不表达支持T细胞，B细胞，NK细胞成熟的受体，获得完全不具备免疫能力的NOD小鼠。
另外，由于NOD小鼠是Ⅰ型糖尿病小鼠，单独敲除JAK3后的纯合子小鼠，生长至6-7周就会患有糖尿病，导致新生仔死亡率很高；该小鼠9-12周时，肚子非常大，根本无法怀孕。因此，RAG1基因敲除的纯合子与JAK3基因敲除的纯合子很难整合在同一只小鼠上。本发明选择敲除的sgRNA靶点还减轻了NOD小鼠糖尿病的症状，推迟了糖尿病的发病时间，为繁殖争取了时间，提高了受孕机率，先有RAG1纯合子小鼠，体内抑制T细胞的成熟，才能会有JAK3纯合子小鼠，否则JAK3小鼠糖尿病发病非常早。
进一步地，所述方法还包括利用引物对F0代NOD小鼠的基因型进行鉴定的步骤。
进一步地，所述鉴定F0代NOD小鼠基因型使用的引物如下：
Rag1正向引物：5'CTGGGAAGCATGGGTGAGC 3'(SEQ ID NO:11)
Rag1反向引物：5'TTGGGCAGTAAGAAAATGTGGAC 3'(SEQ ID NO:12)
Jak3正向引物：5'GGAGCCCGCCAAAGTCAGAACC3'(SEQ ID NO:13)
Jak3反向引物：5'CAGGCCCCATCATGCTCAGGAACT 3'(SEQ ID NO:14)。
进一步地，所述方法还包括检查F0代NOD小鼠的F2代小鼠免疫***的指标和检查病理学组织的步骤。
进一步地，所述检查F0代NOD小鼠的F2代小鼠免疫***的指标采用流式细胞分边方法。
进一步地，所述方法还包括将经基因型鉴定为双基因缺失的F0代NOD小鼠与NOD小鼠杂交，获得RAG1与JAK3双基因敲除杂合F1代子鼠；再将所述F1代子鼠自行交配，获得RAG1与JAK3双基因敲除阳性纯合子的F2代子鼠。
本发明还提供了所述方法在制备肿瘤、移植、免疫学、炎症领域研究的动物模型的应用。
本发明还提供了如SEQ ID NO:1～4所示的DNA片段在靶向敲除小鼠RAG1基因2号外显子中作为sgRNA特异性识别的靶序列的应用；以及如SEQ ID NO:1～4所示的DNA片段在靶向敲除小鼠JAK3基因18～21号外显子中作为sgRNA特异性识别的靶序列的应用。
本发明的有益效果为：NOD遗传背景小鼠中利用CRISPR/Cas9***同时敲除RAG1与JAK3基因在国内外尚属首次，这也是首例RAG1和JAK3双敲基因的纯合子小鼠免疫缺陷小鼠模型，具有很高的原创性和非常重要的基础研究和实际应用价值。
附图说明
图1为针对RAG1基因的外显子2进行敲除的方案示意图；
图2为针对JAK3基因的外显子18～21进行敲除的方案示意图；
图3为F0代双基因敲除NOD的基因鉴定结果示意图(最终显示9号和64号子鼠为RAG1/JAK3双基因敲除阳性小鼠)；
图4为RAG1/JAK3双基因敲除NOD小鼠流式细胞仪检测免疫细胞结果示意图(最终显示该小鼠没有检测出T细胞和B细胞，NK细胞比正常NOD小鼠明显少)；
图5为RAG1基因敲除其他位点的NOD小鼠流式细胞仪检测免疫细胞结果示意图(最终显示该小鼠T细胞，B细胞，NK细胞与正常NOD小鼠比较，没有太大区别)；
图6为JAK3基因敲除其他位点的NOD小鼠流式细胞仪检测免疫细胞结果示意图(最终显示该小鼠T细胞，B细胞，NK细胞与正常NOD小鼠比较，没有太大区别)。
具体实施方式
下为了更加简洁明了的展示本发明的技术方案、目的和优点，下面结合具体实施例详细说明本发明的技术方案。如无特殊说明，本发明实施例中所涉及的试剂均为市售产品，均可以通过商业渠道购买获得。
实施例1RAG1与JAK3基因缺失NOD小鼠的制备方法
1、确定敲除区域
根据RAG1基因结构域选择将外显子2的全部区域敲除，敲除后的RAG1基因序列如SEQ ID NO:1所示。根据JAK3基因结构域选择将外显子18～21的全部区域敲除。
2、确定sgRNA在RAG1与JAK3基因上的识别靶位点序列：
根据如图1所示的敲除方案针对RAG1基因的外显子2进行敲除，两端sgRNA识别位点分别位于小鼠Ragl基因的Intron 1和3'UTR上，根据外显子Intron 1和3'UTR分别设计sgRNA序列结合的靶点：
intron 1序列如下：
AAACAATTATTGAGCACCTAATAGGTACCAGGGACGTTGGGAGATGAAATTAGTCAAAGGCTCTGTGTTCAAAGATGTCAAGGTTTTTGTGGAAAGGGAATTAAATTTCACATATACATGTATTTAAAATCATGCATATATTTAGTATAAGTGTCCCCAAATATTGTCAG
