CN113481238A - 制备IL-2Rg敲除非人动物模型的方法及其应用 - Google Patents
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Abstract
本发明涉及分子生物学与生物医学技术领域,具体而言,涉及一种制备IL‑2Rg敲除非人动物模型的方法及其应用。所述方法包括:使用Crispr‑Cas系统将所述动物IL‑2Rg基因敲除;其中所述Crispr‑Cas系统包括sgRNA和Cas9酶,所述sgRNA包括对应的靶位点序列为SEQ ID NO:1和2所示的sgRNA1和sgRNA2。本发明使用CRISPR/Cas9技术通过特定设计的RNA(sgRNA)介导核酸内切酶Cas9蛋白进行靶向DNA序列识别并造成DNA双链断裂,以同源重组或非同源末端连接方式修复受损DNA,从而实现对目标位点实现基因的定点敲除。
Description
技术领域
本发明涉及分子生物学与生物医学技术领域,具体而言,涉及一种制备IL-2Rg敲除非人动物模型的方法及其应用。
背景技术
白介素(IL)即是由多种细胞产生并作用于多种细胞的一类细胞因子。IL在传递信息,激活与调节免疫细胞,介导T、B细胞活化、增殖与分化及在炎症反应中起重要作用。白介素-2(IL-2)又称T细胞生长因子,主要由活化的CD4+T细胞和CD8+T细胞产生,是所有T细胞亚群的生长因子,并可促进活化B细胞增殖,故为调控免疫应答的重要因子,也参与抗体反应、造血和肿瘤监视。白介素2是一种抗原激活作用发生后产生的多效细胞因子,这种因子在免疫应答中起着重要的作用。白介素2是以T细胞生长因子的身份被发现的,因此它还可以促进CD8+T细胞和NK细胞的裂解活性并对T细胞在应答抗原时的分化起调节作用;它还可以促进未免疫CD4+T细胞分化形成1型辅助性T细胞(Th1)和2型辅助性T细胞(Th2),同时抑制Th17和滤泡辅助性T细胞(Tfh)的分化。还有,IL-2是形成和维护辅助性T细胞以及激活作用诱导性细胞死亡所必需的,生物体可以通过这些作用来调节免疫耐受并对不合适的免疫反应进行限制。IL-2的靶细胞包括T细胞、NK细胞、B细胞及单核-巨噬细胞等。这些细胞表面均可表达白介素-2受体(IL-2R)。IL-2R包含3条多肽链:α链、β链、γ链,α链的胞内区较短,不能向细胞内传递信号,而β链和γ链的胞内区较长,具有传递信号的能力。3种肽链单独与IL-2结合亲和力较低,只有同时表达才能产生高度亲和力。白介素2受体γ链(interleukin2receptor subunit gamma chain,IL2-Rg,或CD132),是多种重要免疫因子的共同受体亚基,包括IL-2、IL-4、IL-7、IL-9、IL-15和IL-21,因此,也被称为受体共同γ链(commongamma chain,γc)。白介素2受体γ链(interleukin 2receptor subunit gamma chain,IL2-Rg,或CD132),是多种重要免疫因子的共同受体亚基,包括IL-2、IL-4、IL-7、IL-9、IL-15和IL-21,因此,也被称为受体共同γ链(common gamma chain,γc)。IL2-Rg是一种表达于大部分淋巴细胞表面的糖蛋白,在哺乳动物中,IL2-Rg基因位于X染色体上。小鼠的IL2-Rg基因位于X染色体,全长3878bp,编码522个氨基酸,与人的蛋白序列同源性可达到99%。该蛋白在不同物种间高度保守。