CN112553194B - Kit基因修饰的非人动物的制备方法和应用 - Google Patents
Kit基因修饰的非人动物的制备方法和应用 Download PDFInfo
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Abstract
本发明涉及基因修饰的非人动物,特别是基因修饰的啮齿动物,尤其是基因修饰的小鼠,具体涉及一种表达突变的KIT蛋白的KIT基因修饰的非人动物。本发明还提供了一种上述非人动物的制备方法及其在生物医药领域的应用。
Description
技术领域
本申请涉及基因修饰的动物、制备方法及应用,具体而言,涉及基于一种表达突变的KIT蛋白的基因修饰的动物、制备方法及其在生物医药领域的应用。
背景技术
实验动物疾病模型对于研究人类疾病发生的病因、发病机制、开发防治技术和开发药物是不可缺少的研究工具。其中,免疫缺陷动物由于缺乏免疫力,容易接受异种细胞或组织,在组织或细胞人源化动物研究、肿瘤药物及其他疾病的治疗机理方面已经得到广泛应用。已有研究表明作为受体鼠,目前普遍使用的免疫缺陷动物排序如下:NOD-PrkdcscidIL-2rγnul小鼠〉NOD-Rag 1-/--IL2rg-/- (NRG)〉Rag 2-/--IL2rg-/- (RG)〉NOD/SCID〉裸鼠,表明NOD-Prkdcscid IL-2rγnul小鼠是目前最佳的移植受体鼠(Ito R et al. Cell MolImmunol. 2012 May;9(3):208-14)。
然而NOD-Prkdcscid IL-2rγnull小鼠仍然存在缺陷,尽管由于该小鼠机体免疫功能严重降低,对人源细胞和组织几乎没有排斥反应,少量细胞即可成瘤(依赖于细胞系或细胞类型),同时也没有B淋巴细胞泄漏,是最适合人源细胞或组织移植的工具小鼠,并已广泛用于新的人源化动物模型研发。但在免疫系统重建前需要进行辐照清髓,该程序需要有放射源且会对小鼠产生多种副作用,包括造血系统、胃肠道、肌肉和神经组织的坏死和凋亡,可导致小鼠消瘦、感染甚至死亡,进而影响药效评价结果。如果小鼠宿主造血干细胞缺失,则可以免于辐照清髓,将会提高药效评价效果同时降低建模时间和成本。
人KIT基因(又称c-KIT)是一种原癌基因,起源于HZ4猫肉瘤病毒,位于人染色体4q12,在4号染色体长臂中心体周围区域,是白色斑点显性基因的等位基因。人KIT基因组DNA全长大约89kb,包括21个外显子。KIT基因编码的蛋白质被称为KIT或c-KIT受体,是一个分子量为145KD的I型跨膜糖蛋白,属于III型蛋白酪氨酸激酶受体超家族成员。分布于细胞膜表面,由胞外结构域、单一跨膜区及胞内酪氨酸激酶区域组成。胞外区含5个免疫球蛋白样的结构域,前三个免疫球蛋白为其配体干细胞生长因子(SCF)的结合位点,后两个在稳定及诱导KIT受体的二聚化方面起重要作用。
KIT与配体SCF共同参与多种细胞信号的传导,如Jak/STAT信号途径、PL3K途径、Ras/Raf/MEK/ERK信号途径等,从而在黑素生成、配子发生和造血系统细胞的存活、增殖和分化过程发挥重要作用。KIT/SCF信号传导途径受KIT调节,KIT基因突变在多种生物中会引起各种不同的表型特征,包括白化、贫血、纯合子死亡、听力受损等,这些表型可归结为发育时相应干细胞群的增殖或迁移缺失,且不同KIT突变体对细胞分化影响程度不同。在小鼠中,当KIT突变纯合时,往往对小鼠产生致死或半致死作用。有研究显示KIT突变会影响宿主造血干细胞的发育,产生不同程度的贫血,因此对于血液系统来说KIT是正常造血所必须的。
由于免疫缺陷模式动物是肿瘤临床治疗研究重要的工具和保障,本发明旨在克服现有技术中的不足,提供一种小鼠细胞中与造血干细胞相关的KIT基因突变的小鼠。该小鼠遗传背景单一,实验结果稳定可靠,不用辐照清髓即可进行人造血干细胞免疫系统重建,且移植后成功率高、寿命长等优点。除可用于人源细胞移植生长以外,还可与多种细胞因子基因人源化小鼠配合用于新的人源化动物模型研发与药效检测应用等方面。
发明内容
本发明的第一方面,提供了一种KIT基因修饰的非人动物的制备方法,所述的非人动物表达突变的KIT蛋白,所述的突变的KIT蛋白包含非人动物KIT蛋白胞内区引入一个或多个氨基酸突变。优选的,所述突变的KIT蛋白影响下游信号传导。
本发明的第二方面,提供了一种KIT基因修饰的非人动物,所述的非人动物表达突变的KIT蛋白,所述的突变的KIT蛋白包含非人动物KIT蛋白胞内区引入一个或多个氨基酸突变。优选的,所述突变的KIT蛋白影响下游信号传导。
其中,所述的下游信号传导包括KIT与配体SCF共同参与的多种细胞信号的传导,如Jak/STAT信号途径、PL3K途径、Ras/Raf/MEK/ERK信号途径等。从而在黑素生成、配子发生和造血系统细胞的存活、增殖和分化过程发挥重要作用。
进一步优选的,所述的非人动物体内造血干细胞的增殖、分化受到抑制。
更进一步优选的,所述的非人动物体内的造血干细胞缺失。
本发明所述的造血干细胞来源于外周血、骨髓、脐带血或胎盘。优选的,所述的造血干细胞来源于骨髓。
优选的,所述突变的KIT蛋白包括胞外区、跨膜区、信号肽和胞内区,其中所述胞内区包含一个或多个氨基酸突变。
优选的,所述突变的KIT蛋白包含SEQ ID NO:2的第626-662位任意一个或多个氨基酸突变的氨基酸序列。进一步优选的,所述突变的KIT蛋白包含SEQ ID NO:2的第660位苏氨酸(T)的突变。在本发明的一个具体实施方式中,所述突变的KIT蛋白包含在NCBI登录号为NC_000071.6的第75640429位苏氨酸(T)的突变。优选包含在NCBI登录号为NC_000071.6的第75640429位苏氨酸(T)突变为甲硫氨酸(M)。
更进一步优选的,所述突变的KIT蛋白包含SEQ ID NO:2的第660位苏氨酸(T)突变为甲硫氨酸(M)。
优选的,所述突变的KIT蛋白包含SEQ ID NO:2的第549-625位和/或第663-975位任意一个或多个氨基酸突变的氨基酸序列。进一步优选的,所述突变的KIT蛋白包含SEQ IDNO:2的第550-560位。
在本发明的一个具体实施方式中,所述突变的KIT蛋白还包含V831M。所述的突变的KIT蛋白还包含胞外区、跨膜区和/或信号肽包含一个或多个氨基酸突变。
优选的,所述胞外区包含一个或多个氨基酸突变。
优选的,所述突变的KIT蛋白包含SEQ ID NO:2的第26-527位任意一个或多个氨基酸突变的氨基酸序列。进一步优选的,所述突变的KIT蛋白包含SEQ ID NO:2的第502-503位的突变。
优选的,所述跨膜区包含一个或多个氨基酸突变。
优选的,所述突变的KIT蛋白包含SEQ ID NO:2的第528-548位任意一个或多个氨基酸突变的氨基酸序列。
优选的,所述信号肽包含一个或多个氨基酸突变。
优选的,所述突变的KIT蛋白包含SEQ ID NO:2的第1-25位任意一个或多个氨基酸突变的氨基酸序列。
在本发明的一个具体实施方式中,所述突变的KIT蛋白选自下列组中的一种:
(A)SEQ ID NO:4所示氨基酸序列的全部或部分;
(B)与SEQ ID NO:4或SEQ ID NO:2所示氨基酸的序列同源性程度为至少90%、91%、92%、93%、94%、95%、96%、97%、98%或至少99%;
(C)编码突变的KIT蛋白的核酸序列在严格条件下,与编码SEQ ID NO:4所示蛋白的核苷酸序列杂交;
(D)与SEQ ID NO:4或SEQ ID NO:2所示氨基酸序列差异不超过10、9、8、7、6、5、4、3、2或不超过1个氨基酸;或,
(E)具有SEQ ID NO:4或SEQ ID NO:2所示的,包括取代、缺失和/或插入一个或多个氨基酸残基的氨基酸序列。
优选的,编码所述突变的KIT蛋白的KIT基因序列包含非人动物KIT基因的第13外显子引入一个或多个突变。优选包含SEQ ID NO:1第2040位的突变。
更进一步优选的,包括SEQ ID NO:1第2040位由C突变为T。
优选的,编码所述突变的KIT蛋白的KIT基因序列还包括SEQ ID NO:1第1945、1963、2023和/或2044位的突变。
进一步优选的,编码所述突变的KIT蛋白的KIT基因序列还包括SEQ ID NO:1第1945位由C突变为T、1963位由G突变为A、2023位由C突变为T和/或2044位由G突变为T。
在本发明的一个具体实施方式中,编码所述突变的KIT蛋白的KIT基因序列转录的mRNA选自下列组中的一种:
(a)编码上述的突变的KIT蛋白;
(b)SEQ ID NO:3所示的序列的部分或全部;
(c)与SEQ ID NO:1或SEQ ID NO:3所示的核苷酸序列的同源性程度为至少90%、91%、92%、93%、94%、95%、96%、97%、98%或至少99%;
(d)在严格条件下,与SEQ ID NO:3所示的核苷酸序列杂交;
(e)与SEQ ID NO:1或SEQ ID NO:3所示的核苷酸序列差异不超过10、9、8、7、6、5、4、3、2或不超过1个核苷酸;或,
(f)具有SEQ ID NO:3所示的核苷酸序列所示的,包括取代、缺失和/或插入一个或多个核苷酸的核苷酸序列。
其中,编码所述突变的KIT蛋白的KIT基因为纯合或杂合的。
优选的,编码所述突变的KIT蛋白的KIT基因还包括特异性诱导物或阻遏物。进一步优选的,所述的特异性诱导物或阻遏物可以为常规可以诱导或阻遏的物质。在本发明的一个具体实施方式中,所述的特异性诱导物选自四环素系统(Tet-Off System/Tet-OnSystem)或他莫昔芬系统(Tamoxifen System)。
优选的,所述KIT基因修饰的非人动物的制备方法或所述KIT基因修饰的非人动物中,至少一个染色体上包含上述编码突变的KIT蛋白的KIT基因。
优选的,所述KIT基因修饰的非人动物的制备方法或所述KIT基因修饰的非人动物中,至少一个细胞表达突变的KIT蛋白。
优选的,使用基因编辑技术进行KIT基因修饰的非人动物的构建,所述的基因编辑技术包括基于胚胎干细胞的基因打靶技术、CRISPR/Cas9技术、锌指核酸酶技术、转录激活子样效应因子核酸酶技术、归巢核酸内切酶或其他分子生物学技术。
优选的,所述的非人动物是免疫缺陷的。
在本发明的一个具体实施方式中,所述的非人动物为小鼠,所述小鼠KIT基因的mRNA序列如SEQ ID NO:1中的全部或部分片段所示,所述小鼠KIT蛋白序列如SEQ ID NO:2中的全部或部分片段所示。
优选的,所述的非人动物为通过在非人动物内源KIT基因座处引入1个或多个突变制备获得。
进一步优选的,所述的非人动物为使用sgRNA和/或靶向载体将非人动物KIT基因座的第13外显子引入一个或多个点突变制备获得。
所述的sgRNA靶向的靶位点序列如SEQ ID NO:5-8任一项所示。
所述的靶向载体包含编码SEQ ID NO:4第626-662位氨基酸的核苷酸序列。优选的,所述的靶向载体还包含5’臂和/或3’臂。所述靶向载体中5’臂为SEQ ID NO:14所示序列的全部或部分。所述靶向载体中3’臂为SEQ ID NO:15所示序列的全部或部分。
在本发明的一个具体实施方式中,所述的制备方法,包括如下制备步骤:
1)将靶向载体和/或sgRNA的载体的体外转录产物、Cas9 mRNA进行混合,将混合液注射至非人动物细胞,所述的细胞为受精卵细胞或者胚胎干细胞;
2)将步骤1)所述细胞在培养液中进行培养;
3)将培养后细胞移植至受体雌性非人哺乳动物的输卵管内,允许所述细胞在所述雌性非人哺乳动物的子宫中发育;其中,所述非人哺乳动物为假孕雌性;
4)鉴定步骤3)的怀孕雌性的后代基因修饰的非人哺乳动物中的种系传递。
优选的,所述受精卵来源于任何非人哺乳动物;进一步优选的,所述受精卵细胞来源于啮齿类动物;更进一步优选的,所述受精卵选自C57BL/6受精卵、FVB/N受精卵、129受精卵、BALB/c受精卵、DBA/1受精卵或DBA/2受精卵。
优选的,所述的胚胎干细胞来源于任何非人哺乳动物。进一步优选的,所述的胚胎干细胞来源于啮齿类动物。更进一步优选的,所述胚胎干细胞选自C57BL/6胚胎干细胞、FVB/N胚胎干细胞、129胚胎干细胞、BALB/c胚胎干细胞、DBA/1胚胎干细胞或DBA/2胚胎干细胞。
本发明的第三方面,提供了采用上述的制备方法获得的KIT基因修饰的非人动物。
本发明的第四方面,提供了一种靶向载体,所述的靶向载体包含编码SEQ ID NO:4第626-662位氨基酸的核苷酸序列。
优选的,所述的靶向载体还包含5’臂和/或3’臂。所述的5’臂与NCBI登录号为NC_000071.6至少具有90%同源性的核苷酸。优选的,5’臂核苷酸长度为955bp。进一步优选的,所述的5’臂至少包含第10-13外显子的全部或部分,且,所述的5’臂核苷酸序列包含突变。最为优选的,5’臂为SEQ ID NO:14所示序列的全部或部分。所述的3’臂与NCBI登录号为NC_000071.6至少具有90%同源性的核苷酸。优选的,3’臂核苷酸长度为947bp。进一步优选的,所述的3’臂至少包含第13外显子及13外显子与14外显子之间的内含子的全部或部分,且,所述的3’臂核苷酸序列包含突变。最为优选的,3’臂为SEQ ID NO:15所示序列的全部或部分。