sgRNA识别靶序列如下：
Ragl-5sgRNA-1：TAATAGGTACCAGGGACGTTGGG(SEQ ID NO:1)
Ragl-5sgRNA-2：CTAATAGGTACCAGGGACGTTGG(SEQ ID NO:2)
3'UTR序列如下：
GAGCCGTTTAGTGAGGCCAGAAGAGCAACAGGAGAAATCAGTTATTTGGAAGCTCAATAACTTGGAGCAGCGGTAGCTGCAGGGGACCAGGGATGCACAGAGATATGTGTGTGCATGCCACTGTGTGCCATGAAAATTGAAGCCAAGGCTGTC
sgRNA识别靶序列如下：
Ragl-3sgRNA-1：CTTGGAGCAGCGGTAGCTGCAGG(SEQ ID NO:3)
Ragl-3sgRNA-2：AGCGGTAGCTGCAGGGGACCAGG(SEQ ID NO:4)
根据如图2所示的敲除方案针对JAK3基因的外显子18～21进行敲除，两端sgRNA识别位点分别位于小鼠Jak3基因的intron 17和intron 21上，根据intron 17和intron 21分别设计sgRNA序列结合的靶点：
Intron 17序列如下：
TCATGGGTGCTGGGATTTGGTTTTATTTTGTTATTCTTAAATTTTGTCGACAAGATGTTCTCATCTGAGGGTATCCAAGTCACAGACTGGCGTCACTGCATGGCTCTGTCTCTCGGGTCC
sgRNA识别靶序列如下：
Jak3-17sgRNA-1：CACAGACTGGCGTCACTGCATGG(SEQ ID NO:5)
Jak3-17sgRNA-2：CGACAAGATGTTCTCATCTGAGG(SEQ ID NO:6)
Intron 21序列如下：
CCCACCCCACAGAGTGATGCTCCACTCGGTTTAGCCACGCCCCCCATTGTTCTGGCTCCATCCTCCTTGACCAGTCTGCAAAGCCCGTCACAGGTCTCTTTTCTAGCTCCGTCCCTCCGCAGGGCCCTGCCTTTCACGCTCTATGGGGTC
sgRNA识别靶序列如下：
Jak3-21sgRNA-1：TTCTAGCTCCGTCCCTCCGCAGG(SEQ ID NO:7)
Jak3-21sgRNA-2：TCTAGCTCCGTCCCTCCGCAGGG(SEQ ID NO:8)
3、体外转录
(1)体外转录获得上述相应的sgRNA mRNA，具体方法如下：
使用T7-ShortScript体外转录试剂盒(AM1354)进行sgRNA的转录：以PrimerStarMax体系(表1)，sgRNA–F、sgRNA-R为引物，测序正确的puc57-sgRNA质粒(1:30稀释)为模板进行PCR，PCR产物进行纯化，制备sgRNA转录模板。，获得体外转录产物；使用Ambion RNA纯化试剂盒(Ambion，AM1909)按照说明书要求回收条带得sgRNA mRNA。
表1：PCR反应体系
| 试剂(TakaraR045) | 体积(μ1) | 规格 |
| Prime STARMaxPremix(2x) | 12 | / |
| ddH<sub>2</sub>O | 10 | / |
| Primer | 1 | 10μM |
| Primer | 1 | 10μM |
| Template | 1 |
(2)获得Cas9核酸酶的mRNA
体外转录获得Cas9核酸酶的mRNA：严格按照体外转录试剂盒的invitrogen试剂盒(invitrogen，AMB1345-5)的使用说明书操作获得体外转录产物以后，使用Ambion RNA纯化试剂盒(Ambion，AM1909)回收Cas9mRNA。
4、RAG1与JAK3基因缺失NOD小鼠模型的建立
将获得的sgRNA mRNA和Cas9 mRNA通过显微注射***注射入NOD内小鼠的受精***内，并将受精卵植入假孕母鼠子宫。出生后的子鼠为RAG1/JAK3双基因敲除鼠的F0代子鼠。基因鉴定选择RAG1/JAK3双基因敲除阳性F0代子鼠和NOD鼠杂交，获得RAG1/JAK3双基因敲除杂合F1代子鼠。再将F1代子鼠进行交配，可获得RAG1/JAK3双基因敲除阳性F2代子鼠。
5、RAG1与JAK3基因缺失NOD小鼠基因组鉴定
基因鉴定的具体步骤包括以下步骤：
(1)鼠尾裂解和基因组DNA模板的准备