研究发现白介素2受体γ链缺失与T细胞和B细胞数量显著减少,缺乏NK细胞密切相关。在人体内,γc的突变可以导致X染色体连锁重症联合免疫缺陷(X-linked severe combined immunodeficiency,X-SCID),表现为T细胞和NK细胞缺失,B细胞数量正常但功能缺陷。X-SCID为X染色体连锁隐性遗传,患者绝大部分为男性,女性多为携带者,其在新生儿中的发病率大约在1:50,000-100,000间。X-SCID患者生长发育迟缓,且因为免疫系统的重度缺陷,极容易受病原微生物感染。通常,患有X-SCID的新生儿大多活不到两岁,但目前干细胞移植和基因疗法已被证明有效。有研究表明,IL-2R g部分突变雄性杂合子小鼠表现出T细胞和B细胞数量显著减少,NK细胞部分减少,肠道相关上皮内淋巴细胞数量严重减少。
有鉴于IL-2的重要作用,关于其的动物模型是本领域亟需的。传统的基因打靶技术制备基因敲除(Knock out)/敲入(Knock in)小鼠是建立在DNA同源重组与胚胎干细胞等技术基础上的分子生物学技术。基因打靶就是通过同源重组技术将外源基因定点整合入靶细胞基因组上某一确定的位点,以达到定点修饰改造染色体上某一基因的目的。例如TALEN(Transcription Activator-Like Effector Nuclease)技术是基于对DNA识别的TALEN臂和人工改造的核酸内切酶的切割域(FokⅠ)的结合对细胞基因组进行修饰而实现的。TALEN的DNA识别域是由一些非常保守的重复氨基酸序列模块组成,每个模块由34个氨基酸组成,其中第12和13位的氨基酸种类为可变的(RVDs),且决定了该模块识别靶向位点的特异性。通过DNA识别域结合到靶位点上,以及Fok I的切割域形成二聚体后,可特异性对目标基因DNA实现切断。在非同源末端连接修复过程中,DNA双链断开后会由于碱基的随机增减造成目标基因功能缺失。传统的基因打靶技术包括TALEN技术构建周期长达12个月,需要首先得到相应的干细胞,然后将干细胞植入囊胚,并生产出嵌合型动物,并通过杂交最终得到纯合型动物,整个过程耗时严重,效率低。此外,目前IL-2Rg-/-敲除小鼠采用的是部分基因敲除,产生的免疫缺陷小鼠缺陷程度不完全。
有鉴于此,特提出本发明。
发明内容
本发明的第一目的在于提供一种制备IL-2Rg敲除非人动物模型的方法,包括:
使用Crispr-Cas系统将所述动物IL-2Rg基因敲除;
其中所述Crispr-Cas系统包括sgRNA和Cas9酶,所述sgRNA包括对应的靶位点序列为SEQ ID NO:1和2所示的sgRNA1和sgRNA2。
可选的,如上所述的方法,所述基因敲除的对象为受精卵。
可选的,如上所述的方法,所述Crispr-Cas系统以sgRNA和Cas9 mRNA的形式转入所述受精卵。
可选的,如上所述的方法,所述Crispr-Cas系统转入所述受精卵的方法为显微注射。
可选的,如上所述的方法,所述方法还包括将处理后受精卵移植到假孕雌性动物体内并产出F0代。
可选的,如上所述的方法,所述非人哺乳动物为免疫缺陷动物。
可选的,如上所述的方法,所述非人哺乳动物为啮齿类动物。
可选的,如上所述的方法,所述非人哺乳动物是小鼠(Mus musculus)。
可选的,如上所述的方法,所述非人哺乳动物是SCID小鼠。
本发明的第二目的在于提供如上所述方法得到的动物在鉴别和/或测试药物中的用途;
所述药物用于预防和/或治疗的适应症为IL-2相关免疫疾病、肿瘤组织移植、移植物抗宿主病中的任一项。