在本发明的一个具体实施方式中,所述的靶向载体包括a)与待改变的转换区5’端同源的DNA片段,即5’臂,其选自KIT基因基因组DNA的100-10000个长度的核苷酸;b)插入或替换的供体DNA序列,其编码供体转换区;c)与待改变的转换区3’端同源的第二个DNA片段,即3’臂,其选自KIT基因基因组DNA的100-10000个长度的核苷酸。
优选的,所述的靶向载体还包括可选择的标记基因。进一步优选的,所述标记基因为负筛选标记的编码基因。更进一步优选的,所述负筛选标记的编码基因为白喉毒素A亚基的编码基因(DTA)。
优选的,所述靶向载体还包括阳性克隆筛选的抗性基因。进一步优选的,所述阳性克隆筛选的抗性基因为新霉素磷酸转移酶编码序列Neo。
优选的,所述靶向载体还包括特异性重组系统。进一步优选的,所述特异性重组系统为Frt重组位点(也可选择常规的LoxP重组系统)。所述的特异性重组系统为2个,分别装在抗性基因的两侧。
优选的,所述的待改变的转换区位于KIT基因的13号外显子。
优选的,所述的插入或替换的供体DNA序列包括KIT基因第13号外显子的全部或部分。
进一步优选的,所述的插入或替换的供体DNA序列编码突变的KIT蛋白的全部或部分。
更进一步优选的,所述的插入或替换的供体DNA序列编码SEQ ID NO:4所示第626-662位氨基酸序列的全部或部分。
在本发明的一个具体实施方式中,所述的插入或替换的供体DNA序列至少包含碱基序列:CAT。
本发明的第五方面,提供了一种靶向KIT基因的sgRNA,所述的sgRNA靶向的靶位点序列如SEQ ID NO:5-8任一项所示。
优选的,所述的sgRNA靶向KIT基因的第13外显子。
优选的,所述的sgRNA靶向的靶位点序列如SEQ ID NO:6所示。
进一步优选的,所述上游序列如SEQ ID NO:10所示。其中,正向寡核苷酸序列如SEQ ID NO:11所示,其由SEQ ID NO:10的5’端加上TAGG得到。
进一步优选的,所述下游序列如SEQ ID NO:12所示。其中,反向寡核苷酸序列如SEQ ID NO:13所示,其由SEQ ID NO:12的5’端加上AAAC得到。
本发明的第六方面,提供了一种编码上述靶向KIT基因的sgRNA的DNA分子。
优选的,所述DNA分子的双链为SEQ ID NO:10和SEQ ID NO:12,或SEQ ID NO:11和SEQ ID NO:13所示。
本发明的第七方面,提供了一种包含上述靶向KIT基因的sgRNA和/或上述DNA分子的载体。
本发明的第八方面,提供了一种包含上述的靶向载体,上述靶向KIT基因的sgRNA,上述DNA分子,和/或,上述的载体的细胞。
本发明的第九方面,提供了一种上述的靶向载体,上述靶向KIT基因的sgRNA,上述DNA分子,上述的载体,或者,上述的细胞在KIT基因修饰中的应用。
本发明的第十方面,提供了一种KIT基因修饰的细胞或细胞株或其制备方法,所述的细胞或细胞株表达突变的KIT蛋白。
优选的,所述的细胞或细胞株选自胚胎干细胞或精子等。
优选的,在细胞或细胞株中,所述编码突变的KIT蛋白的KIT基因为纯合或杂合的。
优选的,所述的细胞或细胞株中至少一个染色体上包含编码突变的KIT蛋白的KIT基因。
优选的,使用基因编辑技术进行KIT基因修饰的细胞或细胞株的构建,所述的基因编辑技术包括基于胚胎干细胞的基因打靶技术、CRISPR/Cas9技术、锌指核酸酶技术、转录激活子样效应因子核酸酶技术、归巢核酸内切酶或其他分子生物学技术。
优选的,所述的细胞或细胞株来自免疫缺陷的非人动物。
优选的,所述细胞或细胞株来自免疫缺陷的非人动物细胞或细胞株。
优选的,所述KIT基因修饰的细胞或细胞株为通过在细胞或细胞株中KIT基因座处引入1个或多个突变制备获得。
进一步优选的,所述KIT基因修饰的细胞或细胞株为使用sgRNA和/或靶向载体将细胞或细胞株KIT基因座的第13外显子引入一个或多个点突变制备获得。所述的sgRNA靶向的靶位点序列如SEQ ID NO:5-8任一项所示。所述的靶向载体包含编码SEQ ID NO:4第626-662位氨基酸的核苷酸序列。优选的,所述的靶向载体还包含5’臂和/或3’臂,所述的5’臂与NCBI登录号为NC_000071.6至少具有90%同源性的核苷酸,优选的,如SEQ ID NO:14所示;所述的3’臂与NCBI登录号为NC_000071.6至少具有90%同源性的核苷酸,优选的,所述的3’臂序列如SEQ ID NO:15所示。
本发明所述的细胞或细胞株不是动物品种,所述KIT基因修饰的细胞或细胞株不会发育为个体。
本发明的第十一方面,提供了一种制备多基因修饰的非人动物的方法,包括如下步骤:
(1)制备上述的KIT基因修饰的非人动物,或者,采用上述的KIT基因修饰的非人动物的制备方法构建的KIT基因修饰的非人动物;
(2)将步骤(1)制备获得的KIT基因修饰的非人动物与其他基因修饰的的非人动物交配、体外授精或直接进行基因编辑,并进行筛选,得到多基因修饰的的非人动物。
优选的,所述的其他基因修饰的非人动物包括但不限于IL2RG基因敲除的非人动物。
优选的,所述多基因修饰的非人动物为双基因修饰的非人动物、三基因修饰的非人动物、四基因修饰的非人动物、五基因修饰的非人动物、六基因修饰的非人动物、七基因修饰的非人动物、八基因修饰的非人动物或九基因修饰的非人动物。
优选的,所述的多基因修饰的动物基因组中修饰的多基因中的每一个基因均可以是纯合或杂合的。
优选的,所述的其他基因修饰的非人动物为非人哺乳动物。更优选的,所述的非人哺乳动物可以为啮齿类动物、猪、兔子、猴子等。最为优选的,所述的非人哺乳动物为啮齿类动物,所述的啮齿类动物为小鼠或大鼠。
本发明的第十二方面,提供了一种上述的制备多基因修饰的非人动物的方法制备得到的多基因修饰的非人动物或其子代。
本发明的第十三方面,提供了一种荷瘤或炎症的动物模型或其制备方法,所述的动物模型来源于上述的制备方法制备的KIT基因修饰的非人动物、上述KIT基因修饰的非人动物、上述的方法构建的多基因修饰的非人动物、或者上述的多基因修饰的非人动物或其子代。
所述动物模型的制备方法包括通过上述制备方法制备KIT基因修饰非人动物或上述的方法制备多基因修饰的非人动物的步骤。
优选的,所述的荷瘤动物模型的制备方法还包括在上述方法制备的非人动物或其后代植入肿瘤细胞的步骤。
本发明的第十四方面,提供了一种细胞或细胞系或原代细胞培养物,所述细胞或细胞系或原代细胞培养物来源于上述的制备方法制备的KIT基因修饰的非人动物、上述KIT基因修饰的非人动物、上述的方法构建的多基因修饰的非人动物、上述的多基因修饰的非人动物或其子代,或者,上述的荷瘤或炎性的动物模型。
本发明的第十五方面,提供了一种组织或器官或其培养物,所述组织或器官或其培养物来源于上述的制备方法制备的KIT基因修饰的非人动物、上述KIT基因修饰的非人动物、上述的方法构建的多基因修饰的非人动物、上述的多基因修饰的非人动物或其子代,或者,上述的荷瘤或炎性的动物模型。优选的,所述组织或器官为脾脏、肿瘤或其培养物。
本发明的第十六方面,提供了一种荷瘤后的瘤组织,所述的瘤组织来源于上述的制备方法制备的KIT基因修饰的非人动物、上述KIT基因修饰的非人动物、上述的方法构建的多基因修饰的非人动物、上述的多基因修饰的非人动物或其子代,或者,上述的荷瘤或炎性的动物模型。
本发明的第十七方面,提供了一种突变的KIT蛋白,所述突变的KIT蛋白包括非人动物KIT蛋白胞内区引入一个或多个氨基酸突变。优选的,所述突变的KIT蛋白影响下游信号传导。
优选的,所述突变的KIT蛋白包括胞外区、跨膜区、信号肽和胞内区,其中所述胞内区包含一个或多个氨基酸突变。
优选的,所述突变的KIT蛋白包含SEQ ID NO:2的第626-662位任意一个或多个氨基酸突变的氨基酸序列。进一步优选的,所述突变的KIT蛋白包含SEQ ID NO:2的第660位苏氨酸(T)的突变。更进一步优选的,所述突变的KIT蛋白包含SEQ ID NO:2的第660位苏氨酸(T)突变为甲硫氨酸(M)。
在本发明的一个具体实施方式中,所述突变的KIT蛋白包含在NCBI登录号为NC_000071.6的第75640429位苏氨酸(T)的突变。优选包含在NCBI登录号为NC_000071.6的第75640429位苏氨酸(T)突变为甲硫氨酸(M)。
优选的,所述突变的KIT蛋白包含SEQ ID NO:2的第549-625位和/或第663-975位任意一个或多个氨基酸突变的氨基酸序列。进一步优选的,所述突变的KIT蛋白包含SEQ IDNO:2的第550-560位。
在本发明的一个具体实施方式中,所述突变的KIT蛋白包含V831M。
所述的突变的KIT蛋白还包含胞外区、跨膜区和/或信号肽包含一个或多个氨基酸突变。
优选的,所述胞外区包含一个或多个氨基酸突变。优选的,所述突变的KIT蛋白包含SEQ ID NO:2的第26-527位任意一个或多个氨基酸突变的氨基酸序列。进一步优选的,所述突变的KIT蛋白包含SEQ ID NO:2的第502-503位的突变。
优选的,所述跨膜区包含一个或多个氨基酸突变。优选的,所述突变的KIT蛋白包含SEQ ID NO:2的第528-548位任意一个或多个氨基酸突变的氨基酸序列。
优选的,所述信号肽包含一个或多个氨基酸突变。优选的,所述突变的KIT蛋白包含SEQ ID NO:2的第1-25位任意一个或多个氨基酸突变的氨基酸序列。
在本发明的一个具体实施方式中,所述突变的KIT蛋白选自下列组中的一种:
(A)SEQ ID NO:4所示氨基酸序列的全部或部分;
(B)与SEQ ID NO:4或SEQ ID NO:2所示氨基酸的序列同源性程度为至少90%、91%、92%、93%、94%、95%、96%、97%、98%或至少99%;
(C)在严格条件下,与编码SEQ ID NO:4所示蛋白的核苷酸序列杂交;
(D)与SEQ ID NO:4或SEQ ID NO:2所示氨基酸序列差异不超过10、9、8、7、6、5、4、3、2或不超过1个氨基酸;或,
(E)具有SEQ ID NO:4或SEQ ID NO:2所示的,包括取代、缺失和/或插入一个或多个氨基酸残基的氨基酸序列。
本发明的第十八方面,提供了一种编码突变的KIT蛋白的KIT基因,所述的KIT基因序列为非人动物KIT基因的第13外显子包含一个或多个突变。
优选的,所述的KIT基因序列为SEQ ID NO:1包括第2040位的突变。
进一步优选的,所述的KIT基因序列包括SEQ ID NO:1第2040位由C突变为T。更进一步优选的,所述编码突变的KIT蛋白的KIT基因序列还包括SEQ ID NO:1第1945、1963、2023和/或2044位的突变。
优选的,所述的KIT基因序列还包括SEQ ID NO:1第1945位由C突变为T、1963位由G突变为A、2023位由C突变为T和/或2044位由G突变为T。
在本发明的一个具体实施方式中,所述的KIT基因序列选自下列组中的一种:
(a)编码突变的KIT蛋白的KIT基因编码上述的突变的KIT蛋白;
(b)转录的mRNA序列为SEQ ID NO:3所示的序列的部分或全部;
(c)与SEQ ID NO:1或SEQ ID NO:3所示的核苷酸序列的部分或全部的同源性程度为至少90%、91%、92%、93%、94%、95%、96%、97%、98%或至少99%;
(d)在严格条件下,与SEQ ID NO:3所示的核苷酸序列杂交;
(e)与SEQ ID NO:1或SEQ ID NO:3所示的核苷酸序列差异不超过10、9、8、7、6、5、4、3、2或不超过1个核苷酸;或,
(f)具有SEQ ID NO:3所示的核苷酸序列所示的,包括取代、缺失和/或插入一个或多个核苷酸的核苷酸序列。
优选的,SEQ ID NO:3所示序列为KIT基因修饰的非人动物KIT DNA的非模板链、编码链或有义链。
本发明的第十九方面,提供了一种细胞、组织、器官或荷瘤后的瘤组织,所述的细胞、组织、器官或荷瘤后的瘤组织表达上述突变的KIT蛋白。
本发明所述的非人动物为非人哺乳动物,进一步优选的,所述的非人哺乳动物为啮齿类动物、猪、兔子、猴子等任何可以进行基因修饰的制备KIT基因修饰的非人动物的非人哺乳动物,更进一步优选的,所述的非人哺乳动物为啮齿类动物,最为优选的,所述的啮齿类动物为小鼠或大鼠。
本发明所述的非人动物为免疫缺陷非人哺乳动物,进一步优选的,所述的非人动物为免疫缺陷的啮齿类动物、猪、兔子、猴子等任何可以进行基因修饰的制备KIT基因修饰的非人动物的非人哺乳动物,更进一步优选的,所述的非人动物为免疫缺陷的啮齿类动物,更为优选的,所述的非人动物为免疫缺陷的小鼠或大鼠,最为优选的,所述的非人动物为NOD-Prkdcscid IL-2rγnul小鼠、NOD-Rag 1-/--IL2rg-/- (NRG)〉Rag 2-/--IL2rg-/- (RG)、NOD/SCID、或者裸鼠。
本发明的第二十方面,提供了一种表达上述突变的KIT蛋白的构建体。
本发明的第二十一方面,提供了一种包含上述构建体的细胞。
本发明的第二十二方面,提供了一种包含上述细胞的组织。
