CN113604473A - 一种可诱导自然杀伤细胞缺陷小鼠模型的构建方法及应用 - Google Patents
一种可诱导自然杀伤细胞缺陷小鼠模型的构建方法及应用 Download PDFInfo
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Abstract
本发明公开了一种针对小鼠Ncr1基因的gRNA,利用所述gRNA,进一步将包含重组左臂、核糖体接入位点(IRES)、白喉毒素受体(DTR)和绿色荧光蛋白(EGFP)融合编码序列、重组右臂的同源重组载体同源重组到小鼠Ncr1基因,构建了一个Ncr1‑IRES‑DTR‑EGFP基因修饰小鼠模型。该模型可以实现小鼠内源Ncr1基因驱动白喉毒素受体和绿色荧光蛋白在NK细胞的表达,利用该模型,可以实现小鼠自然杀伤细胞缺陷的可诱导,实现小鼠NK细胞的可诱导缺失,并通过EGFP荧光表达标记小鼠NK细胞。本发明公开的构建可诱导自然杀伤细胞缺陷小鼠模型的方法以及采用所述方法建立的模型对于NK细胞功能研究等领域具有重要的意义。
Description
技术领域
本发明属于生物技术领域,具体涉及一种可诱导自然杀伤细胞缺陷小鼠模型的构建方法及应用。
背景技术
自然杀伤细胞(natural killer cell)是一种细胞质中具有大颗粒的细胞,简称NK细胞(NK cell),也称作大颗粒淋巴细胞(LGL, Large Granular Lymphocytes)。由骨髓淋巴样干细胞发育而成,主要分布于外周血和脾脏,在淋巴结和其他组织中也有少量存在。自然杀伤细胞可通过直接和陌生细胞接触,分泌穿孔素及肿瘤坏死因子,以细胞膜破裂的方式杀死目标细胞。因为其非专一性的细胞毒杀作用而被命名。
NK细胞是机体重要的免疫细胞,不仅与抗肿瘤、 抗病毒感染和免疫调节有关,而且在某些情况下参与超敏反应和自身免疫性疾病的发生,能够识别靶细胞、杀伤介质。在抗肿瘤研究中,NK细胞扮演关键角色,NK细胞有两方面抗癌作用,一是上述的对肿瘤细胞的直接杀伤,通过释放后穿孔素和颗粒酶或通过死亡受体杀死肿瘤细胞;二是它通过分泌细胞因子和趋化因子扮演免疫系统的调节细胞角色,激活T细胞等的杀伤作用。目前用于肿瘤免疫治疗的NK细胞策略有:体外活化的自体或异体NK细胞治疗;联合NK细胞和单抗药(如免疫检查点抑制剂)来诱导抗体特异的细胞毒性;构建CAR-NK细胞免疫疗法等。
在体内特异性的剔除某一类细胞,是研究该类细胞在体内功能的一种有效方法。为了更好的研究NK细胞的作用和功能,研究人员开发了多种NK细胞缺陷小鼠模型,如Beige小鼠、NSG小鼠、Perforin基因缺失小鼠等。Beige小鼠是最早发现的NK细胞功能缺陷小鼠模型之一,缺陷原因是因为小鼠自发点突变,造成其在天然细胞毒性和抗体依赖性细胞毒性中均缺乏NK细胞裂解细胞的功能;NSG小鼠是一种T,B,NK细胞均缺失的小鼠重度免疫缺陷小鼠类型,该模型因为Il2rg基因缺陷导致NK细胞缺失;Perforin基因敲除小鼠模型的NK细胞,对MHC-I分子缺失细胞的杀伤能力显著下降。这些NK细胞缺陷模型无论是NK细胞杀伤能力的缺陷,还是NK细胞数量的减少,都广泛应用于NK细胞发育、驯化机制研究和NK细胞生物医药研发,但这些模型中NK细胞的缺陷是小鼠生来就有的免疫缺陷,这种长期的免疫缺陷可能导致机体本身适应性的代偿和小鼠本身其他免疫方面的异常,导致研究结果的误读。因此,迫切需求有一种可诱导性的NK细胞缺陷小鼠模型,其可以在诱导剂的作用下,在特定时间内造成NK细胞缺失,在其他时间段内,NK细胞维持正常状态,用于NK细胞的研究。
白喉毒素(DT)和其受体(DTR)介导的细胞毒性是一种非常高效的细胞杀伤系统。白喉毒素是人类发现的第一种细菌毒素,其通过受体进入细胞之后,由于其含有 ADP 依赖的核糖体转移酶活性,能催化 NAD+的 ADP 核糖基向延长因子(EF-2)转移,生成烟酰胺,使EF-2失活,从而阻断细胞蛋白质的合成,最后导致细胞死亡。真核细胞细胞质中单个分子的白喉毒素活性就足以杀死细胞,这种杀伤机制严格依赖于其受体-肝素结合 EGF 样生长因子前体(proHB-EGF,也称为 DTR)介导的内吞作用。人类和灵长类动物细胞天然对 DT 敏感,但鼠细胞对白喉毒素的杀伤不敏感。原因在于灵长类和人类的HB-EGF分子作为白喉毒素受体,可以和白喉毒素结合从而发挥细胞毒性作用,但是小鼠表达的HB-EGF分子由于序列差异,不会和白喉毒素结合。因此,小鼠细胞对白喉毒素的抗性约是人类细胞的105倍。已有的研究表明,如果将灵长类动物的DTR转入小鼠细胞可使小鼠对DT产生敏感。因此,可以借助细胞类型特异的启动子驱动灵长类DTR在小鼠特定细胞类型表达,进而通过给予小鼠白喉毒素,实现对特定细胞类型的可诱导杀伤。
目前研究发现NK细胞表面有多种受体分子,如CD56、NCR1、NKp30等,其中NCR1目前被认为是有关NK细胞的最可靠的标志物。NCR1在多种物种(人类、小鼠、非人类灵长类动物)的所有NK细胞(CD49b+和CD49b−)都有表达。2007年法国Eric Vivier课题组利用Ncr1启动子构建了Ncr1-DTR转基因小鼠,在NK细胞中驱动DTR表达,实现对NK细胞的选择性杀伤(Thierry Walzer,et.al. Identification, activation, and selective in vivoablation of mouse NK cells via NKp46. PNAS, February 27, 2007 104 (9) 3384-3389)。转基因方法小鼠模型制备,本身具有插入位点不确定,表达细胞类型和表达量不确定等缺陷,后期需要进行大量的建系和品系鉴定工作,根据文献报道,Eric Vivier课题组从6个品系founder中筛选到一个相对符合要求的DTR品系。针对这些问题,本发明通过定点敲入的方式,将IRES-DTRGFP表达元件定点插入到Ncr1基因位点,构建了Ncr1-DTR小鼠模型,实现了在特定时间段内对NK细胞的杀伤。该模型利用Ncr1内源基因启动子驱动DTRGFP表达,因为插入位点确定、驱动表达的基因确定,不需要进行品系founder筛选,模型制备方法明确、高效。