与现有技术相比,本发明的有益效果为:
现有的IL-2Rg-/-敲除小鼠是采用部分敲除外显子1的方法,容易出现免疫泄漏,NK细胞发育未能完全阻断,免疫缺陷不完全。本发明采用全基因敲除的方法,目标的编码区跨度大,虽然CRISPR技术能够大片段地剪掉DNA序列,实现大跨度的全身性基因敲除;但如果首尾两个外显子位点之间距离过大,仍会导致不仅构建模型的难度大、重组率低下,而且可能影响小鼠的最终使用效果。本发明使用CRISPR/Cas9技术通过特定设计的RNA(sgRNA)介导核酸内切酶Cas9蛋白进行靶向DNA序列识别并造成DNA双链断裂,以同源重组或非同源末端连接方式修复受损DNA,从而实现对目标位点实现基因的定点敲除。
附图说明
为了更清楚地说明本发明具体实施方式或现有技术中的技术方案,下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图是本发明的一些实施方式,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1为本发明利用CRISPR/Cas9技术建立I2rg全基因敲除小鼠的示意图;
图2为本发明一个实施例中sgRNA1溶液和sgRNA2溶液中sgRNA电泳检测结果;
图3为本发明一个实施例中F0代小鼠电泳鉴定结果;
图4为本发明一个实施例中不同IL2-Rg基因型小鼠的肝脏和脾脏中C57BL/6J蛋白的Western blot检测结果;
图5为本发明一个实施例中SCID-lL12Rg全敲除小鼠与IL-2Rg-/-部分敲除和裸鼠免疫细胞含量比较;
图6为本发明一个实施例中PDX模型建模成功率比较。
具体实施方式
现将详细地提供本发明实施方式的参考,其一个或多个实例描述于下文。提供每一实例作为解释而非限制本发明。实际上,对本领域技术人员而言,显而易见的是,可以对本发明进行多种修改和变化而不背离本发明的范围或精神。例如,作为一个实施方式的部分而说明或描述的特征可以用于另一实施方式中,来产生更进一步的实施方式。
除非另有定义,本文所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。本文中在本发明的说明书中所使用的术语只是为了描述具体的实施例的目的,不是旨在于限制本发明。本文所使用的术语“和/或”包括一个或多个相关的所列项目的任意的和所有的组合。
本发明涉及一种制备IL-2Rg敲除非人动物模型的方法,包括:
使用Crispr-Cas系统将所述动物IL-2Rg基因敲除;
其中所述Crispr-Cas系统包括sgRNA和Cas9酶,所述sgRNA包括对应的靶位点序列为SEQ ID NO:1和2所示的sgRNA1和sgRNA2。
本发明涉及一种基于CRISPR/Cas9基因敲除技术建立IL-2Rg基因敲除动物模型的方法,此处的“基因敲除”仅指针对目的基因的Cas9,对目的基因基因组的特定区域敲除,引发目的基因表达缺陷,从而达到基因敲除的效果。
sgRNA1和sgRNA2覆盖了全长的IL2-Rg,优于IL2-Rg部分敲除的设计。且SEQ IDNO:1和2所对应的靶点,脱靶效率低,敲除效率高。IL2-Rg是多种重要免疫因子的共同受体亚基,在免疫系统中发挥了重要作用。在人体内,它的突变或缺失可导致严重的免疫疾病。而我们可以利用IL-2Rg在多种免疫细胞发育分化增殖中的重要性,在动物例如SCID小鼠基础上敲除小鼠的全长IL2-Rg基因,构建免疫缺陷小鼠用于免疫肿瘤学等方面的研究。本发明的IL2-Rg基因突变的方法和动物模型,为研究肿瘤以及肿瘤免疫的药效学评价提供了便捷、可靠、经济的动物模型。