本发明的第二十三方面,提供了来源于上述制备方法制备的KIT基因修饰的非人动物、上述KIT基因修饰的非人动物、上述的细胞或细胞株、上述制备方法得到的细胞或细胞株、上述的方法构建的多基因修饰的非人动物、上述的多基因修饰的非人动物或其子代、上述的荷瘤或炎性的动物模型、上述的细胞或细胞系或原代细胞培养物、上述的组织或器官或其培养物、上述的细胞、组织、器官、荷瘤后的瘤组织、上述突变的KIT蛋白、上述编码突变的KIT蛋白的KIT基因、上述的构建体、上述的细胞或者上述的组织在需要涉及人类细胞的免疫过程的产品开发,制造人类抗体,或者作为药理学、免疫学、微生物学和医学研究的模型系统中的应用;或在生产和利用动物实验疾病模型,用于人源细胞移植、免疫系统重建、病原学研究和/或用于开发新的诊断策略和/或治疗策略中的应用;或在筛选、验证、评价或研究KIT基因功能、靶向人的抗体、靶向人的药物、药效研究,免疫相关疾病药物以及抗肿瘤或抗炎症药物,筛选和评估人用药及药效研究方面的用途。
优选的,所述药物筛选或药效评价的方法不是治疗方法。该方法用来筛选药物,对候选药物的药效进行检测和比较,以确定哪些候选药物可以作为药物,哪些不能作为药物,或者,比较不同药物的药效敏感程度,即治疗效果不是必然的,只是一种可能性。
本发明的第二十四方面,提供了一种人免疫系统重建的方法,所述的方法包括向个体施加人造血干细胞,以在非人动物体内重建免疫系统,所述的个体选自上述制备方法制备的KIT基因修饰的非人动物、上述KIT基因修饰的非人动物、上述的方法构建的多基因修饰的非人动物、上述的多基因修饰的非人动物或其子代或者上述的荷瘤或炎性的动物模型。
优选的,所述的注射为尾静脉注射。
优选的,所述注射人造血干细胞的量为1.5×105人造血干细胞(HSC)。
优选的,所述的免疫系统重建的标注为hCD45占裂解红细胞之后总活细胞的比例≥25%。
优选的,所述的方法还包括注射人造血干细胞后每间隔一段时间取外周血(PB)进行检测验证是否成功以及观察非人动物存活情况。其中,在本发明的一个具体实施方式中,所述的一段时间为4周。
优选的,所述取外周血(PB)进行检测验证包括检测表达人白细胞表面分子标记(CD45+)的细胞、T细胞(CD3+)、B细胞(CD19+)和髓系细胞(CD33+)的发育情况。
本发明的第二十五方面,提供了一种采用上述人免疫系统重建的方法获得的人免疫系统重建的非人动物或其子代。
本发明的第二十六方面,提供了一种抗体筛选的方法,所述的方法包括向上述人免疫系统重建的非人动物或其子代移植人肿瘤细胞后,给予待筛选的抗体,对给予抗体的非人动物或子代进行检测、效果评价。
优选的,所述抗体筛选的方法不是治疗方法。该方法用来筛选或评价抗体,对候选抗体的药效进行检测和比较,以确定哪些候选抗体可以作为药物,哪些不能作为药物,或者,比较不同抗体的药效敏感程度,即治疗效果不是必然的,只是一种可能性。
本发明的第二十七方面,提供了一种人用药物筛选或评价的方法,所述的方法包括向上述人免疫系统重建的非人动物或其子代体内移植人肿瘤细胞,向移入人肿瘤细胞的动物给予候选药物,对给予候选药物的个体进行药效检测和/或比较。
优选的,所述药物筛选或评价的方法不是治疗方法。该方法用来筛选或评价药物,对候选药物的药效进行检测和比较,以确定哪些候选药物可以作为药物,哪些不能作为药物,或者,比较不同药物的药效敏感程度,即治疗效果不是必然的,只是一种可能性。
优选的,所述候选药物为单抗或双特异性抗体或两种及两种以上药物的联合使用。
优选的,所述检测包括测定肿瘤细胞的大小和/或增殖速率;优选的,所述检测的方法包括游标卡尺测量、流式细胞检测和/或动物活体成像检测。
优选的,所述的检测包括评估个体体重、脂肪量、活化途径、神经保护活性或代谢变化,所述的代谢变化包括食物消耗或水消耗的变化。
本发明的第二十八方面,提供了一种治疗方案的评价方法,所述的评价方法包括向上述人免疫系统重建的非人动物或其子代植入人肿瘤细胞,向植入人肿瘤细胞的个体施加治疗方案,对施加治疗方案后的个体进行肿瘤抑制效果检测和评价。
优选的,所述的治疗方案为CAR-T。
优选的,所述的评价方法不是治疗方法。该方法对治疗方案的效果进行检测和评价,以确定该治疗方案是否有治疗效果,即治疗效果不是必然的,只是一种可能性。
采用本申请所述的制备方法获得的KIT基因修饰的非人动物,无需辐照清髓即可成功重建人免疫系统,且非人动物的状态、存活率及重建后的人白细胞比例均显著优于B-NDG小鼠,而且可以更好的支持T细胞、单核细胞、粒细胞等发育。
本发明所述“治疗(treating)”(或“治疗(treat)”或“治疗(treatment)”)表示减缓、中断、阻止、控制、停止、减轻、或逆转一种体征、症状、失调、病症、或疾病的进展或严重性,但不一定涉及所有疾病相关体征、症状、病症、或失调的完全消除。术语“治疗(treating)”等是指在疾病已开始发展后改善疾病或病理状态的体征、症状等等的治疗干预。
本发明所述的“突变”包括基因突变和蛋白突变,所述的基因突变或蛋白突变均为可遗传突变。其中,所述的基因突变包括碱基置换、移码、缺失或插入。优选的,所述的基因突变为碱基置换。所述的基因突变可以是自发的也可以是诱发的。所述的蛋白突变包括将野生型氨基酸用其他天然氨基酸、其他经修饰的天然氨基酸或非天然氨基酸替换等。
本发明所述的“非天然氨基酸”为非在蛋白质中天然发现的包含氨基和羧基的化合物。优选的,所述的非天然氨基酸为本领域已知的任何非天然氨基酸。进一步优选的,所述的非天然氨基酸包括但不限于N-乙基天冬氨酰、羟基赖氨酸、3-羟基脯氨酸、2-氨基丁酸、β-丙氨酸、β-氨基丙酸、2-氨基己二酸、3-氨基己二酸、4-氨基丁酸、6-氨基己酸、2-氨基庚酸、别-异亮氨酸、异锁链赖氨酸、4-羟基脯氨酸、别-羟基赖氨酸、2-氨基异丁酸、N-甲基甘氨酸、N-甲基异亮氨酸、3-氨基异丁酸、6-N-甲基赖氨酸、2,4-二氨基丁酸、N-甲基缬氨酸、鸟氨酸、正亮氨酸、正缬氨酸、锁链素、2,2’-二氨基庚二酸、2,3-二氨基丙酸、N-乙基甘氨酸或2-氨基庚二酸等等。
本发明所述的“经修饰的天然氨基酸”为侧链被化学修饰的氨基酸。例如:翻译后修饰的氨基酸,或者,侧链包含新型官能团(如硫氢基、氨基或羧基),或者,侧链包含产生信号的部分(如荧光基团或放射性标记)。
本发明所述的“突变的KIT蛋白”是指与野生型KIT蛋白具有至少55、60、65、70、75、80、85、90、91、92、93、94、95、96、97、98、99、99.5或99.9%或更多、或可来自其间的任何范围、但小于100%的同一性并且当非人动物表达这种“突变的KIT蛋白”时可以影响下游信号传导,使该非人动物体内造血干细胞的增殖、分化被抑制。优选的,所述的KIT基因修饰的非人动物体内的造血干细胞缺失。
本发明所述的“同源性”,是指在使用蛋白序列或核苷酸序列的方面,本领域技术人员可以根据实际工作需要对序列进行调整,使使用序列与现有技术获得的序列相比,具有(包括但不限于)1%,2%,3%,4%,5%,6%,7%,8%,9%,10%,11%,12%,13%,14%,15%,16%,17%,18%,19%,20%,21%,22%,23%,24%,25%,26%,27%,28%,29%,30%,31%,32%,33%,34%,35%,36%,37%,38%,39%,40%,41%,42%,43%,44%,45%,46%,47%,48%,49%,50%,51%,52%,53%,54%,55%,56%,57%,58%,59%,60%,70%,80%,81%,82%,83%,84%,85%,86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98%,99%,99.1%,99.2%,99.3%,99.4%,99.5%,99.6%,99.7%,99.8%,99.9%的同源性。
在一个方面,所述非人动物是哺乳动物。在一个方面,所述非人动物是小型哺乳动物,例如跳鼠科或鼠总科超家族。在一个实施方式中,所述基因修饰的动物是啮齿动物。在一个实施方式中,所述啮齿动物选自小鼠、大鼠和仓鼠。在一个实施方式中,所述啮齿动物选自鼠家族。在一个实施方式中,所述基因修饰的动物来自选自丽仓鼠科(例如小鼠样仓鼠)、仓鼠科(例如仓鼠、新世界大鼠和小鼠、田鼠)、鼠总科(真小鼠和大鼠、沙鼠、刺毛鼠、冠毛大鼠)、马岛鼠科(登山小鼠、岩小鼠、有尾大鼠、马达加斯加大鼠和小鼠)、刺睡鼠科(例如多刺睡鼠)和鼹形鼠科(例如摩尔大鼠、竹大鼠和鼢鼠)家族。在一个特定实施方式中,所述基因修饰的啮齿动物选自真小鼠或大鼠(鼠总科)、沙鼠、刺毛鼠和冠毛大鼠。在一个实施方式中,所述基因修饰的小鼠来自鼠科家族成员。在一个实施方式中,所述动物是啮齿动物。在一个特定实施方式中,所述啮齿动物选自小鼠和大鼠。在一个实施方式中,所述非人动物是小鼠。
在一个特定实施方式中,所述非人动物是啮齿动物,其为选自BALB/c、A、A/He、A/J、A/WySN、AKR、AKR/A、AKR/J、AKR/N、TA1、TA2、RF、SWR、C3H、C57BR、SJL、C57L、DBA/2、KM、NIH、ICR、CFW、FACA、C57BL/A、C57BL/An、C57BL/GrFa、C57BL/KaLwN、C57BL/6、C57BL/6J、C57BL/6ByJ、C57BL/6NJ、C57BL/10、 C57BL/10ScSn、C57BL/10Cr和C57BL/Ola的C57BL、C58、CBA/Br、CBA/Ca、CBA/J、CBA/st、CBA/H品系的小鼠。
除非特别说明,本发明的实践将采取细胞生物学、细胞培养、分子生物学、转基因生物学、微生物学、重组DNA和免疫学的传统技术。这些技术在以下文献中进行了详细的解释。例如:Molecular Cloning A Laboratory Manual,2ndEd.,ed. By Sambrook,FritschandManiatis (Cold Spring Harbor Laboratory Press:1989);DNA Cloning,Volumes I and II (D.N.Glovered.,1985);Oligonucleotide Synthesis (M.J.Gaited.,1984);Mullisetal. U.S. Pat.No.4,683,195;Nucleic Acid Hybridization(B.D.Hames& S.J.Higginseds.1984);Transcription And Translation (B.D.Hames&S.J.Higginseds.1984);Culture Of Animal Cells (R.I.Freshney,AlanR.Liss,Inc.,1987);Immobilized Cells And Enzymes (IRL Press,1986);B.Perbal,A PracticalGuide To Molecular Cloning(1984);the series,Methods In ENZYMOLOGY (J.Abelsonand M.Simon,eds.-in-chief,Academic Press,Inc.,New York),specifically,Vols.154 and 155 (Wuetal.eds.) and Vol.185,″Gene Expression Technology″(D.Goeddel,ed.);Gene Transfer Vectors For Mammalian Cells (J.H.Miller andM.P.Caloseds.,1987,Cold Spring Harbor Laboratory);Immunochemical Methods InCell And Molecular Biology (Mayer and Walker,eds.,Academic Press,London,1987);Handbook Of Experimental Immunology,Volumes V (D.M.Weir andC.C.Blackwell,eds.,1986);and Manipulating the Mouse Embryo,(Cold SpringHarbor Laboratory Press,Cold Spring Harbor,N.Y.,1986)。
以上只是概括了本发明的一些方面,不是也不应该认为是在任何方面限制本发明。
本说明书提到的所有专利和出版物都是通过参考文献作为整体而引入本发明的。本领域的技术人员应认识到,对本发明可作某些改变并不偏离本发明的构思或范围。下面的实施例进一步详细说明本发明,不能认为是限制本发明或本发明所说明的具体方法的范围。
附图说明
以下,结合附图来详细说明本发明的实施例,其中:
图1:小鼠和人的KIT基因对比示意图(非按比例)。
图2:sgRNA的相对活性检测结果,其中,kit-NC为阴性对照,kit-PC为阳性对照,纵坐标为sgRNA的相对活性值。
图3:F0代小鼠示例性PCR检测结果,其中,M为Marker,KIT-9为在F0代测序验证正确的小鼠中随机抽选的进行PCR检测的小鼠鼠尾PCR检测结果,H2O为水对照。
图4:F1代小鼠示例性PCR检测结果,其中,M为Marker,H2O为水对照,WT为野生型对照,F1-1、F1-2、F1-3、F1-5、F1-6和F1-9为阳性小鼠编号。