发明内容
本发明的目的在于提供一种构建可诱导性NK细胞缺陷小鼠模型的构建方法及应用。该方法利用Ncr1分子是小鼠NK细胞特异性标记分子这一特性,通过将白喉毒素受体和绿色荧光蛋白定点插入到Ncr1分子基因表达框,构建了一个Ncr1-IRES-DTR-EGFP(简称:Ncr1-DTR)基因修饰小鼠模型,该模型可以实现小鼠内源Ncr1基因驱动白喉毒素受体和绿色荧光蛋白在NK细胞的表达。利用该模型,发明人可以通过EGFP荧光表达标记小鼠NK细胞;可以通过在特定时间点或时间段内给予小鼠白喉毒素将NK细胞杀死,造成NK细胞缺失,对NK细胞在特定生理、病理过程中的作用进行研究。
为实现本发明,采用了如下技术方案。
本发明公开了一种针对小鼠Ncr1基因的gRNA,所述gRNA的靶位点序列如SEQ IDNO:1-10所示。
优选的,所述gRNA的靶位点序列如SEQ ID NO: 5所示。
本发明公开了一种用于构建可诱导自然杀伤细胞缺陷小鼠模型的试剂盒,所述试剂盒包括:
(1)针对小鼠Ncr1基因的gRNA,所述gRNA的靶位点序列如SEQ ID NO:1-10所示;
(2)包含白喉毒素受体编码序列的同源重组载体;
(3)Cas9 mRNA。
优选的,所述试剂盒中的gRNA的靶位点序列如SEQ ID NO: 5所示。
优选的,所述试剂盒包括的同源重组载体为针对小鼠Ncr1基因的同源重组载体,所述载体从左到右顺序包括:重组左臂、核糖体接入位点序列、白喉毒素受体编码序列与绿色荧光蛋白编码序列融合序列元件、重组右臂。
优选的,所述载体顺序包括:重组左臂、核糖体接入位点序列、白喉毒素受体编码序列与绿色荧光蛋白编码序列融合序列元件、重组右臂。
优选的,所述重组左臂、核糖体接入位点序列、白喉毒素受体编码序列与绿色荧光蛋白编码序列融合序列元件、重组右臂的序列分别如SEQ ID NO:11-14所示。
优选的,所述同源重组载体为PBR322。
优选的,所述试剂盒还包括针对同源重组左臂的鉴定引物对P1和P2和针对同源重组右臂的鉴定引物对P3和P4。
优选的,所述P1和P2的序列分别如SEQ ID NO:15-16所示,所述P3和P4的序列分别如SEQ ID NO:17-18所示。
本发明公开了针对小鼠Ncr1基因的基因编辑的靶标序列,所述靶标序列分别如SEQ ID NO:1-10所示。
优选的,所述基因编辑为CRISPR-Cas9基因编辑系统。
本发明公开了针对小鼠Ncr1基因的gRNA,所述gRNA对应的靶位点序列分别如SEQID NO:1-10所示。
优选的,所述靶位点序列为SEQ ID NO:5所示。
本发明公开了所述的gRNA在制备可诱导自然杀伤细胞缺陷小鼠模型的试剂盒中的应用。
优选的,所述gRNA对应的靶位点序列分别如SEQ ID NO:1-10所示。
优选的,所述靶位点序列为SEQ ID NO:5所示。
优选的,所述gRNA的序列为auggaacuga aggcaacucc ugg。
本发明公开了一种构建可诱导自然杀伤细胞缺陷小鼠模型的方法,所述方法包括:
(1)取小鼠的受精卵;
(2)将包含白喉毒素受体编码序列的同源重组载体、Cas9 mRNA以及所述的gRNA混合后,进行受精卵显微注射;
(3)将显微注射后的受精卵经培养箱短暂培养后,移植至受体母鼠的输卵管,获得基因修饰小鼠F0代小鼠;
(4)对F0代小鼠抽提基因组,分别利用针对同源左臂的鉴定引物对和同源右臂的鉴定引物对进行同源重组鉴定。
优选的,所述gRNA的靶位点序列如SEQ ID NO:1-10所示。
优选的,所述gRNA的靶位点序列如SEQ ID NO: 5所示。
优选的,所述同源重组载体为针对小鼠Ncr1基因的同源重组载体,所述载体从左到右顺序包括:重组左臂、核糖体接入位点序列、白喉毒素受体编码序列与绿色荧光蛋白编码序列融合序列元件、重组右臂。
优选的,所述重组左臂、核糖体接入位点序列、白喉毒素受体编码序列与绿色荧光蛋白编码序列融合序列元件、重组右臂的序列分别如SEQ ID NO:11-14所示。
优选的,所述同源重组载体为PBR322。
优选的,所述试剂盒还包括针对同源重组左臂的鉴定引物对P1和P2和针对同源重组右臂的鉴定引物对P3和P4。
优选的,所述P1和P2的序列分别如SEQ ID NO:15-16所示,所述P3和P4的序列分别如SEQ ID NO:17-18所示。
本发明筛选获得实现高效率基因编辑的gRNA靶位点,进一步将包含重组左臂、核糖体接入位点(IRES)、白喉毒素受体(DTR)和绿色荧光蛋白(EGFP)融合序列、重组右臂的同源重组载体同源重组到小鼠Ncr1基因,构建了一个Ncr1-IRES-DTR-EGFP(简称:Ncr1-DTR)基因修饰小鼠模型,该模型可以实现小鼠内源Ncr1基因驱动白喉毒素受体和绿色荧光蛋白在NK细胞的表达。利用该模型,发明人可以通过EGFP荧光表达标记小鼠NK细胞;可以通过在特定时间点或时间段内给予小鼠白喉毒素将NK细胞杀死,造成NK细胞缺失,结果表明,给予Ncr1-DTR基因敲入小鼠白喉毒素可以诱导性的杀死NK细胞,造成NK细胞缺陷;在当前剂量下,3次连续给药可以维持1周时间内的NK细胞含量处于正常含量的19.2%-24%的低水平,NK细胞的缺失可以在给药结束16天后恢复正常水平。
本发明公开的构建可诱导自然杀伤细胞缺陷小鼠模型的方法以及采用所述方法建立的模型对于NK细胞功能研究等领域具有重要的意义。
附图说明
图1 为小鼠Ncr1基因结构和Ncr1-DTR基因修饰后的Ncr1基因结构。
图2为gRNA活性T7核酸内切酶I酶切检测电泳图。数字为对应的gRNA,Con为对照,M为DNA marker。
图3 为Ncr1-DTR小鼠模型构建策略示意图及鉴定结果。其中,A为Ncr1-DTR小鼠构建策略示意图;P1,P2,P3,P4为重组PCR鉴定引物位置示意;B为F0代小鼠左臂同源重组鉴定电泳图;C为F0代左臂重组阳性F0代小鼠的右臂同源重组鉴定电泳图。数字为小鼠编号;M,为1kb DNA marker。