在本发明中,所述sgRNA可以包括SEQ ID NO:1和2中至少一种所示的核酸片段,或者与SEQ ID NO:1和2所示核酸片段实质上相同的核酸片段。
“实质上相同的核酸片段”是指在严格条件下能够与SEQ ID NO:1和2所对应的靶序列杂交的核酸片段。这样的核酸片段可以相较于SEQ ID NO:1和2替换、增加或减少1、2、3或更多个核酸碱基或碱基类似物【例如4-乙酰胞嘧啶、8-羟基-N6-甲基腺苷、吖丙啶基胞嘧啶、假异胞嘧啶、5-(羧基羟基甲基)尿嘧啶、5-氟尿嘧啶、5-溴尿嘧啶、Q核苷等】,或者是部分碱基具有某些修饰(例如甲基化修饰,通常这种修饰对gRNA与靶核酸的杂交是无关紧要的)。本发明中使用的“严格条件”是公知的,包括诸如在含400mM NaCl、40mM PIPES(pH6.4)和1mM EDTA的杂交液中于65℃杂交12~16小时,然后在65℃下用含0.1%SDS、和0.1%SSC的洗涤液洗涤15~60分钟。这对于本领域技术人员来说是熟悉的。
在一些实施方式中,所述Cas9来自化脓性链球菌或肺炎链球菌。
在一些实施方式中,所述基因敲除的对象为受精卵。
在一些实施方式中,所述Crispr-Cas系统以sgRNA和Cas9 mRNA的形式转入所述受精卵。
在一些实施方式中,所述Crispr-Cas系统通过载体介导转入所述基因敲除的对象。在一些实施方式中,常规的基于病毒的系统可以包括用于基因转移的逆转录病毒、慢病毒、腺病毒、腺相关和单纯疱疹病毒载体;上述病毒通常是复制缺陷的;优选慢病毒。
所述sgRNA和Cas9 mRNA及其相关生物材料,也属于本发明的保护范围。
所述sgRNA相关的生物材料为F1)至F7)中的任一种:
F1)编码所述sgRNA的核酸分子;
F2)含有F1)所述核酸分子的载体;
F3)含有F1)所述核酸分子的重组微生物;
F4)含有F1)所述核酸分子的转基因动物细胞系;
F5)含有F1)所述核酸分子的转基因动物组织;
F6)含有F1)所述核酸分子的转基因动物器官。
在一些方面,当前公开的主题提供包括将所述Crispr-Cas系统,或者相关的生物材料转入细胞的方法,具体可以将包括将上述提到的一种或多种多核酸,例如一种或多种本文所述的载体,其一种或多种转录物,和/或由其转录的一种或多种蛋白质递送至宿主细胞的方法。在一些方面,当前公开的主题还提供由这些方法产生的细胞,以及包含这些细胞或由这些细胞产生的生物体。上述产物可组成本发明所述的药用组合物。常规的基于病毒和非病毒的基因转移方法可用于在哺乳动物细胞或靶组织中引入核酸。这种方法可用于将编码CRISPR系统组分的核酸施用到培养中或宿主生物体中的细胞。非病毒载体递送系统包括DNA质粒,RNA(例如本文所述载体的转录物),裸核酸和与递送媒介物(例如脂质体)复合的核酸。病毒载体递送系统包括DNA和RNA病毒,其在递送至细胞后具有附加或整合的基因组。
非病毒递送核酸的方法包括脂转染,核转染,微注射,生物射弹,病毒体,脂质体,免疫脂质体,聚阳离子或脂质,核酸缀合物,裸DNA,人工病毒粒子和DNA的试剂增强摄取。
病毒递送核酸的方法可以直接施用到所述主体或可以用于体外处理细胞,且可以任选地将修饰的细胞施用到所述主体。用载体转染方法可以在宿主基因组中整合,通常导致插入的转基因的长期表达。另外,已经在许多不同细胞类型和靶组织中观察到高转导效率。