图5:免疫重建实验的生存曲线图,其中,纵坐标为存活率(%),横坐标为天数(d)。
图6:免疫重建实验过程中,KIT B-NDG与B-NDG小鼠体内的人白细胞(CD45+)占总活细胞的比例(%)结果。
图7:免疫重建实验过程中,KIT B-NDG与B-NDG小鼠中,重建成功(hCD45占裂解红细胞之后总活细胞的比例≥25%)小鼠占该组存活小鼠的百分比。
图8:流式分析外周血中人T细胞的发育情况,具体为KIT B-NDG与B-NDG小鼠体内的人CD3+CD45+ T细胞占人CD45+细胞的比例(%)。
图9:流式分析外周血中人B细胞的发育情况,具体为KIT B-NDG小鼠与B-NDG小鼠体内的人B细胞(CD19+)占人CD45细胞的比例(%)。
图10:流式分析外周血中人NK细胞的发育情况,具体为KIT B-NDG小鼠与B-NDG小鼠体内的人NK(CD56+)细胞占人CD45+细胞的比例(%)。
图11:流式分析外周血中人髓系细胞的发育情况,具体为KIT B-NDG小鼠与B-NDG小鼠体内的人髓系细胞(CD33+)占人CD45+细胞的比例(%)。
图12:流式分析外周血中人单核细胞的发育情况,具体为KIT B-NDG小鼠与B-NDG小鼠体内的人单核细胞(CD14+)占人CD33+细胞的比例(%)。
图13:流式分析外周血中人粒细胞的发育情况,具体为KIT B-NDG小鼠与B-NDG小鼠体内的人粒细胞(CD66b+)占人CD33+细胞的比例(%)。
具体实施方式
下面结合具体实施例来进一步描述本发明,本发明的优点和特点将会随着描述而更为清楚。但这些实施例仅是范例性的,并不对本发明的范围构成任何限制。本领域技术人员应该理解的是,在不偏离本发明的精神和范围下可以对本发明技术方案的细节和形式进行修改或替换,但这些修改和替换均落入本发明的保护范围内。
在下述每一实施例中,设备和材料是从以下所指出的几家公司获得:
NOD/scid小鼠购自北京华阜康生物科技股份有限公司;
NOD-Prkdcscid IL-2rgnull(B-NDG)小鼠来源百奥赛图公司,货号为B-CM-002;
Cas9mRNA来源SIGMA,货号CAS9MRNA-1EA;UCA试剂盒来源北京百奥赛图基因生物技术有限公司,货号为BCG-DX-001;
MEGAshortscript™Kit(Ambion体外转录试剂盒)购自Thermo Fisher,货号为AM1354;
EcoRI、BamHI和BbsI酶购自NEB,货号分别为R3101M、R3136M、R0539L。
实施例1:KIT基因突变小鼠的构建
小鼠KIT基因(NCBI Gene ID:16590,Primary source:MGI:96677,UniProt ID:P05532,位于5号染色体NC_000071.6的第75574987至75656722位,基于转录本NM_021099.3(SEQ ID NO:1)及其编码蛋白NP_066922.2(SEQ ID NO:2)),人、鼠KIT基因示意图如图1所示。
为了达到本发明的目的,可在内源小鼠KIT基因座处将1个或多个点突变引入编码序列,产生KIT突变基因,其编码的蛋白序列与野生型蛋白相比具有1个或多个氨基酸突变。
具体来说,根据KIT基因特点,可以通过基因编辑技术,在KIT基因座的第13外显子区域引入多个点突变,使得该小鼠体内表达的突变体KIT蛋白第660位氨基酸从T突变为M,所述突变后的DNA部分序列如SEQ ID NO:3所示,氨基酸序列如SEQ ID NO:4所示。
引入CRISPR/Cas系统进行基因编辑,该系统中靶序列决定了sgRNA的靶向特异性和诱导Cas9切割目的基因的效率,因此,高效特异的靶序列选择和设计是构建sgRNA表达载体的前提。设计并合成识别靶位点的sgRNA序列。靶位点位于KIT基因第13号外显子上,各sgRNA在KIT上的靶位点序列如下:
kit-sgRNA1靶位点序列(SEQ ID NO:5):5’- CCACCGTGCATGCGCCAAGCAGG-3’
kit-sgRNA2靶位点序列(SEQ ID NO:6):5’- CCTGCTTGGCGCATGCACGGTGG-3’
kit-sgRNA3靶位点序列(SEQ ID NO:7):5’- CTGCTTGGCGCATGCACGGTGGG-3’
kit-sgRNA4靶位点序列(SEQ ID NO:8):5’- GAACCTGCTTGGCGCATGCACGG-3’
利用UCA试剂盒检测多个sgRNA的活性,从结果可见sgRNA具有不同活性,其中,kit-sgRNA1和kit-sgRNA3相对活性较低,这可能是由于靶位点序列的特殊性导致,但根据我们的实验及其具体活性值,kit-sgRNA1和kit-sgRNA3的相对活性值仍显著高于阴性对照组值,仍可判断kit-sgRNA1和kit-sgRNA3是具有活性的,并且活性满足基因打靶实验要求,具体检测结果参见图2。从中随机选择kit-sgRNA2进行后续实验。在其5’端及互补链上分别加上酶切位点得到正向寡核苷酸和反向寡核苷酸(序列见表1),退火后将退火产物分别连接至pT7-sgRNA质粒(质粒先用BbsI线性化),获得表达载体pT7-sgRNA2。
pT7-sgRNA载体由质粒合成公司合成含有T7启动子及sgRNA scaffold的片段DNA(SEQ ID NO:9)并依次通过酶切(EcoRI及BamHI)连接至骨架载体(来源Takara,货号3299)上,经专业测序公司测序验证,结果表明获得了目的质粒。
表1 sgRNA序列
构建靶向小鼠KIT基因的靶向载体,靶向载体上含有包含KIT基因突变的13号外显子、突变位点上游的5’同源臂(SEQ ID NO:14),其第1-952位核苷酸序列与NCBI登录号为NC_000071.6的第75640475-75641426位核苷酸序列有3处不同,即第860位C突变为T、第878位G突变为A、第938位C突变为T,这些突变不影响蛋白表达;以及突变位点下游的3’同源臂(SEQ ID NO:15),其与NCBI登录号为NC_000071.6的第75641430-75642376位核苷酸序列有1处不同,即第4位G突变为T,该突变不影响蛋白表达。5’同源臂第953-955位核苷酸序列为CAT。
靶向载体构建可采用常规方法进行,如酶切连接、直接合成等。构建好的靶向载体通过酶切进行初步验证后,再送测序公司进行测序验证。将测序验证正确的靶向载体质粒用于后续实验。
取小鼠的原核期受精卵,例如,NOD-Prkdcscid IL-2rγnull(B-NDG)小鼠,或NOD/scid小鼠的受精卵,利用显微注射仪将pT7-sgRNA2质粒的体外转录产物(使用Ambion体外转录试剂盒,按照说明书方法进行转录)、靶向载体与Cas9 mRNA预混好后注射至小鼠受精卵细胞质或细胞核中。按照《小鼠胚胎操作实验手册(第三版)》(安德拉斯・纳吉,化学工业出版社,2006)中的方法进行受精卵的显微注射,注射后的受精卵转移至培养液中短暂培养,然后移植至受体母鼠的输卵管中发育,将获得的小鼠(F0代)通过杂交和自交,扩大种群数量,建立稳定的KIT基因突变小鼠品系。
使用NOD-Prkdcscid IL-2rγnull(如,B-NDG)小鼠受精卵进行注射时,因其具有较高的免疫缺陷程度,得到的KIT基因突变小鼠背景明确且具有高度免疫缺陷。选择NOD/scid小鼠的受精卵进行注射,得到经鉴定正确的突变体可进一步的与NOD-Prkdcscid IL-2rγnull小鼠进行交配或体外授精,对其后代进行筛选,根据孟德尔遗传规律,可有一定几率得到KIT基因突变与IL-2rg基因敲除的杂合动物模型(NOD/scid背景),再将杂合子相互交配可以得到重度免疫缺陷的KIT基因突变小鼠。
可通过常规检测方法鉴定F0代小鼠体细胞的基因型,例如,通过PCR方法鉴定F0代鼠尾,将鉴定正确的PCR条带回收测序,确认是否发生了点突变的精确重组。将测序鉴定正确的F0代阳性小鼠进行二次剪尾PCR鉴定并再次测序核实,将测序正确的F0代小鼠与野生型C57BL/6小鼠交配可得到F1代小鼠(示例性的F0代PCR结果见图3)。使用同样的PCR方法对F1代小鼠进行基因型鉴定,部分F1代小鼠实验结果见图4,结果显示编号F1-1、F1-2、F1-3、F1-5、F1-6和F1-9的小鼠均为阳性小鼠。进一步经过测序确认这6只小鼠为阳性杂合子且无随机插入。这表明使用本方法能构建出可稳定传代且发生目标基因精确点突变的基因工程小鼠,且所有小鼠及子代全身毛发均为白色,与野生型小鼠相比,除体型稍小外,未观察到明显异常。
小鼠PCR分析包括下述引物对:
KIT-MUT-F1:5’- ACTGTTGGTTGGTCTTCCCACTGAC-3’(SEQ ID NO:16),
KIT-MUT-R1:5’-AGCCTAGTAGGGAAGTAACCAGGGA-3’(SEQ IDNO:17)。
实施例2:含有KIT基因突变的免疫缺陷小鼠免疫重建及验证
选择6周龄的KIT基因突变小鼠(B-NDG背景,n=13)和B-NDG小鼠(n=19),B-NDG小鼠经辐照(2.0Gy)清髓预处理后,与未经辐照的KIT基因突变小鼠均经尾静脉注射1.5×105人造血干细胞(HSC),以在小鼠体内重建免疫系统,重建成功的标准为hCD45+占裂解红细胞之后总活细胞的比例≥25%。注射移植后每四周取外周血(PB)进行流式细胞检测以评估重建是否成功,观察小鼠状态并记录的存活情况。
实验结果显示,未经辐照的KIT基因突变小鼠(后简称“KIT B-NDG小鼠”)与经辐照的B-NDG小鼠(后简称“B-NDG小鼠”)在整个实验周期生存情况较好,实验结束时(第20周,具体为注射后第136天)时KIT B-NDG小鼠存活率为46.2%,B-NDG小鼠存活率为31.6%,存在明显差异(参见图5),且观察发现KIT B-NDG小鼠状态一直较好,而B-NDG小鼠状态在实验结束时相对较差,主要表现在B-NDG小鼠活动度减少、个别出现弓背,体毛稀疏等。
流式检测结果显示第4周起可以在所有小鼠体内检测到表达人白细胞表面分子标记(CD45+)的细胞,在12周时,与B-NDG小鼠相比,在KIT B-NDG小鼠体内观察到的人白细胞的平均百分比(61.13%±15.55)高7倍(8.84%±4.00),从整个实验周期的数据来看,KIT B-NDG小鼠体内的人白细胞的比例、重建成功的比例自第8周起显著高于B-NDG小鼠(见图6、7)。进一步通过流式分析外周血中T细胞(CD3+)、B细胞(CD19+)和髓系细胞(CD33+)的发育情况,从16周起还检测了NK细胞(CD56+)、单核细胞(CD14+)和粒细胞(CD66b+)的发育(图8至图13)。检测结果表明KIT B-NDG小鼠与B-NDG小鼠体内的各类细胞分化比例比较接近,表明改造后的小鼠允许人造血干细胞的直接稳定移植。在KIT B-NDG小鼠体内,自16周起T细胞占人白细胞的比例、单核细胞及粒细胞占髓系细胞的比例均高于B-NDG小鼠(见表2,及图8、12、13),表明改造后的小鼠能更好的支持T细胞、单核细胞、粒细胞等发育。流式检测门控策略:人白细胞被定位为完整的、单一的、活的细胞,hCD45+,mCD45-。在人白细胞群中,T细胞(CD3+)被定为完整的、单一的、活的,hCD45+,mCD45-,hCD3+,hCD19-;B细胞被定为完整的、单一的、活的,hCD45+,mCD45-,hCD3-,hCD19+;NK细胞被定为完整的、单一的、活的,hCD45+,mCD45-,hCD3-,hCD56+。髓系细胞被定为完整的、单一的、活的,hCD45+,mCD45-,hCD33+。髓系细胞群中,单核细胞被定为完整的、单一的、活的,hCD45+,mCD45-,hCD33+,hCD14+;粒细胞被定为完整的、单一的、活的,hCD45+,mCD45-,hCD33+,hCD66b+。
以上数据表明经本方法改造的KIT基因人源化小鼠可以不经清髓直接进行HSC免疫重建,且在体内能够有效的促进人源细胞的发育,提高人组织和细胞的移植率。
重建后的人源化免疫系统小鼠可用于建立异种移植肿瘤模型,这类模型可广泛应用于针对人用药物的筛选、药效学研究等,提高药物的临床转化率。具体来说,可以在KITB-NDG小鼠移植CD34+细胞8-16周时移植肿瘤组织,待肿瘤生长至一定体积后分组、给药,定期测量肿瘤体积、小鼠体重和死亡情况,可用于评估、筛选待测药物或其组合的药效、安全性等。
表2 T细胞占人白细胞的比例重建情况
实施例3:双重或多重基因修饰的小鼠的制备及应用
利用本方法制得的KIT基因突变小鼠还可以制备双基因或多基因修饰的小鼠。如,前述实施例1中,显微注射使用的受精卵可选择来源于含有其它基因修饰的小鼠,或者,也可选择对KIT基因突变小鼠的受精卵细胞进行基因编辑,可以进一步得到KIT与其它基因修饰的双基因或多基因修饰的小鼠模型。也可将本方法得到的KIT小鼠纯合或杂合子与其它基因修饰纯合或杂合小鼠交配,对其后代进行筛选,根据孟德尔遗传规律,可有一定几率得到KIT与其它基因修饰的双基因或多基因修饰的杂合小鼠,再将杂合子相互交配可以得到双基因或多基因修饰的纯合子,利用这些双基因或多基因修饰的小鼠可用于植入异种细胞、组织及人类病理、药物筛选等方面的研究。