鼠尾加入90μl裂解液A(500mM NaOH)，95℃保温30分钟。然后加入裂解液B(50mMTris-HCl，pH 8.2)，混匀后10000rpm离心1分钟，上清液即为基因鉴定PCR的模板。
(2)基因鉴定PCR引物序列和产物大小如下表2所示：
表2：
| Rag1正向引物 | 5'CTGGGAAGCATGGGTGAGC 3'(SEQ ID NO:11) |
| Rag1反向引物 | 5'TTGGGCAGTAAGAAAATGTGGAC 3'(SEQ ID NO:12) |
| 产物长度 | 野生型：5.6Kb；敲除突变型：1.8Kb |
| Jak3正向引物 | 5'GGAGCCCGCCAAAGTCAGAACC3'(SEQ ID NO:13) |
| Jak3反向引物 | 5'CAGGCCCCATCATGCTCAGGAACT 3'(SEQ ID NO:14) |
| 产物长度 | 野生型：2.9Kb；敲除突变型：1.4Kb |
基因鉴定PCR体系及条件如表3、4所示：
表3：
表4：
(3)PCR产物用1％琼脂糖凝胶电泳分离并拍照记录。
F0代小鼠基因鉴定结果如图3所示，其中9号和64号子鼠为RAG1/JAK3双基因敲除阳性NOD小鼠。
6、RAG1与JAK3基因缺失NOD小鼠免疫***指标评价
获得的RAG1与JAK3基因缺失NOD小鼠具有免疫***不健全的病理表型，会引起小鼠免疫***的紊乱(即：无发育成熟的T、B细胞、NK细胞)，对于确认品系制作有效性至关重要。以流式细胞分边术检测小鼠免疫指标(主要为T/B/NK细胞)，判定小鼠的免疫***指标。收集野生和RAG1/JAK3双基因敲除阳性小鼠外周血淋巴细胞，经裂解红细胞、Fc受体阻断后，细胞用CD45/CD3/CD19/CD4/CD8/CD49b抗体染色，进行流式细胞术分析(外周血白细胞FACS鉴定(所有细胞均为CD45+)；B细胞：CD3-CD19+；T细胞：CD3+CD19-；T helper：CD3+CD4+CD8-；T cytotoxic：CD3+CD4-CD8+；NK：CD3-CD49b+)。
结果如图4所示，RAG1/JAK3双敲除NOD小鼠的外周血中，没有发现T细胞、B细胞，NK细胞比正常NOD小鼠明显少。这表明本发明构建的NOD小鼠具有严重的免疫缺陷生理特征。
最后所应当说明的是，以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制，尽管参照较佳实施例对本发明作了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的实质和范围。
序列表
<110> 广州欣意生物技术有限公司
<120> 一种NOD遗传背景双基因缺陷小鼠模型的制备方法及应用
<141> 2021-05-11
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<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 6
cgacaagatg ttctcatctg agg 23
<210> 7
<211> 23
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 7
ttctagctcc gtccctccgc agg 23
<210> 8
<211> 23
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 8
tctagctccg tccctccgca ggg 23
<210> 9
<211> 852
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 9
cctttcaagt agtgagtaat tagtttcttt gggtttgtag ctttatcatc tctttatgac 60
ccgttcagaa gaaataaaca accaatctcc cggaagaatg ctttaaaaat ctagaaatat 120
attgtccagt tagtgtaatg aggctaatac aatgtgagaa atattacttt tctctggttt 180
agtatccagt ttatcattgc atttattcac aaacaattat tgagcaccta ataggtacca 240
gggatgcaca gagatatgtg tgtgcatgcc actgtgtgcc atgaaaattg aagccaaggc 300