图4为Ncr1-DTR小鼠NK细胞可诱导剔除效果检测。其中,A为实验时间轴;B为流式细胞术检测小鼠外周血NK细胞含量结果。
具体实施方式
以下实施例进一步说明本发明的内容,但不应理解为对本发明的限制。在不背离本发明精神和实质的情况下,对本发明方法、步骤或条件所作的修改或替换,均属于本发明的范围。
若未特别指明,实施例中所用的技术手段为本领域技术人员所熟知的常规手段。
实施例1
Ncr1-DTR小鼠模型构建
为了避免破坏小鼠内源Ncr1的表达进而造成正常情况下的NK细胞功能缺陷,发明人Ncr1-DTR小鼠模型的构建设计策略为将IRES-DTRGFP元件插入到小鼠Ncr1基因终止密码子后。野生型Ncr1基因结构和Ncr1-DTR基因修饰后基因结构如图1所示。
1)gRNA靶位点筛选
gRNA靶序列决定了其靶向特异性和诱导Cas9切割目的基因的效率。Cas9切割目的基因的效率越高,发生同源重组的效率就越高。因此,高效特异的靶序列选择和设计是成功构建Ncr1-DTR小鼠模型的前提。
根据重组方案,在插入位点附近设计并合成识别靶位点1-10(SEQ ID NO:1-10)的gRNA(gRNA1-gRNA10)。使用T7核酸内切酶 I检测试剂盒检测,从酶切后凝胶电泳结果可见不同的gRNA活性不同,检测结果参见图2所示。从中优先选择靶向位点5的gRNA5为后续小鼠受精卵注射实验的gRNA。
2)同源重组载体构建
根据获得的gRNA位点及插入位点信息,构建同源重组载体,载体结构如策略图3所示,从左到右结构依次为:重组左臂、核糖体接入位点(IRES)、白喉毒素受体(DTR)和绿色荧光蛋白(EGFP)融合序列、重组右臂,序列依次为SEQ ID NO:11-14。重组左臂和重组右臂,通过以小鼠基因组为模板,PCR扩增获得,其余片段通过全基因合成获得。构建过程中,先通过PCR获得重组左臂、基因敲入片段(IRES-DTRGFP)和重组右臂,利用In-Fusion的方法将3个DNA片段连入PBR322载体,获得最终的PBR322-Ncr1-DTR重组载体。载体经酶切及测序验证正确后,进行后续的小鼠制备。
3)Ncr1-DTR基因敲入小鼠构建:取C57BL/6小鼠的受精卵,按照《小鼠胚胎操作实验手册(第三版)》中的方法将PBR322-Ncr1-DTR重组载体、Cas9 mRNA和gRNA混合后,进行受精卵显微注射,注射后的受精卵经培养箱短暂培养后,移植至受体母鼠的输卵管,获得基因修饰小鼠F0代小鼠。F0代小鼠出生后,剪尾抽提基因组,分别利用针对同源重组左臂的鉴定引物对(引物P1和P2)和右臂的鉴定引物对(引物P3和P4),对是否发生正确同源重组进行鉴定,引物位置示意如图3中的A中的P1,P2,P3,P4所示。
左臂同源重组鉴定条件如下:
引物序列信息如下:
引物名称 | 序列信息(5’→3’) |
P1 | AGCCTTGCACCTACCGACCCTACT (SEQ ID NO: 15) |
P2 | TGTGGCCATATTATCATCGTGTTT (SEQ ID NO: 16) |
PCR反应体系相同,具体如下:
PCR反应组成 | 体积 (µl) |
ddH2O | 13.2 |
PCR Buffer | 2 |
2.5 mM dNTP | 2 |
引物1(20pmol/µl) | 0.5 |
引物2(20pmol/µl) | 0.5 |
DNA Polymerase | 0.8 |
genomic DNA | 1 |
总计 | 20 |
PCR反应程序相同,具体如下:
步骤 | 温度(℃) | 时间 | 备注 |
1 | 94 | 3 min | |
2 | 98 | 15 sec | |
3 | 60 | 15 sec | |
4 | 68 | 3 min | 重复步骤2-4共34个循环 |
5 | 68 | 5 min | |
6 | 12 | 10 min |
右臂同源重组鉴定条件如下:
引物序列信息如下:
引物名称 | 序列信息(5’→3’) |
P3 | CAGCCGCTACCCCGACCACA (SEQ ID NO: 17) |
P4 | CCCCCTCTTGCCTTCTTACTCC (SEQ ID NO: 18) |
PCR反应体系相同,具体如下:
PCR反应组成 | 体积 (µl) |
ddH2O | 13.2 |
PCR Buffer | 2 |
2.5 mM dNTP | 2 |
引物1(20pmol/µl) | 0.5 |
引物2(20pmol/µl) | 0.5 |
DNA Polymerase | 0.8 |
genomic DNA | 1 |
总计 | 20 |
PCR反应程序相同,具体如下:
步骤 | 温度(℃) | 时间 | 备注 |
1 | 94 | 3 min | |
2 | 98 | 15 sec | |
3 | 60 | 15 sec | |
4 | 68 | 3 min | 重复步骤2-4共34个循环 |
5 | 68 | 5 min | |
6 | 12 | 10 min |
共计出生23只F0代小鼠,对小鼠左臂和右臂同源重组鉴定结果如图3中的B和图3中的C所示,左臂和右臂正确同源重组的F0代小鼠,左臂和右臂鉴定均可以扩增出4.0 kb大小的条带,阴性小鼠均无PCR产物可以扩增出,结果显示左臂正确同源重组的小鼠为:1、2、4、5、7、10、17、18、19、20、21号(图3中的B);对左臂正确重组小鼠的右臂PCR鉴定结果显示:1、2、5、7、10、17、18、19、20、21号(图3中的C)为阳性,4号小鼠右臂PCR鉴定为重组阴性。对获得的PCR产物通过测序进一步确认,1、2、5、7、10、17、18、19、20、21号小鼠为双臂正确同源重组的阳性小鼠,阳性小鼠比例43.5%,说明当前的方法是一个同源重组效率很高的方法。
F0代小鼠成年后与野生型C57BL/6小鼠交配,获得F1代小鼠。F1代小鼠出生后,剪尾抽提基因组,用和鉴定F0代小鼠同源重组相同的条件,分别针对同源重组左臂(引物P1和P2)和右臂(引物P3和P4)进行PCR鉴定,确认获得的 F1代小鼠是否是正确同源重组的Ncr1-DTR基因敲入小鼠的阳性后代,该阳性小鼠为F1代杂合子小鼠。F1代杂合子小鼠可以通过自交获得Ncr1-DTR基因敲入纯合子小鼠。