采用其它基因编辑系统和制备方法也可以得到本发明的非人哺乳动物,包括但不限于基于胚胎干细胞的基因打靶技术、锌指核酸酶(ZFN)技术、转录激活子样效应因子核酸酶(TALEN)技术、归巢核酸内切酶(兆碱基大范围核酶)或其他分子生物学技术。本实施例以传统的基因同源重组技术为例,以部分替换设计为例,阐述如何采用其它方法制备获得KIT基因点突变小鼠。
鉴于本发明的目的之一是在小鼠KIT基因的第13号外显子区域引入多个点突变,最终使得KIT蛋白的第660位氨基酸从T突变为M。为此,发明人设计了包含5’同源臂、3’同源臂和含有突变体基因片段的靶向载体,在靶向载体上构建了用于阳性克隆筛选的抗性基因,如新霉素磷酸转移酶编码序列Neo,并在抗性基因的两侧装上两个同向排列的位点特异性重组系统,如Frt或LoxP重组位点。进一步的,还在靶向载体3’同源臂下游构建了具有负筛选标记的编码基因,如白喉毒素A亚基的编码基因(DTA)。靶向载体构建可采用常规方法进行,如酶切连接等。将构建正确的靶向载体转染小鼠胚胎干细胞,如C57BL/6小鼠的胚胎干细胞,利用阳性克隆筛选标记基因对得到的靶向载体转染细胞进行筛选,并利用Southern Blot技术进行DNA重组鉴定。将筛选出的正确阳性克隆按照《小鼠胚胎操作实验手册(第三版)》中的方法将阳性克隆细胞(黑色鼠)通过显微注射进入已分离好的囊胚中(白色鼠),注射后的嵌合囊胚转移至培养液中短暂培养,然后移植至受体母鼠(白色鼠)的输卵管,可生产F0代嵌合体鼠(黑白相间)。通过提取鼠尾基因组和PCR检测,挑选基因正确重组的F0代嵌合鼠用于后续繁殖和鉴定。将F0代嵌合鼠与野生型鼠交配获得F1代鼠,通过提取鼠尾基因组和PCR检测,挑选可以稳定遗传的基因重组阳性F1代杂合子小鼠。再将F1代杂合小鼠互相交配即可获得基因重组阳性F2代纯合子鼠。此外,可将F1代杂合鼠与Flp或Cre工具鼠交配去除阳性克隆筛选标记基因(neo等)后,再通过互相交配即可得到基因突变纯合子小鼠。对获得的F1代杂合或F2代纯合鼠进行基因型和表型检测的方法与前述实施例1一致。
以上详细描述了本发明的优选实施方式,但是,本发明并不限于上述实施方式中的具体细节,在本发明的技术构思范围内,可以对本发明的技术方案进行多种简单变型,这些简单变型均属于本发明的保护范围。
另外需要说明的是,在上述具体实施方式中所描述的各个具体技术特征,在不矛盾的情况下,可以通过任何合适的方式进行组合,为了避免不必要的重复,本发明对各种可能的组合方式不再另行说明。
此外,本发明的各种不同的实施方式之间也可以进行任意组合,只要其不违背本发明的思想,其同样应当视为本发明所公开的内容。
序列表
<110> 北京百奥赛图基因生物技术有限公司
<120> KIT基因修饰的非人动物的制备方法和应用
<130> 1
<160> 17
<170> SIPOSequenceListing 1.0
<210> 1
<211> 5189
<212> DNA/RNA
<213> 小鼠(Mouse)
<400> 1
ggtgcacttg ggcgagagct gtagcagaga gaggagctca gagtctagcg cagccaccgc 60
gatgagaggc gctcgcggcg cctgggatct gctctgcgtc ctgttggtcc tgctccgtgg 120
ccagacagcc acgtctcagc catctgcaag tccaggggag ccgtctccgc catccatcca 180
tccagcacaa tcagagttaa tagttgaagc tggcgacacc ctcagcctga cgtgcattga 240
tcccgacttt gtcagatgga ctttcaagac ctatttcaat gaaatggttg agaataaaaa 300
aaatgaatgg atccaggaaa aagccgaggc cactcgcacg ggcacataca cgtgcagcaa 360
cagcaatggc ctcacgagtt ctatttacgt gtttgttaga gatcctgcca aacttttcct 420
ggttggcctt cccttgtttg gcaaagaaga cagcgacgcg ctggtccgct gccctctgac 480
agacccacag gtgtccaatt attccctcat cgagtgtgat gggaaatctc tccccacgga 540
cctgacgttt gtcccaaacc ccaaggctgg catcaccatc aaaaacgtga agcgcgccta 600
ccaccggctc tgtgtccgct gtgctgctca gcgtgacggt acatggctgc attctgacaa 660
attcaccctc aaagtgcggg cagccatcaa ggctatccct gttgtgtctg tgcctgaaac 720
aagtcacctc cttaagaaag gggacacatt tacggtggtg tgcaccataa aagatgtgtc 780
tacatccgtg aactccatgt ggctaaagat gaaccctcag cctcagcaca tagcccaggt 840
aaagcacaat agctggcacc ggggtgactt caattatgaa cgccaggaga cgctgactat 900
cagctcggca agagttgacg attctggagt gttcatgtgt tatgccaata atacttttgg 960
atcagcaaat gtcacaacaa ccttgaaagt agtagaaaaa ggattcatca acatctcccc 1020
tgtgaagaac actacagtat ttgtaaccga tggagaaaac gtagatttgg ttgttgaata 1080
cgaggcctac cccaaacccg agcaccagca gtggatatat atgaacagga cctcggctaa 1140
caaagggaag gattatgtca aatctgataa caaaagcaac atcagatatg tgaaccaact 1200
tcgcctgacc agattaaaag gcacagaagg aggcacttat acctttctgg tgtccaactc 1260
tgatgccagt gcttccgtga cattcaacgt ttacgtgaac acaaaaccag aaatcctgac 1320
gtacgacagg ctcataaatg gcatgctcca gtgtgtggca gagggattcc cggagcccac 1380
aatagattgg tatttttgta caggagcaga gcaaaggtgt accactcctg tctcaccagt 1440
ggacgtacag gtccagaatg tatctgtgtc accatttgga aaactggtgg ttcagagttc 1500
catagactcc agcgtcttcc ggcacaacgg cacggtggag tgtaaggcct ccaacgatgt 1560
gggcaagagt tccgccttct ttaactttgc atttaaagag caaatccagg cccacactct 1620
gttcacgccg ctgctcattg gctttgtggt tgcagctggc gcgatgggga tcattgtgat 1680
ggtgctcacc tacaaatatt tgcagaaacc catgtatgaa gtacaatgga aggttgtcga 1740
ggagataaat ggaaacaatt atgtttacat agacccgacg caacttcctt atgatcacaa 1800
atgggagttt cccagaaaca ggctgagttt tggaaagaca ttgggagctg gtgccttcgg 1860
gaaggtcgtt gaggccactg catatggctt gattaagtcg gatgctgcca tgacagttgc 1920
cgtgaagatg ctcaaaccaa gtgcccattt aacagaaaga gaggccctaa tgtcggaact 1980
gaaggtcctg agctacctgg gcaatcacat gaatattgtg aacctgcttg gcgcatgcac 2040
ggtgggaggg cccaccctgg tcattacaga atattgttgc tatggtgatc ttttgaattt 2100
tttgagaagg aagcgtgact cgtttatttt ctcaaagcaa gaagagcagg cagaagcggc 2160
actttataag aaccttctgc actcaacgga gccttcctgt gacagttcaa atgaatatat 2220
ggacatgaag cctggcgttt cctacgtggt gccaaccaag acagacaaga ggagatccgc 2280
aagaatagac tcgtacatag aaagagacgt gactcctgcc atcatggaag atgacgagct 2340
ggctctggac ctggatgatt tgctgagctt ctcctaccag gtggccaagg gcatggcgtt 2400
cctcgcctcc aagaattgta ttcacagaga tttggcagcc aggaatatcc tcctcactca 2460
cgggcggatc acaaagattt gcgatttcgg gctagccaga gacatcagga atgattcgaa 2520
ttacgtggtc aaaggaaatg cacgactgcc cgtgaagtgg atggcaccag agagcatttt 2580
cagctgcgtg tacacatttg aaagtgatgt ctggtcctat gggattttcc tctgggagct 2640
cttctcctta ggaagcagcc cctacccagg gatgccggtc gactccaagt tctacaagat 2700
gatcaaggaa ggcttccgga tggtcagccc ggagcacgcg cctgccgaaa tgtatgacgt 2760
catgaagact tgctgggacg ctgacccctt gaaaaggcca acattcaagc aggttgtcca 2820
acttattgag aagcagatct cggacagcac caagcacatt tactccaact tggcaaactg 2880
caaccccaac ccagagaacc ccgtggtggt ggaccattcc gtgagggtca actcggtggg 2940
cagcagcgcc tcttctacgc agcccctgct cgtgcacgaa gatgcctgag cagaaaccca 3000
agtccaacag gctttgctgc tgtctccgac cccgtccttc tggcttctgt gatggttact 3060
tggtttccct ttgacttgca tcctattcca gggtagcgag ttccccaccc cacctccaac 3120
cccactgtga ttccgccttt acgagcacac actttagtgg ccgatggcct tttcttttct 3180
gccatcagcc accgtcccgc tgcgaaggtc cgaactgtat gtatatattt tcccaatagc 3240
aaagtagctc ctactgtaaa cagaaggact cctcctgctt tagaggagaa gggaagggcg 3300
gggtgaaact ggatgcccag agttcttccc ccagtgctcc cctgagtgta tttgaaaagt 3360
atggccagta gttcacttga agaatagatg tagtcccatt tggccctgag agccatcctt 3420
aatgatggga gatatatgta gcaagactag aaaaggaaag ccaagccctt tgtgtagaaa 3480
gcagaccatt cttagaacag agggcaacgg ggcatcggaa gtctggtcac gctaagaaga 3540
ccgaggctga gaaggaacaa gccaggggaa gcgtgaacaa tgatgctctg ctctgggctg 3600
ccgctcgggc ttctgtacaa ctgacctggt ttctcagtac tttgctgtct gggagtagca 3660