上述方法中,所述sgRNA1和所述sgRNA2均由各自的靶序列转录的RNA与sgRNA的骨架序列连接而成。所述sgRNA的骨架序列具体可由TS450载体(Addgene)中sgRNA的骨架序列对应的DNA序列转录。
在一些实施方式中,所述Cas9来自化脓性链球菌或肺炎链球菌。
在一些实施方式中,所述Crispr-Cas系统转入所述受精卵的方法为显微注射。
在一些实施方式中,所述方法还包括将处理后受精卵移植到假孕雌性动物体内并产出F0代。
在一些实施方式中,所述方法还包括将正确敲除CNGA1基因的F0代动物与野生型动物交配获得F1代杂合子动物。
在一些实施方式中,所述方法还包括将所述F1代杂合子动物自交获得F2代纯合子动物。
非人哺乳动物非限制性地包括牛、马、乳牛、猪、绵羊、山羊、大鼠、小鼠、狗、猫、兔、骆驼、驴、鹿、貂、鸡、鸭、鹅、火鸡、斗鸡等。在一些实施方式中,所述非人哺乳动物为啮齿类动物。
在一些实施方式中,所述非人哺乳动物是免疫缺陷动物。
在一些实施方式中,所述非人哺乳动物是小鼠(Mus musculus)。
小鼠品系可以选择BALB/c、C57BL、C3H/He、昆明小鼠、ICR、NIH、CFW、LACA、nude小鼠或Scid小鼠等。
在一些实施方式中,所述非人哺乳动物为SCID小鼠。
根据本发明的再一方面,本发明还涉及如上所述方法得到的动物在鉴别和/或测试药物中的用途;
所述药物用于预防和/或治疗的适应症为IL-2相关免疫疾病、肿瘤组织移植、移植物抗宿主病中的任一项。
在一些实施方式中,所述肿瘤包括但不限于胰腺癌、膀胱癌、结肠直肠癌、乳腺癌、前列腺癌、肾癌、肝细胞癌、肺癌、卵巢癌、宫颈癌、胃癌、食管癌、头颈部癌、黑素瘤、神经内分泌癌、CNS癌、脑肿瘤、骨癌和软组织肉瘤。
在一些实施方式中,所述肿瘤选自晚期或转移性恶性实体瘤。
在一些实施方式中,所述药物通过口服、皮下注射、肌肉注射、静脉输液等非手术的方式进行给药。
下面结合具体实施,进一步阐述本发明。应理解,这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法,通常按照常规条件,例如Sambrook等人,分子克隆:实验室手册(New York:Cold Spring Harbor LaboratoryPress,1989)中所述的条件,或按照制造厂商所建议的条件。除非另外说明,否则百分比和份数按重量计算。
实施例1IL2-Rg基因定点突变SCID小鼠模型的构建
本发明利用CRISPR/Cas9方法对SCID小鼠模型的IL2-Rg基因(NCBI gene ID:16186),进行定点突变。
技术路线如图1所示,具体操作方法如下:
图1利用CRISPR/Cas9技术建立IL2-Rg全基因敲除小鼠的示意图
本发明所有动物饲养与繁殖于SPF(Specific Pathogen Free)级实验动物设施。
本发明SCID小鼠来源于中国食品药品鉴定研究院国家啮齿类实验动物资源库
一、靶DNA的确定
根据IL2-Rg基因序列,确定利用CRISPR/Cas9方法突变IL2-Rg基因时所用两个sgRNA(分别记为sgRNA1和sgRNA2)的靶序列,sgRNA1的靶序列为为X染色体的第101264950-101264972位,PAM序列为TGG,sgRNA2的靶序列为为X染色体的第101268119-101268141位。PAM序列为AGG。外显子1-8被确定为靶位点。有效的敲除区域大小为3878bp.