实施例2
为了验证NK细胞表达DTR后是否可以为白喉毒素所剔除,发明人分别在实验第1,3,5天通过腹腔注射给予野生型小鼠和Ncr1-DTR小鼠白喉毒素,剂量为0.015mg/kg;给予白喉毒素同时,分别在实验第1,3,7,14天对外周血中NK细胞含量进行检测(第1,3天采血时间点为给药前),相关时间点如图4中的A所示。给药不同时间点,流式细胞检测代表结果如图4中的B所示:在Ncr1-DTR小鼠组,第1天Ncr1-DTR小鼠腹腔给予1次白喉毒素,到第3天(第2次给药前)NK细胞的含量即从给药前的2.35%下降到0.39%;3次给药结束后,到实验第7天NK细胞比例为0.44%,到实验第14天(最后一次给药9天后)NK细胞比例逐步恢复到1.27%,到实验第21天(最后一次给药16天后),NK比例恢复到2.24%的正常水平;而野生型C57BL/6小鼠在给药期间及给药后,NK细胞水平维持在正常状态。该结果表明,给予Ncr1-DTR基因敲入小鼠白喉毒素可以诱导性的杀死NK细胞,造成NK细胞缺陷;在当前剂量下,3次连续给药可以维持1周时间内的NK细胞含量处于正常含量的19.2%-24%的低水平,NK细胞的缺失可以在给药结束16天后恢复正常水平。
综上所述,发明人提供了一种可诱导NK细胞缺陷小鼠模型的构建方法,利用筛选到的高活性gRNA靶位点,基于CRISPR/Cas9系统介导的同源重组构建Ncr1-DTR小鼠模型;发明人对该模型白喉毒素对NK细胞的可诱导性杀伤造成的NK细胞缺陷进行了验证,该模型可以应用于特定生理、病理条件下的NK细胞功能研究等领域。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明技术原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
序列表
<110> 广东南模生物科技有限公司
上海南方模式生物科技股份有限公司
上海砥石生物科技有限公司
<120> 一种可诱导自然杀伤细胞缺陷小鼠模型的构建方法及应用
<130> 2021
<160> 18
<170> SIPOSequenceListing 1.0
<210> 1
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
aatgcagcat tactttgaag agg 23
<210> 2
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
aagaaatgca atatctatga tgg 23
<210> 3
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
aatatctatg atggaactga agg 23
<210> 4
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
catagagctc acaaggcccc agg 23
<210> 5
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
atggaactga aggcaactcc tgg 23
<210> 6
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
tggaactgaa ggcaactcct ggg 23
<210> 7
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
ggaactgaag gcaactcctg ggg 23
<210> 8
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
ggtacagcat agagctcaca agg 23
<210> 9
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
aagatctctt tctttatcca ggg 23
<210> 10
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
gtgagctcta tgctgtaccc tgg 23
<210> 11
<211> 3003
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
atttactcag accatccatt tagtgcacag caccttctgc agtcattcag caaaacaccc 60
tggcctttgt gaaacttaga ccccaagaaa caaaccaaga gtcaacaaac caaggaccaa 120
taagtaggtt ctctagcgca tacagaggaa gtgatgatgt aggacaagaa aatgtcttaa 180
atcaatcagg agtggagata gcaatatgaa agagggtctc ccagagtggg acgtctttaa 240
gagaagggca actggaaaaa acgcatggaa gtacagaggg agagagattt atagccatgt 300
ggagacagcc taaggcagga agatgattta gattcttagc catgtggaga cagcctaagg 360
caggaaggtg atttagattc ttagccttgt ggagacagcc taaggcagga aggtgattta 420
ggttcttagc catgtgggga cagcctaagg caggaaggtg acttaggtga tagagaacag 480