ttggaatcaa ggcctcctcc ctagtcagcc tttgtatata ctcatctata cgttgtatgc 3720
gttcatactt tggaggaggg atttcccaca agctttcgtt tctgtgtaca gccctggcat 3780
tagacctact gtgtgtaaga atagattaag agccatacat atttgaagga aacagttaaa 3840
tgttttttgg ttgtggttgt tgttgttgtt gttttaaaga aaaaaatgta tatgctaagc 3900
acaatcttta taagacctct tagccaacat acttgctctg tctacacttc ggaacaagcc 3960
ttccatgtca gagtggcttt gcaggcagga gaactgaggc tgtttgaaaa ggttaccaca 4020
ggatggagaa aacagtgcag tcctggtttg gattctcaca tagcagggag cacaagttaa 4080
actcagcctt ttataggcac gtcccggaca tcgggccagt atctattcaa gtgtgtatgt 4140
gtgtgcatgc gtgtgtctat gcgtgtgggt gagttgtgtt gggaaacttg ccctgcatcc 4200
ctgagggtcc tccttcagga cccaagacgt aacagcttct gtcaccgctc ctgtctctcc 4260
agtttccctg catgtcgctc actgtctaga atttactcaa agccgccaca gaggcttagc 4320
ggagtgaagt gccgaaggac ctctttattt ggagtcctcc tgtatttaac aacactctta 4380
tcgtagaccc attcattaga ccttatgtaa tgctgccaat ccagggaaac agatttaaag 4440
tgtaccccgt agacagggcc cagaggttcc cttgtccttg ccctccccca caccacccat 4500
gatcactgtc caacataaag ggttcagtgt gtacgtggtc atgtgttgtc cttacaggat 4560
tcaggtatgt tgccttcacg gttttcccca ccccctcctg ccctttatcc tttaggccgt 4620
gtggccatga acctggaaga agtgatcgtt tgcacttgag tgctacactc ttgcaccttt 4680
ccaaagtaag ctggtttgga ggtcctgttg tcatgtacga gactgtcacc agttaccgcg 4740
ctctgtttga aacatgtctt tgtattccta atgacttcag ttagagtaag gagaatagct 4800
gttaatatgg atgtcaggta cttaaggggc cacaccattg agaattttgt cttggatatt 4860
cttgaaagtt tatattttta taattttttt tacatcagat gtcagatgtt tctttcagtt 4920
gcttgatgtt tggaattatt atgtggcttt ttttgtaaat attgaaatgt agcaataatg 4980
tcttttgaat attcctgagc ccatgagtcc ctgaaaatat tttttatata tacagtaact 5040
ttatgtgtaa ataatacgct gtgcaagttt aaacatgtca cgttacatgt gggttttttc 5100
tgatatgttg tccaactgtt gacagttctg aagaattcta ataaaaatgt aaatatataa 5160
atcaaaaaaa aaaaaaaaaa aaaaaaaaa 5189
<210> 2
<211> 975
<212> PRT
<213> 小鼠(Mouse)
<400> 2
Met Arg Gly Ala Arg Gly Ala Trp Asp Leu Leu Cys Val Leu Leu Val
1 5 10 15
Leu Leu Arg Gly Gln Thr Ala Thr Ser Gln Pro Ser Ala Ser Pro Gly
20 25 30
Glu Pro Ser Pro Pro Ser Ile His Pro Ala Gln Ser Glu Leu Ile Val
35 40 45
Glu Ala Gly Asp Thr Leu Ser Leu Thr Cys Ile Asp Pro Asp Phe Val
50 55 60
Arg Trp Thr Phe Lys Thr Tyr Phe Asn Glu Met Val Glu Asn Lys Lys
65 70 75 80
Asn Glu Trp Ile Gln Glu Lys Ala Glu Ala Thr Arg Thr Gly Thr Tyr
85 90 95
Thr Cys Ser Asn Ser Asn Gly Leu Thr Ser Ser Ile Tyr Val Phe Val
100 105 110
Arg Asp Pro Ala Lys Leu Phe Leu Val Gly Leu Pro Leu Phe Gly Lys
115 120 125
Glu Asp Ser Asp Ala Leu Val Arg Cys Pro Leu Thr Asp Pro Gln Val
130 135 140
Ser Asn Tyr Ser Leu Ile Glu Cys Asp Gly Lys Ser Leu Pro Thr Asp
145 150 155 160
Leu Thr Phe Val Pro Asn Pro Lys Ala Gly Ile Thr Ile Lys Asn Val
165 170 175
Lys Arg Ala Tyr His Arg Leu Cys Val Arg Cys Ala Ala Gln Arg Asp
180 185 190
Gly Thr Trp Leu His Ser Asp Lys Phe Thr Leu Lys Val Arg Ala Ala
195 200 205
Ile Lys Ala Ile Pro Val Val Ser Val Pro Glu Thr Ser His Leu Leu
210 215 220
Lys Lys Gly Asp Thr Phe Thr Val Val Cys Thr Ile Lys Asp Val Ser
225 230 235 240
Thr Ser Val Asn Ser Met Trp Leu Lys Met Asn Pro Gln Pro Gln His
245 250 255
Ile Ala Gln Val Lys His Asn Ser Trp His Arg Gly Asp Phe Asn Tyr
260 265 270
Glu Arg Gln Glu Thr Leu Thr Ile Ser Ser Ala Arg Val Asp Asp Ser
275 280 285
Gly Val Phe Met Cys Tyr Ala Asn Asn Thr Phe Gly Ser Ala Asn Val
290 295 300
Thr Thr Thr Leu Lys Val Val Glu Lys Gly Phe Ile Asn Ile Ser Pro
305 310 315 320
Val Lys Asn Thr Thr Val Phe Val Thr Asp Gly Glu Asn Val Asp Leu
325 330 335
Val Val Glu Tyr Glu Ala Tyr Pro Lys Pro Glu His Gln Gln Trp Ile
340 345 350
Tyr Met Asn Arg Thr Ser Ala Asn Lys Gly Lys Asp Tyr Val Lys Ser
355 360 365
Asp Asn Lys Ser Asn Ile Arg Tyr Val Asn Gln Leu Arg Leu Thr Arg
370 375 380
Leu Lys Gly Thr Glu Gly Gly Thr Tyr Thr Phe Leu Val Ser Asn Ser
385 390 395 400
Asp Ala Ser Ala Ser Val Thr Phe Asn Val Tyr Val Asn Thr Lys Pro
405 410 415
Glu Ile Leu Thr Tyr Asp Arg Leu Ile Asn Gly Met Leu Gln Cys Val
420 425 430
Ala Glu Gly Phe Pro Glu Pro Thr Ile Asp Trp Tyr Phe Cys Thr Gly
435 440 445
Ala Glu Gln Arg Cys Thr Thr Pro Val Ser Pro Val Asp Val Gln Val
450 455 460
Gln Asn Val Ser Val Ser Pro Phe Gly Lys Leu Val Val Gln Ser Ser
465 470 475 480
Ile Asp Ser Ser Val Phe Arg His Asn Gly Thr Val Glu Cys Lys Ala
485 490 495
Ser Asn Asp Val Gly Lys Ser Ser Ala Phe Phe Asn Phe Ala Phe Lys
500 505 510
Glu Gln Ile Gln Ala His Thr Leu Phe Thr Pro Leu Leu Ile Gly Phe
515 520 525
Val Val Ala Ala Gly Ala Met Gly Ile Ile Val Met Val Leu Thr Tyr
530 535 540
Lys Tyr Leu Gln Lys Pro Met Tyr Glu Val Gln Trp Lys Val Val Glu
545 550 555 560
Glu Ile Asn Gly Asn Asn Tyr Val Tyr Ile Asp Pro Thr Gln Leu Pro
565 570 575
Tyr Asp His Lys Trp Glu Phe Pro Arg Asn Arg Leu Ser Phe Gly Lys
580 585 590
Thr Leu Gly Ala Gly Ala Phe Gly Lys Val Val Glu Ala Thr Ala Tyr
595 600 605
Gly Leu Ile Lys Ser Asp Ala Ala Met Thr Val Ala Val Lys Met Leu
610 615 620
Lys Pro Ser Ala His Leu Thr Glu Arg Glu Ala Leu Met Ser Glu Leu
625 630 635 640
Lys Val Leu Ser Tyr Leu Gly Asn His Met Asn Ile Val Asn Leu Leu
645 650 655
Gly Ala Cys Thr Val Gly Gly Pro Thr Leu Val Ile Thr Glu Tyr Cys
660 665 670
Cys Tyr Gly Asp Leu Leu Asn Phe Leu Arg Arg Lys Arg Asp Ser Phe
675 680 685
Ile Phe Ser Lys Gln Glu Glu Gln Ala Glu Ala Ala Leu Tyr Lys Asn
690 695 700
Leu Leu His Ser Thr Glu Pro Ser Cys Asp Ser Ser Asn Glu Tyr Met
705 710 715 720
Asp Met Lys Pro Gly Val Ser Tyr Val Val Pro Thr Lys Thr Asp Lys
725 730 735
Arg Arg Ser Ala Arg Ile Asp Ser Tyr Ile Glu Arg Asp Val Thr Pro
740 745 750
Ala Ile Met Glu Asp Asp Glu Leu Ala Leu Asp Leu Asp Asp Leu Leu
755 760 765
Ser Phe Ser Tyr Gln Val Ala Lys Gly Met Ala Phe Leu Ala Ser Lys
770 775 780
Asn Cys Ile His Arg Asp Leu Ala Ala Arg Asn Ile Leu Leu Thr His