靶位点序列:gRNA-A1:TGGGGGGAATCTCGCTGACG-TGG(SEQ ID NO:1);
gRNA-B1:GAGCAGCTGAAGGACTAAGA-AGG(SEQ ID NO:2)。
靶点确认:依据打靶细胞的基因组设计扩增靶位点的高特异性引物,PCR扩增获得含靶位点片段;
在靶位点中选择扩增片段唯一的限制性内切酶进行酶切电泳鉴定;酶切鉴定正确后,将PCR扩增产物
测序鉴定;通过酶切、测序鉴定,确认打靶鉴定引物的特异性;
基因合成分别与靶位点序列相同和互补的两条引物,即多聚核苷酸单链(Oligo),分别为S Oligo和AS Oligo;将反应组分加入1.5mL EP管中,置于沸水中加热10分钟后,冷却至室温,瞬间离心,获得编码guide RNA(gRNA)的含粘性末端的、线性化guide DNA序列(即L-gDNA)备用;
S Oligo序列:5’-AAACTACAGAAGTAAGCGTG-ACC-3’;AS Oligo序列:5’-ACCCCCCTTAGAGCGACTGC-ACC-3’。
通过DNA连接酶(如Takara公司Solution I),将L-pT7载体和L-gDNA连接成完整载体pT7-gDNA,转化、涂板、挑单克隆、摇菌、提取质粒DNA、酶切鉴定、质粒测序,筛选测序正确质粒备用;
gRNA体外转录
①以测序正确的pT7-gDNA质粒(1-30ng)为模版,以T7-S和Tracr-Rev为引物扩增gDNA基因片段,电泳、胶回收、溶解于30μL无(RNA)酶水中备用;
T7-S引物序列:5’-GAAATTAATACGACTCACTATA-3’
Tracr-Rev引物序列:5’-AAAAAAAGCACCGACTCGGTGCCAC-3’
②通过体外转录试剂盒T7Kit(Ambion公司)将PCR扩增后的gDNA基因片段体外转录成gRNA,利用mirVanaTM miRNA Isolation Kit试剂盒(Ambion公司)从转录体系中回收纯化体外转录的gRNA,用20μL无酶水溶解,-80℃保存备用。
电泳检测sgRNA1溶液和sgRNA2溶液中sgRNA,结果如图2所示。
二、Cas9体外转录
①用BamH I限制性内切酶剪切Cas9载体,使载体线性化,电泳、跑胶、回收,用20μL无酶水溶解备用;
②用T7Kit体外转录带帽的Cas9mRNA,电泳、跑胶、回收Cas9 mRNA。
③利用mirVanaTM miRNA Isolation Kit试剂盒(Ambion公司)从转录体系中回收纯化体外转录的Cas9mRNA,用10-20μL无酶水溶解,-80℃保存备用。
实施例2 F0代小鼠的制备
SCID小鼠超排:公鼠周龄:10-11周,雌鼠周龄:8周。母鼠于第一天13:00注射PMSG(7.5IU/只),第三天13:00注射HCG(7.5IU/只),第三天17:00每只雌鼠与2只雄鼠合笼,第四天8:00-9:00
将步骤二得到的sgRNA1溶液、sgRNA2溶液和Cas9mRNA溶液等体积混合后得到混合液(胚胎操作培养基为20mmol/L的HEPES,pH 7.4~7.8,非必需氨基酸为0.1mmol/L,必需氨基酸为0.1-0.6mmol/L),用小鼠胚胎培养基培养24小时后(培养基调整为:丙酮酸浓度提高至0.35mmol/L,谷氨酰胺浓度调至1mmol/L,葡萄糖浓度调至0.1mmol/L),取50nL混合液注射到雄性小鼠SCID的精子和雌性小鼠SCID的卵子体外受精0.5天形成的受精卵中,取注射后存活的受精卵移植到代孕母鼠体内,胚胎移植的小鼠出生即为F0代小鼠。
实施例3 F0代小鼠的鉴定
待F0代小鼠出生3周后剪尾利用引物TTTGATGTCTTCATTCGCAC-TGG和引物GCCTGGAGTGGTGTGTCTAA-AGG进行PCR扩增,并对PCR产物进行测序,鉴定得到数个阳性F0代小鼠。结果如3所示。
实施例4 IL2-Rg蛋白的鉴定
采用IL2-Rg抗体(利用IL2-Rg蛋白作为抗原免疫小鼠得到的抗体)对野生型BALb/c(基因型为IL2-Rg+/+)小鼠和缺失3878bp基因的杂合子小鼠(基因型为IL2-Rg-/+)进行Western blot实验,分别选择IL2-Rg-/+基因型小鼠的肝脏和脾脏以及野生型C57BL/6J的肝脏和脾脏作为样品进行实验。利用β-Actin作为内参。结果显示IL2-Rg基因在IL2-Rg-/+基因型小肝脏和脾脏组织中的蛋白表达量显著低于野生型BALb/c小鼠,结果如图4所示。上述结果说明IL2-Rg基因在SCID小鼠中的蛋白表达量显著下降,即IL2-Rg基因敲除小鼠模型成功构建。