ttgcgaggcc aatgcaattg aaaacacttg tgtggactag cttcattttt ttaaattttt 540
attaattgag aattgcatac aatattctaa tcatattcaa ctcaccataa gttcatcccc 600
ctctttaccc aacttttgtg tcttccttct tttttaattc atcaaataca atttttgctc 660
tgtatatctc ttgggtatgt agcctgccac cagagcatgg ccaacctacc aagggacaat 720
gaccccatca gctaaaaaaa aagaaaaaaa aagaaaaatg ccaagagcta ctcatactgt 780
gctggaactt tgagcctata ttctctttcc atgttgggag tgagtctagc ttgcacaggt 840
cttatgaatg ctaacataac agctgtgagt atatatgtgc aactgctctg ttgtagtatc 900
aagacactag gtccttgtgt catctaccac ctctggctct tacgaattta ccaatccttc 960
cctgaattta caatattccc tgatccttag gaggaaggat gtgctaaagc ccatttaggg 1020
caaggacttc aacagagtct tattctcggg tttctgtctt agttaccttc ttctctggtg 1080
ataatttctg ttctctgata aaacattctg gctaaaagca actctgaaaa aggaaaggac 1140
ttattcagct tctacttcta ggtctcatgc catcacaggg aggaagtcaa gataagaagt 1200
catgcagcta gttacaccac atccacagtc aaatacattt gtgctacctg ttttagctct 1260
aatcaggtgt tcggtcctgt atcattcaca cactcctgcc caggaagtgg tgtcatacac 1320
aaaaggctgg gtcttcacac atcaattaat tatcaataca atcctgcata tactcatagg 1380
ccaacgtgat ctaaataatt cctcacttga cctctctttt caggcaattc tagattgtgg 1440
caaactgaca tttaaaacta ctacccatct tataattggg ttgtttggtg attagctgct 1500
tgcagccggg tgtggtggcg cacgccttta atcccagcac ttgggaggca gaggcaggcg 1560
gatttctgag ttcgaggcca gcctggtcta caaagtgagt tccaggacag ccagagctac 1620
acagagaaac cctgtctcaa aaaaaccaaa aaaaaaaaaa aaaaaaaatg attagctgct 1680
tgcattctct atatatttta gatattagac ctctaacaga tatgggatta ctgaagattg 1740
ttttccaaat ctgcaggtta ctgatttgtc ttattatcga tgtcctttgc cttacagaag 1800
ctttccagtt tcatgaggtc ccatttatca attcttgatc ttaaagcatg agccactggt 1860
gttctgttta tgaaatttcc cccatgccaa tgaattcaaa gctctttccc actttctctt 1920
ctattagatt cggtgtatct gattttatat tgaggtcctt ggtccatatg gacttgagct 1980
ttgtgacaaa tatgggtcta ttttcatttt tctacatacg gacagctagt tagaccagca 2040
ccatttattg aagatgcttt atcttttcca ttgtatattt ttggcttctt tttttcaaag 2100
atcaagtgtc cataagtgtg tagttttatt tctgggtctt caattctatt ccattgatca 2160
acatgtctgt ctctgtacca ataccatgca gtttttatca ctattgctct gtagtacagc 2220
ttgaggtcag ggatggtgat ttccccaagc tgttcttttc taattaagaa ttgttttcgc 2280
tattctgggt tttttgcctt tccagatgaa tttgagaatt gctctttcca tgtctttgaa 2340
taattgtgtt gggattttga ttaggattgc attgaatcta tgaattacct ttagtaagat 2400
ggccattttt actatgttaa ttctgccaat cctcagatta ttgggagttt gacctttcaa 2460
ccaatgaatc aggattacaa aagggtaagt gaaagtctaa aaccatagtc tctggggaag 2520
gtacagggaa aagcaggaag gtcataaaag aaggagggag gccccaggga gtggggagtt 2580
ctggtggggt agggacatcc ttgtgacaag gggggatcga ggaggtatga gatgtggatg 2640
tgggaaggag acaaaatctg gagtataaaa aaaattaaat aaaaatttta aaatgaagaa 2700