785 790 795 800
Gly Arg Ile Thr Lys Ile Cys Asp Phe Gly Leu Ala Arg Asp Ile Arg
805 810 815
Asn Asp Ser Asn Tyr Val Val Lys Gly Asn Ala Arg Leu Pro Val Lys
820 825 830
Trp Met Ala Pro Glu Ser Ile Phe Ser Cys Val Tyr Thr Phe Glu Ser
835 840 845
Asp Val Trp Ser Tyr Gly Ile Phe Leu Trp Glu Leu Phe Ser Leu Gly
850 855 860
Ser Ser Pro Tyr Pro Gly Met Pro Val Asp Ser Lys Phe Tyr Lys Met
865 870 875 880
Ile Lys Glu Gly Phe Arg Met Val Ser Pro Glu His Ala Pro Ala Glu
885 890 895
Met Tyr Asp Val Met Lys Thr Cys Trp Asp Ala Asp Pro Leu Lys Arg
900 905 910
Pro Thr Phe Lys Gln Val Val Gln Leu Ile Glu Lys Gln Ile Ser Asp
915 920 925
Ser Thr Lys His Ile Tyr Ser Asn Leu Ala Asn Cys Asn Pro Asn Pro
930 935 940
Glu Asn Pro Val Val Val Asp His Ser Val Arg Val Asn Ser Val Gly
945 950 955 960
Ser Ser Ala Ser Ser Thr Gln Pro Leu Leu Val His Glu Asp Ala
965 970 975
<210> 3
<211> 188
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 3
gctttgtttt gctaaaatac atcactttcc cttttagcaa gtgctcattt aacagaaaga 60
gaagccctaa tgtcggaact gaaggtcctg agctacctgg gcaatcacat gaatattgtg 120
aatctgcttg gcgcatgcat ggttggaggt gagtctcgag ttgccatctc tctggttatc 180
aaagtatg 188
<210> 4
<211> 975
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 4
Met Arg Gly Ala Arg Gly Ala Trp Asp Leu Leu Cys Val Leu Leu Val
1 5 10 15
Leu Leu Arg Gly Gln Thr Ala Thr Ser Gln Pro Ser Ala Ser Pro Gly
20 25 30
Glu Pro Ser Pro Pro Ser Ile His Pro Ala Gln Ser Glu Leu Ile Val
35 40 45
Glu Ala Gly Asp Thr Leu Ser Leu Thr Cys Ile Asp Pro Asp Phe Val
50 55 60
Arg Trp Thr Phe Lys Thr Tyr Phe Asn Glu Met Val Glu Asn Lys Lys
65 70 75 80
Asn Glu Trp Ile Gln Glu Lys Ala Glu Ala Thr Arg Thr Gly Thr Tyr
85 90 95
Thr Cys Ser Asn Ser Asn Gly Leu Thr Ser Ser Ile Tyr Val Phe Val
100 105 110
Arg Asp Pro Ala Lys Leu Phe Leu Val Gly Leu Pro Leu Phe Gly Lys
115 120 125
Glu Asp Ser Asp Ala Leu Val Arg Cys Pro Leu Thr Asp Pro Gln Val
130 135 140
Ser Asn Tyr Ser Leu Ile Glu Cys Asp Gly Lys Ser Leu Pro Thr Asp
145 150 155 160
Leu Thr Phe Val Pro Asn Pro Lys Ala Gly Ile Thr Ile Lys Asn Val
165 170 175
Lys Arg Ala Tyr His Arg Leu Cys Val Arg Cys Ala Ala Gln Arg Asp
180 185 190
Gly Thr Trp Leu His Ser Asp Lys Phe Thr Leu Lys Val Arg Ala Ala
195 200 205
Ile Lys Ala Ile Pro Val Val Ser Val Pro Glu Thr Ser His Leu Leu
210 215 220
Lys Lys Gly Asp Thr Phe Thr Val Val Cys Thr Ile Lys Asp Val Ser
225 230 235 240
Thr Ser Val Asn Ser Met Trp Leu Lys Met Asn Pro Gln Pro Gln His
245 250 255
Ile Ala Gln Val Lys His Asn Ser Trp His Arg Gly Asp Phe Asn Tyr
260 265 270
Glu Arg Gln Glu Thr Leu Thr Ile Ser Ser Ala Arg Val Asp Asp Ser
275 280 285
Gly Val Phe Met Cys Tyr Ala Asn Asn Thr Phe Gly Ser Ala Asn Val
290 295 300
Thr Thr Thr Leu Lys Val Val Glu Lys Gly Phe Ile Asn Ile Ser Pro
305 310 315 320
Val Lys Asn Thr Thr Val Phe Val Thr Asp Gly Glu Asn Val Asp Leu
325 330 335
Val Val Glu Tyr Glu Ala Tyr Pro Lys Pro Glu His Gln Gln Trp Ile
340 345 350
Tyr Met Asn Arg Thr Ser Ala Asn Lys Gly Lys Asp Tyr Val Lys Ser
355 360 365
Asp Asn Lys Ser Asn Ile Arg Tyr Val Asn Gln Leu Arg Leu Thr Arg
370 375 380
Leu Lys Gly Thr Glu Gly Gly Thr Tyr Thr Phe Leu Val Ser Asn Ser
385 390 395 400
Asp Ala Ser Ala Ser Val Thr Phe Asn Val Tyr Val Asn Thr Lys Pro
405 410 415
Glu Ile Leu Thr Tyr Asp Arg Leu Ile Asn Gly Met Leu Gln Cys Val
420 425 430
Ala Glu Gly Phe Pro Glu Pro Thr Ile Asp Trp Tyr Phe Cys Thr Gly
435 440 445
Ala Glu Gln Arg Cys Thr Thr Pro Val Ser Pro Val Asp Val Gln Val
450 455 460
Gln Asn Val Ser Val Ser Pro Phe Gly Lys Leu Val Val Gln Ser Ser
465 470 475 480
Ile Asp Ser Ser Val Phe Arg His Asn Gly Thr Val Glu Cys Lys Ala
485 490 495
Ser Asn Asp Val Gly Lys Ser Ser Ala Phe Phe Asn Phe Ala Phe Lys
500 505 510
Glu Gln Ile Gln Ala His Thr Leu Phe Thr Pro Leu Leu Ile Gly Phe
515 520 525
Val Val Ala Ala Gly Ala Met Gly Ile Ile Val Met Val Leu Thr Tyr
530 535 540
Lys Tyr Leu Gln Lys Pro Met Tyr Glu Val Gln Trp Lys Val Val Glu
545 550 555 560
Glu Ile Asn Gly Asn Asn Tyr Val Tyr Ile Asp Pro Thr Gln Leu Pro
565 570 575
Tyr Asp His Lys Trp Glu Phe Pro Arg Asn Arg Leu Ser Phe Gly Lys
580 585 590
Thr Leu Gly Ala Gly Ala Phe Gly Lys Val Val Glu Ala Thr Ala Tyr
595 600 605
Gly Leu Ile Lys Ser Asp Ala Ala Met Thr Val Ala Val Lys Met Leu
610 615 620
Lys Pro Ser Ala His Leu Thr Glu Arg Glu Ala Leu Met Ser Glu Leu
625 630 635 640
Lys Val Leu Ser Tyr Leu Gly Asn His Met Asn Ile Val Asn Leu Leu
645 650 655
Gly Ala Cys Met Val Gly Gly Pro Thr Leu Val Ile Thr Glu Tyr Cys
660 665 670
Cys Tyr Gly Asp Leu Leu Asn Phe Leu Arg Arg Lys Arg Asp Ser Phe
675 680 685
Ile Phe Ser Lys Gln Glu Glu Gln Ala Glu Ala Ala Leu Tyr Lys Asn
690 695 700
Leu Leu His Ser Thr Glu Pro Ser Cys Asp Ser Ser Asn Glu Tyr Met
705 710 715 720
Asp Met Lys Pro Gly Val Ser Tyr Val Val Pro Thr Lys Thr Asp Lys
725 730 735
Arg Arg Ser Ala Arg Ile Asp Ser Tyr Ile Glu Arg Asp Val Thr Pro
740 745 750
Ala Ile Met Glu Asp Asp Glu Leu Ala Leu Asp Leu Asp Asp Leu Leu
755 760 765
Ser Phe Ser Tyr Gln Val Ala Lys Gly Met Ala Phe Leu Ala Ser Lys
770 775 780
Asn Cys Ile His Arg Asp Leu Ala Ala Arg Asn Ile Leu Leu Thr His
785 790 795 800
Gly Arg Ile Thr Lys Ile Cys Asp Phe Gly Leu Ala Arg Asp Ile Arg
805 810 815
Asn Asp Ser Asn Tyr Val Val Lys Gly Asn Ala Arg Leu Pro Val Lys
820 825 830
Trp Met Ala Pro Glu Ser Ile Phe Ser Cys Val Tyr Thr Phe Glu Ser
835 840 845
Asp Val Trp Ser Tyr Gly Ile Phe Leu Trp Glu Leu Phe Ser Leu Gly
850 855 860
Ser Ser Pro Tyr Pro Gly Met Pro Val Asp Ser Lys Phe Tyr Lys Met
865 870 875 880
Ile Lys Glu Gly Phe Arg Met Val Ser Pro Glu His Ala Pro Ala Glu
885 890 895
Met Tyr Asp Val Met Lys Thr Cys Trp Asp Ala Asp Pro Leu Lys Arg
900 905 910
Pro Thr Phe Lys Gln Val Val Gln Leu Ile Glu Lys Gln Ile Ser Asp