tgtcaaagga cggccaagca tgctaggaaa gaaatttgtc ttgggcatgg tttttttttc 360
cacttcaatt tattatattt tgtgctgaga tctatgcagt ttttttgcca tgtcatattt 420
tttggtcata ttttatagga ctattttgta aatttcacat atgttgattt atcctacaag 480
atttaaagga gcctttgagg tttctctaac aaccctgtac atgtgtagga cgtggtggcc 540
tttccaaggg atctcagtgc attttctcgt tgctacattt atgcataaat aaattaaacc 600
aagagaggtt tcagaaaatc atccttttcc aggtttaaat ggcctcagta ttgtcacctt 660
cagtgtctag gaggtcatct ggtgtcataa atttttcaat aatgaagtta gagaatgcac 720
ctgttagcta cagctaccta ttaagtataa taaagtcatg cttgcttagt tatctagagc 780
cagctctggt tgtttatttt tactttttaa ttaagagtca tttatagcct tagtgctttt 840
ctatgtaatc tc 852
<210> 10
<211> 958
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 10
ggggggcggc aggggcggag agggtccctg agctgggtcc aggaagtttg ctggttctag 60
cagtcagcgt gaactaagac agctgctgca acaaacctaa gacccgatct gcatttgttt 120
tgcagtgtag cccaggttgg tgttttacaa tgtagcccaa actagcatct caggctggat 180
ttctccctgc ttcaccatca tgggtgctgg gatttggttt tattttgtta ttcttaaatt 240
ttgtcgacaa gatgttctca tctgagggta tccaagtcac agaccctgcc tttcacgctc 300
tatggggtcc gcccctctta gcgtcttcta cttctccgtc catcttactc tgctttcccg 360
tcgctggctc ctccccttat gacatttacg cctatctgcc caggcacttt tccagctgcc 420
tgtcatccaa tgctgaccca ctcaaagccc cgtccttaac acttagagtc cctctctcag 480
cctggccacg cccacaggtc accagacagc ctctgagtcg ccttctgggg gccgtgctag 540
taggaagtct cctgttcacg cccctttcct caccccagac tggccatact tcacattgac 600
gactccctcc ccaggccact gtttcacacc ctgcccactc tcctcactgg ccacaccaca 660
cctctgtccg gctctcaggt atgccccgga gtccctatct gacaacatct tctcccgcca 720
atctgacgtg tggagcttcg gagtggtgtt gtacgagctc ttcacctact gcgacaagag 780
ctgcagccca tccgctgtgc gtcgcctgcc ccatccctcg gtcctccctc ttgatctcca 840
aatcccctcc tgacctctag cccctatcct gaccccagcc cttcctcctg acctccagat 900
catctcctga cctcagtcct ccctccggaa tcccagccct tcctcctcat ctccagat 958
<210> 11
<211> 19
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 11
ctgggaagca tgggtgagc 19
<210> 12
<211> 23