gagggagaat ggggtgggtg tagcctctat cagcagtcac cctgtcttct gaatccagac 2760
tccgccttct gggatcacac aacccagaat ctcattcgaa ttggtctggc atgcataatc 2820
ctgataactc tagtatggct tttgactgaa gactggctca gcaagaggaa agatcatgaa 2880
gaggccaaca gattaacaaa ttgggaatgc aggagaagat ggagaatgca gcattacttt 2940
gaagaggaac aaagaaatgc aatatctatg atggaactga aggcaactcc tggggccttg 3000
tga 3003
<210> 12
<211> 588
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
gcccctctcc ctcccccccc cctaacgtta ctggccgaag ccgcttggaa taaggccggt 60
gtgcgtttgt ctatatgtta ttttccacca tattgccgtc ttttggcaat gtgagggccc 120
ggaaacctgg ccctgtcttc ttgacgagca ttcctagggg tctttcccct ctcgccaaag 180
gaatgcaagg tctgttgaat gtcgtgaagg aagcagttcc tctggaagct tcttgaagac 240
aaacaacgtc tgtagcgacc ctttgcaggc agcggaaccc cccacctggc gacaggtgcc 300
tctgcggcca aaagccacgt gtataagata cacctgcaaa ggcggcacaa ccccagtgcc 360
acgttgtgag ttggatagtt gtggaaagag tcaaatggct ctcctcaagc gtattcaaca 420
aggggctgaa ggatgcccag aaggtacccc attgtatggg atctgatctg gggcctcggt 480
gcacatgctt tacatgtgtt tagtcgaggt taaaaaaacg tctaggcccc ccgaaccacg 540
gggacgtggt tttcctttga aaaacacgat gataatatgg ccacaacc 588
<210> 13
<211> 1365
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
atgaagctgc tgccgtcggt ggtgctgaag ctccttctgg ctgcagttct ttcggcactg 60
gtgactggcg agagcctgga gcagcttcgg agagggctag ctgctggaac cagcaacccg 120
gacccttcca ctggatctac ggaccagctg ctacgcctag gaggcggccg ggaccggaaa 180
gtccgtgact tgcaagaggc agatctggac cttttgagag tcactttatc ctccaagcca 240
caagcactgg ccacaccaag caaggaggag cacgggaaaa gaaagaagaa aggcaaggga 300
ctagggaaga agagggaccc atgtcttcgg aaatacaagg acttctgcat ccacggagaa 360
tgcaaatatg tgaaggagct ccgggctccc tcctgcatct gccacccagg ttaccatgga 420
gagaggtgtc atgggctgag cctcccagtg gaaaatcgct tatataccta tgaccataca 480
actatcctgg ctgtggtggc cgtggtgctg tcctctgtct gtctgctggt catcgtgggg 540
cttctcatgt ttaggtacca taggagaggt ggttatgatg tggaaaacga agagaaagtg 600
aagttgggca tgactaattc ccacccggat ccaccggtcg ccaccatggt gagcaagggc 660
gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc 720
cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg 780
aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctg 840
acctacggcg tgcagtgctt cagccgctac cccgaccaca tgaagcagca cgacttcttc 900
aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc 960
aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag 1020
ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac 1080
tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat caaggtgaac 1140
ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca ctaccagcag 1200
aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagcacccag 1260
tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg 1320
accgccgccg ggatcactct cggcatggac gagctgtaca agtaa 1365
<210> 14
<211> 3000
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
gctctatgct gtaccctgga taaagaaaga gatcttttca gaaatgaagg ggtaggagtg 60
ttaccaagtt tggcaaagca ttctaaagga cactgtgaga ggaagcctct aacagacctc 120
tctgtctctg ctaagcttta agtactttaa cgcttattta ctgcaagtta tacttttgat 180
cactctggtc tttcctagaa ataacttcgt tccaatgtga tggccatggg aatatttcct 240
tcctacttta ggtcttatcg tcatttcttt ttctaatttt atttatttta tgttgccggg 300
aattgaactc aagttgtctg gaggagcaac aagtgctctt aactacaaac catatctcca 360
gctccatcct caatatttta tattgtgtgt gcatgtctgt aggatgcatg tgtgtgcaca 420
catatgtaca tatattctat gccattaaaa tgaaggtcag agaacacctc tgtgaagtca 480
gttttctctt tctttttttc ttttcttttt tttttttttg ttgttttttt ttttgttttt 540
ttttttgttt ttttgaggca gggtttctct gtgtagccct ggctgtcctg gaactcactc 600
tgtagaccag gctggcctcg aactcagaaa tccgactgcc tctgcctccc gagtgctggg 660
attaaaggcg tgcaccacca cgcccggctg tgaagtcagt tttctatttc cactttctaa 720
caattctttt atttttgaga atataattta aacatctgtg tttttttttt aaagttatat 780
ctcctccacc cttcactttc ttctcttcaa acactcccat atacccctct tactctcttt 840
caaattcatg gcctctagtt tgtgacaaga aaaatgtcat cttaagcaaa aacactctct 900
ctttctctct ctctctcttg ctcttgctct ctctggtcta tggatctaga tataaagtct 960
gtcttgtttc tgccatgatg ctcatagggt agttctctaa agctgtgaac aagcccccaa 1020
ttaaatactt tcttttataa gagttacttt ggtcatagac tttcgtctcg tcactagaac 1080
agtaactaag gcagactgtc ttagttaggg ttttattgtt atgaaaagac ataatgacca 1140
atgaaactct tataaaggaa aacatttaat tggggctgac ttacagttca ggggtttagt 1200
cctttatcat cacggcagga agcatggtgg catgctagaa gacaaggtat tagagaagaa 1260
gctaagaatc ctataccttg atccacaagc aacaggagac tgtttgccac aatgggcata 1320
ggtggagcat aggagacctc aaagcctgcc cccacagtga cacacttcct ccaacaaggc 1380
cacacctact ccaacaaggt cacacttcag aatagtgcca ctccctatgg gccaagcatt 1440
caaatacatt agtctatggg ggctattaat attcaaacca ccactcagaa tacttctcgc 1500
cctctttcat tcatggatct ggacctcagt cctccatgat attgcccaac acaactatgt 1560
tatgtgctga ctccccaaag ccctgcagat tgtcttcttt ccaccattgc ttcttcttta 1620
actgctattg taataatcac taataagctc ccaataacac acaatgagat ctgcacctct 1680
tgttggttct agctaacact ttgcccactt cttgaatcac ttctctagtt tctcgctgct 1740
tatctccaaa tggttatgat actgttatgt tttctgtcag gaaccatcta ggggcagcct 1800
accttttttg catggcctca gatgagaggc tctgagaggc aaagtcccaa atacatttca 1860
cctcaggatt tcttcttgca gctgatatgc ttttttccta tcacgatcac atagcctgac 1920
acttcctggg catcagcaca ccctattggg cagatttcct gaagatcaac atgaagatga 1980
actttcatga attaatgcta tttagaagaa ttgattattt tatagagttc cagatttgat 2040