915 920 925
Ser Thr Lys His Ile Tyr Ser Asn Leu Ala Asn Cys Asn Pro Asn Pro
930 935 940
Glu Asn Pro Val Val Val Asp His Ser Val Arg Val Asn Ser Val Gly
945 950 955 960
Ser Ser Ala Ser Ser Thr Gln Pro Leu Leu Val His Glu Asp Ala
965 970 975
<210> 5
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
ccaccgtgca tgcgccaagc agg 23
<210> 6
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
cctgcttggc gcatgcacgg tgg 23
<210> 7
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
ctgcttggcg catgcacggt ggg 23
<210> 8
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
gaacctgctt ggcgcatgca cgg 23
<210> 9
<211> 132
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
gaattctaat acgactcact atagggggtc ttcgagaaga cctgttttag agctagaaat 60
agcaagttaa aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgct 120
tttaaaggat cc 132
<210> 10
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
ggcctgcttg gcgcatgcac gg 22
<210> 11
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
caccggcctg cttggcgcat gcacgg 26
<210> 12
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
ccgtgcatgc gccaagcagg cc 22
<210> 13
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
aaacccgtgc atgcgccaag caggcc 26
<210> 14
<211> 952
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
tggttccctt ccttgcagag caaatccagg cccacactct gttcacgccg ctgctcattg 60
gctttgtggt tgcagctggc gcgatgggga tcattgtgat ggtgctcacc tacaaatatt 120
tgcaggtgag cattgaattg ttctcttcct ggggacgcca aggcggcagg gcaggcactg 180
attgttcagc gggtgacaca tctttctttt cctttctcct ccagaaaccc atgtatgaag 240
tacaatggaa ggttgtcgag gagataaatg gaaacaatta tgtttacata gacccgacgc 300
aacttcctta tgatcacaaa tgggagtttc ccagaaacag gctgagtttt ggtcagtgtg 360
aggcaggggc tttccacgaa gaagcccttt tgtgtacgcg taaccgtgat tttttttttt 420
taaggaaccc gcagtggctt cctttgtctt gtttcaccta aaacaagaag tgctttctgt 480
caaactgcac aggagttggc agggttagca gaaagagccc cgtagagaag tggctccaga 540
cacacatgtc ttccattgcc ttgttgattt gggaacctca caaactgttg gttggtcttc 600
ccactgacat gaccctgcct tcttccttcc taaaggaaag acattgggag ctggtgcctt 660
cgggaaggtc gttgaggcca ctgcatatgg cttgattaag tcggatgctg ccatgacagt 720
tgccgtgaag atgctcaaac gtaagtgttt cgggggcact caccccctgt acccaccacc 780
agtttatcta ataggttctt cccccctgcc cccacgcttt gttttgctaa aatacatcac 840
tttccctttt agcaagtgct catttaacag aaagagaagc cctaatgtcg gaactgaagg 900
tcctgagcta cctgggcaat cacatgaata ttgtgaatct gcttggcgca tg 952
<210> 15
<211> 947
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
ggttggaggt gagtctcgag ttgccatctc tctggttatc aaagtatgat ttaggcagtg 60
cctagctata agctacttga gagatttttt ttttttaacc aagcattttg ttctattttg 120
ctaggtttcg ttttccacag tgttcctcag aatgtagatt tttataattt ctatgctggg 180
agccttcaga ctcatgtctt gtcatgattt tgcaatgtag gttgtagttg ttccgctgtg 240
tggtcgagtg ttggaactac tcatcacagt tgtgtttggt acccattact cagattctta 300
tttctcccct ccctctccac agtcctaagg acctctattc tgccctcagt ggatcaacat 360
tttaaactcc cacaagcttt tgggaacaca caaggtttct ctttatgcag agccacttac 420
accccgcctt tcctcattag gcattatatg tgtgtattga attataatac tctccccctt 480
aaatggcata agcagtgggg ggcaggtagc tttatataat gcggggggcc gtaatcagtc 540
atttgagagt taatgttgat tttccttcta taactgagag tggccccagc ctcccagttc 600
tcaactgtac accaagtaga tccagctttt cactcccaca aaagcaagct ggcggctcta 660
gcccctaagt cagagtgcca ccaagggtgc gcaggcacac tgttctacag aacaaagctg 720
tgtagggtga gctggtgctg tctgtcctga gagagccatg ggatggtagg cagcttagac 780
aacgtctccg gacagcccac agacccggca ctctgcacac catgaattgt tcatgcgaca 840
gtatctaccc atggcaatta gttgcctgtt aagttcggaa ctgcgctttt aaacatcctg 900
aggaaccact cgtgttttca agtagtcatg ctcagaaaaa cgggccg 947
<210> 16
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
actgttggtt ggtcttccca ctgac 25
<210> 17
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
agcctagtag ggaagtaacc aggga 25
Claims (14)
1.一种KIT基因修饰的非人动物的制备方法,其特征在于,所述的非人动物表达突变的KIT蛋白,所述突变的KIT蛋白如SEQ ID NO:4所示,编码所述突变的KIT蛋白的KIT基因序列为SEQ ID NO:1第2040位由C突变为T、1945位由C突变为T、1963位由G突变为A、2023位由C突变为T和2044位由G突变为T;
所述的构建方法包括使用靶向载体,所述的靶向载体包含编码SEQ ID NO:4所示第626-662位氨基酸序列的核苷酸序列,所述的靶向载体还包含5’臂和3’臂,所述的5’臂为与待改变的转换区5’端同源的DNA片段,所述的3’臂为与待改变的转换区3’端同源的DNA片段;
所述的待改变的转换区位于KIT基因的13号外显子;
所述的非人动物为NOD-Prkdcscid IL-2rγnull小鼠。
2.根据权利要求1所述的制备方法,其特征在于,编码所述突变的KIT蛋白的KIT基因包含SEQ ID NO:3。
3.根据权利要求1-2任一所述的制备方法,其特征在于,所述KIT基因修饰的非人动物为使用sgRNA和靶向载体将非人动物KIT基因座的第13外显子引入一个或多个点突变制备获得;所述的sgRNA靶向的靶位点序列如SEQ ID NO:5-8任一项所示,所述的靶向载体包含编码SEQ ID NO:4第626-662位氨基酸的核苷酸序列。
4.一种靶向载体,其特征在于,所述的靶向载体包含编码SEQ ID NO:4第626-662位氨基酸的核苷酸序列,所述的靶向载体还包含5’臂和3’臂,所述的5’臂如SEQ ID NO:14所示;所述的3’臂如SEQ ID NO:15所示。
5.一种靶向KIT基因的sgRNA,其特征在于,所述的sgRNA靶向的靶位点序列如SEQ IDNO:5-8任一项所示。
6.一种制备多基因修饰的非人动物的方法,其特征在于,包括如下步骤:
(1)采用权利要求1-3任一所述的制备方法构建的KIT基因修饰的非人动物;
(2)将步骤(1)制备获得的KIT基因修饰的非人动物与其他基因修饰的非人动物交配、体外授精或直接进行基因编辑,并进行筛选,得到多基因修饰的非人动物。
7.一种编码突变的KIT蛋白的KIT基因,其特征在于,所述的KIT基因序列为SEQ ID NO:1第2040位由C突变为T、1945位由C突变为T、1963位由G突变为A、2023位由C突变为T和2044位由G突变为T。
8.根据权利要求7所述的KIT基因,其特征在于,所述的KIT基因序列包含SEQ ID NO:3。
9.一种细胞、组织、器官或荷瘤后的瘤组织,其特征在于,所述的细胞、组织、器官或荷瘤后的瘤组织表达突变的KIT蛋白,所述突变的KIT蛋白如SEQ ID NO:4所示;编码所述突变的KIT蛋白的KIT基因序列为SEQ ID NO:1第2040位由C突变为T、1945位由C突变为T、1963位由G突变为A、2023位由C突变为T和2044位由G突变为T。
10.根据权利要求9所述的细胞、组织、器官或荷瘤后的瘤组织,其特征在于,编码所述突变的KIT蛋白的KIT基因包含SEQ ID NO:3。
11.来源于权利要求1-3任一所述的制备方法制备的KIT基因修饰的非人动物、权利要求6所述的方法构建的多基因修饰的非人动物、权利要求7或8所述的KIT基因、权利要求9或10所述的细胞、组织、器官或荷瘤后的瘤组织的应用,其特征在于,所述的应用为非疾病的诊断或治疗目的,所述的应用包括:
A)在需要涉及人类细胞的免疫过程的产品开发中的应用;
B)作为药理学、免疫学、微生物学和医学研究的模型系统中的应用;
C)在生产和利用动物实验疾病模型,用于人源细胞移植、免疫系统重建或病原学研究中的应用;
D)在筛选、验证、评价或研究KIT基因功能中的应用;
E)在筛选、验证、评价或研究靶向人的药物、药效研究中的应用;
F)在筛选和评估人用药及药效研究方面的应用。
12.根据权利要求11所述的应用,其特征在于,A)中所述的涉及人类细胞的免疫过程的产品开发包括制造人类抗体。
13.根据权利要求11所述的应用,其特征在于,E)中所述的靶向人的药物包括靶向人的抗体。
14.根据权利要求11所述的应用,其特征在于,E)中所述的靶向人的药物包括免疫相关疾病药物、抗肿瘤或抗炎症药物。
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