实施例5与免疫缺陷IL2-Rg-/-部分敲除小鼠和裸鼠相比,SCID/IL2-Rg-/-全敲除小鼠在B细胞,T细胞及NK细胞在血液中含量的比较
取小鼠的外周血,裂红后用T、B、NK细胞相应的抗体孵育(分别是抗CD3、CD19和CD56的抗体),通过流式细胞仪分析。图5中,SCID/IL2-Rg-/-全敲除小鼠的B细胞、T细胞及NK细胞水平显著低于IL2-Rg-/-部分敲除小鼠和裸鼠。
实施例6 PDX模型在不同免疫缺陷小鼠的建模成功率
选取术前未接受任何抗肿瘤治疗且接受手术切除的结直肠癌病人,标本离体后立即切取部分肿瘤组织。肿瘤样本切碎后在消化液中根据纤维化程度进行不同时间的孵育,直至无组织碎片。而后肿瘤细胞经100u细胞筛过滤。将细胞团或单细胞悬液添加VEGF,EGF,bFGF,ROCK-1抑制剂,胰岛素及氢化可的松,并1:1添加Matrigel。将肿瘤细胞接种于免疫缺陷小鼠右侧肩部皮下。观察至肿瘤生长至800-1000mm3,收集肿瘤。图6表明SCID/IL2-Rg-/-全敲除小鼠的PDX模型建模成功率显著高于包括IL2-Rg-/-部分敲除鼠在内的其它免疫缺陷小鼠。
以上所述实施例的各技术特征可以进行任意的组合,为使描述简洁,未对上述实施例中的各个技术特征所有可能的组合都进行描述,然而,只要这些技术特征的组合不存在矛盾,都应当认为是本说明书记载的范围。
以上所述实施例仅表达了本发明的几种实施方式,其描述较为具体和详细,但并不能因此而理解为对发明专利范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变形和改进,这些都属于本发明的保护范围。因此,本发明专利的保护范围应以所附权利要求为准。
序列表
<110> 创模生物科技(北京)有限公司
<120> 制备IL-2Rg敲除非人动物模型的方法及其应用
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 23
<212> DNA
<213> artificial sequence
<400> 1
tggggggaat ctcgctgacg tgg 23
<210> 2
<211> 23
<212> DNA
<213> artificial sequence
<400> 2
gagcagctga aggactaaga agg 23
Claims (10)
1.制备IL-2Rg敲除非人动物模型的方法,其特征在于,包括:
使用Crispr-Cas系统将所述动物IL-2Rg基因敲除;
其中所述Crispr-Cas系统包括sgRNA和Cas9酶,所述sgRNA包括对应的靶位点序列为SEQ ID NO:1和2所示的sgRNA1和sgRNA2。
2.根据权利要求1所述的方法,其特征在于,所述基因敲除的对象为受精卵。
3.根据权利要求2所述的方法,其特征在于,所述Crispr-Cas系统以sgRNA和Cas9 mRNA的形式转入所述受精卵。
4.根据权利要求2或3所述的方法,其特征在于,所述Crispr-Cas系统转入所述受精卵的方法为显微注射。
5.根据权利要求4所述的方法,其特征在于,所述方法还包括将处理后受精卵移植到假孕雌性动物体内并产出F0代。
6.根据权利要求1~3、5任一项所述的方法,其特征在于,所述非人哺乳动物为免疫缺陷动物。
7.根据权利要求1~3、5任一项所述的方法,其特征在于,所述非人哺乳动物为啮齿类动物。
8.根据权利要求7所述的方法,其特征在于,所述非人哺乳动物是小鼠(Musmusculus)。
9.根据权利要求8所述的方法,其特征在于,所述非人哺乳动物是SCID小鼠。
10.权利要求1~9任一项所述方法得到的动物在鉴别和/或测试药物中的用途;
所述药物用于预防和/或治疗的适应症为IL-2相关免疫疾病、肿瘤组织移植、移植物抗宿主病中的任一项。
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