tcaaataatc tcagaaagaa catttcaaga gctgtttttt gttgtttgct agaaagatat 2100
aataaagtta ctatcaagga tagaatgcac ccactcccca taataataag taaggctgca 2160
taataagaaa tacaatgagc tttcatattg cactaaaagg agatataggc ttgggaaaaa 2220
tttgtgttca aggaaaagca tccaggtaca aggatgaagc cccaagtgtg cactctgaga 2280
aaacaaggcc atgtaaaact gttgctttga acttagaatg agcatataga atgaatcact 2340
gctttgaact tagcatcaac atctttttgt aactcccatt ttgtctaaca ttttatctct 2400
gcggtaactt tctaaaatcc tttctctata agaaaataaa taaaaatgat ggaatggctt 2460
ggctctatgc cagatacatc tgtccatcca tctgtgcatc tgtctgtcca tccatccatc 2520
cattcacctg tctgtccatc catccatatg tagtgatgta gccattttct gcttcttctg 2580
gtttgggtgt gtgttctttc tgtgtgaatg actctttctt tcctttctgg ttcacaaaga 2640
gttaaccagc ctgacaagtg aggggctctt gggctactag aaagcatgct agcaataaat 2700
cctttactta atcccttgct gttaagcaac aggtgttaat tcttcagaag ccattggctt 2760
ctggccccag aataagaaag aaccaaagag aatagatatc aatactaagt aatctttata 2820
tttgcaaaac cactctccac cagccatcat ctgccttccg gagaggactc agccagggta 2880
agctccagcc agttttcaag cttcttcaac tccacttcta tttaatatac ttttattagt 2940
tttatttttt atgtgaagag tgtgtatgtg tgcgcacaca tgctcacatg cctgtgaagg 3000
<210> 15
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
agccttgcac ctaccgaccc tact 24
<210> 16
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
tgtggccata ttatcatcgt gttt 24
<210> 17
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
cagccgctac cccgaccaca 20
<210> 18
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
ccccctcttg ccttcttact cc 22
Claims (10)
1.一种针对小鼠Ncr1基因的gRNA,其特征在于,所述gRNA的靶位点序列如SEQ ID NO:5所示。
2.一种用于构建可诱导自然杀伤细胞缺陷小鼠模型的试剂盒,其特征在于,所述试剂盒包括:
(1)针对小鼠Ncr1基因的gRNA,所述gRNA的靶位点序列如SEQ ID NO:5所示;
(2)包含白喉毒素受体编码序列的同源重组载体;
(3)Cas9 mRNA。
3.根据权利要求2所述的试剂盒,其特征在于,所述试剂盒包括的同源重组载体为针对小鼠Ncr1基因的同源重组载体,所述载体从左到右顺序包括:重组左臂、核糖体接入位点序列、白喉毒素受体编码序列与绿色荧光蛋白编码序列融合序列元件、重组右臂。
4.根据权利要求3所述的试剂盒,其特征在于,所述重组左臂、核糖体接入位点序列、白喉毒素受体编码序列与绿色荧光蛋白编码序列融合序列元件、重组右臂的序列分别如SEQID NO:11-14所示。
5.根据权利要求2-4任一权利要求所述的试剂盒,其特征在于,所述同源重组载体为PBR322。
6.根据权利要求2-4任一权利要求所述的试剂盒,其特征在于,所述试剂盒还包括针对重组左臂的鉴定引物对P1和P2和针对重组右臂的鉴定引物对P3和P4。
7.根据权利要求6所述的试剂盒,其特征在于,所述P1和P2的序列分别如SEQ ID NO:15-16所示,所述P3和P4的序列分别如SEQ ID NO:17-18所示。
8.权利要求1所述的gRNA在制备可诱导自然杀伤细胞缺陷小鼠模型的试剂盒中的应用。
9.一种构建可诱导自然杀伤细胞缺陷小鼠模型的方法,其特征在于,所述方法包括:
(1)取小鼠的受精卵;
(2)将包含白喉毒素受体编码序列的同源重组载体、Cas9 mRNA以及权利要求1所述的gRNA混合后,进行受精卵显微注射;
(3)将显微注射后的受精卵经培养箱短暂培养后,移植至受体母鼠的输卵管,获得基因修饰小鼠F0代小鼠;
(4)对F0代小鼠抽提基因组,分别利用针对同源左臂的鉴定引物对和同源右臂的鉴定引物对进行同源重组鉴定。
10.根据权利要求9所述的方法,其特征在于,所述同源重组载体为针对小鼠Ncr1基因的同源重组载体,所述载体从左到右顺序包括:重组左臂、核糖体接入位点序列、白喉毒素受体编码序列与绿色荧光蛋白编码序列融合序列元件、重组右臂。
Priority Applications (1)
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