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 12
ttgggcagta agaaaatgtg gac 23
<210> 13
<211> 22
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 13
ggagcccgcc aaagtcagaa cc 22
<210> 14
<211> 24
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 14
caggccccat catgctcagg aact 24
Claims (10)
1.一种NOD遗传背景双基因免疫缺陷小鼠模型的制备方法，其特征在于，利用基因编辑技术敲除NOD小鼠的RAG1基因的第2号外显子全部编码区DNA片段以及JAK3基因上包括18-21号外显子序列在内的DNA片段；敲除的后的RAG1基因序列如SEQ ID NO:9所示，敲除的后的JAK3基因序列如SEQ ID NO:10所示。
2.如权利要求1所述的制备方法，其特征在于，所述基因编辑技术为CRISPR-Cas9基因编辑工具。
3.如权利要求2所述的方法，其特征在于，包括以下步骤：
(1)针对NOD小鼠的RAG1基因的第2号外显子设计两端sgRNA识别位点，其识别序列如下：
Ragl-5sgRNA-1：TAATAGGTACCAGGGACGTTGGG(SEQ ID NO:1)
Ragl-5sgRNA-2：CTAATAGGTACCAGGGACGTTGG(SEQ ID NO:2)
Ragl-3sgRNA-1：CTTGGAGCAGCGGTAGCTGCAGG(SEQ ID NO:3)
Ragl-3sgRNA-2：AGCGGTAGCTGCAGGGGACCAGG(SEQ ID NO:4)；
(2)针对NOD小鼠的JAK3基因的第18号至21号外显子设计两端sgRNA识别位点，其识别序列如下：
Jak3-17sgRNA-1：CACAGACTGGCGTCACTGCATGG(SEQ ID NO:5)
Jak3-17sgRNA-2：CGACAAGATGTTCTCATCTGAGG(SEQ ID NO:6)
Jak3-21sgRNA-1：TTCTAGCTCCGTCCCTCCGCAGG(SEQ ID NO:7)
Jak3-21sgRNA-2：TCTAGCTCCGTCCCTCCGCAGGG(SEQ ID NO:8)；
(3)利用体外转录技术获得相应sgRNA mRAN和Cas9 mRNA；
(4)将sgRNA mRAN和Cas9 mRNA共同注射入NOD小鼠受精***内，然后将所述受精卵移植入受体母鼠产生RAG1与JAK3双基因缺失NOD小鼠模型。
4.如权利要求3所述的制备方法，其特征在于，所述方法还包括利用引物对F0代NOD小鼠的基因型进行鉴定的步骤。
5.如权利要求4所述的制备方法，其特征在于，所述鉴定F0代NOD小鼠基因型使用的引物如下：
Rag1正向引物：5'CTGGGAAGCATGGGTGAGC 3'(SEQ ID NO:11)
Rag1反向引物：5'TTGGGCAGTAAGAAAATGTGGAC 3'(SEQ ID NO:12)
Jak3正向引物：5'GGAGCCCGCCAAAGTCAGAACC3'(SEQ ID NO:13)
Jak3反向引物：5'CAGGCCCCATCATGCTCAGGAACT 3'(SEQ ID NO:14)。
6.如权利要求5所述的制备方法，其特征在于，所述方法还包括检查F0代NOD小鼠的免疫***的指标和检查病理学组织的步骤。
7.如权利要求6所述的制备方法，其特征在于，所述检查F0代NOD小鼠的F2代小鼠免疫***的指标采用流式细胞分边方法。
8.如权利要求7所述的制备方法，其特征在于，所述方法还包括将经基因型鉴定为双基因缺失的F0代NOD小鼠与NOD小鼠杂交，获得RAG1与JAK3双基因敲除杂合F1代子鼠；再将所述F1代子鼠自行交配，获得RAG1与JAK3双基因敲除阳性纯合子的F2代子鼠。
9.如权利要求1～8任一项权利要求所述的方法在制备肿瘤、移植、免疫学、炎症领域研究的动物模型的应用。
10.如SEQ ID NO:1～4所示的DNA片段在靶向敲除小鼠RAG1基因2号外显子中作为sgRNA特异性识别的靶序列的应用；如SEQ ID NO:1～4所示的DNA片段在靶向敲除小鼠JAK3基因18～21号外显子中作为sgRNA特异性识别的靶序列的应用。
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