CN115058456B - Hprt基因敲除的动物模型的构建方法和应用 - Google Patents
Hprt基因敲除的动物模型的构建方法和应用 Download PDFInfo
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Abstract
本发明属于动物模型构建领域,具体涉及HPRT基因敲除的动物模型的构建方法和应用,本发明针对HPRT基因第3外显子设计一条sgRNA,将sgRNA序列与T7启动子相连,获得体外转录载体,利用体外转录试剂盒,以构建的体外转录载体为模板,制备显微注射使用的Cas9mRNA和gRNA,随后将Cas9mRNA和gRNA混和后,注射到单细胞期兔受精卵中,通过PCR扩增及Sanger测序获得HPRT基因敲除的家兔模型,本发明构建HPRT基因敲除的动物模型的方法简单、高效,相对于现有的小鼠模型,本发明模型动物为家兔,家兔较小鼠具有更丰富的神经系统,在模拟人类神经系统疾病上相对更有优势。
Description
技术领域
本发明属于生物医药领域,涉及动物模型构建领域,具体涉及HPRT基因敲除的动物模型的构建方法和应用。
背景技术
Lesch-Nyhan综合症(Lesch-Nyhan syndrome,LNS),也称为自毁容貌症,是由编码次黄嘌呤磷酸核糖转移酶(hypoxanthine phosphoribosyltransferase,HPRT)缺失引起的X-连锁隐性遗传的先天性嘌呤代谢缺陷病。缺乏该酶使得次黄嘌呤和鸟嘌呤不能转换为IMP和GMP,而是降解为尿酸,高尿酸盐血症引起早期肾脏结石,逐渐出现痛风症状。该病症是一种主要影响男性的人类神经系统疾病,患者出现智力低下,有特征性的强迫性自身毁伤行为。
尽管已有大量关于HPRT缺陷病LNS的研究,对该病的治疗仍未取得理想效果。为了推动HPRT缺陷的治疗,科研工作者创建了HPRT敲除的大小鼠模型。HPRT敲除的大小鼠模型的构建是通过培养胚胎干细胞,获得HPRT活性丧失的细胞系,进而构建出嵌合鼠。其研究结果显示神经症状的产生与多巴胺缺乏具有相关性,并提出许多基因的异常转录可能影响LNS表型,而且HPRT缺乏细胞模型的建立和对HPRT缺乏人类神经干细胞的研究也证实了这一设想。这些大小鼠模型均表现出代谢缺陷表型(例如,通过从头合成途径增加嘌呤合成),但缺乏任何神经行为表型。因此,这些模型通常用于研究体内LNS的神经化学和代谢方面,而不是疾病的行为方面。另外,由于大小鼠的体型及神经系统与人类相差较大,并不能很好的模拟人类的相关疾病特征。
发明内容
针对现有技术存在的问题,本发明旨在提供一种高效且简单的方法获得HPRT基因敲除的家兔模型,以期能更好的模拟LNS神经方面的表型,为LNS疾病发病机制及治疗方式探索提供理想的动物模型。
基于上述目的,本发明采用的技术方案如下:
第一方面,本发明提供一种HPRT基因敲除的动物模型的构建方法,包括如下步骤:
(1)靶向家兔HPRT基因的Cas9靶位点的选择;
(2)构建HPRT-gRNA体外转录载体;
(3)制备gRNA和cas9mRNA;
(4)将gRNA和cas9mRNA混合注射到单细胞期兔受精卵胞质中,体外培养后植入母兔子宫;
(5)F0代家兔出生与鉴定,所得纯合子即为HPRT基因敲除的动物模型。
优选地,步骤(1)中所述Cas9靶位点为针对兔HPRT基因第3外显子的sgRNA,所述sgRNA的靶向识别序列如SEQ ID NO.1所示。
优选地,步骤(2)构建HPRT-gRNA体外转录载体的方法如下:
依据所述Cas9靶位点合成相应引物,其序列分别如SEQ ID NO.4和SEQ ID NO.5所示,将引物退火后连入至BbsI酶切后的gRNA克隆骨架载体pT7-gRNA中,连接、转化、挑取克隆并测序鉴定得到正确的HPRT-gRNA体外转录载体。
优选地,在步骤(3)制备cas9mRNA的体系中添加有polyA聚合酶。
优选地,步骤(3)制备sgRNA包括如下步骤:
利用如SEQ ID NO.6和SEQ ID NO.7所示引物对,以步骤(2)构建好的HPRT-gRNA体外转录载体为模板,使用高保真酶PCR,得到gRNA体外转录模板;经PCR电泳确认为单一条带后回收PCR产物,收集gRNA。
本发明针对HPRT基因第3外显子设计一条sgRNA,sgRNA的靶位点碱基长度为18-20bp左右,将sgRNA序列与T7启动子相连,获得体外转录载体,利用体外转录试剂盒,以构建的体外转录载体为模板,制备显微注射使用的Cas9mRNA和gRNA,接着将Cas9 mRNA和gRNA混和后,注射到单细胞期兔受精卵中,通过PCR扩增及Sanger测序获得HPRT基因敲除的家兔模型,本发明构建HPRT基因敲除的动物模型的方法简单、高效,相对于现有的小鼠模型,本发明模型动物为家兔,家兔较小鼠具有更丰富的神经系统,在模拟人类神经系统疾病上相对更有优势。
第二方面,本发明提供一种HPRT基因敲除的动物模型,所述动物模型由上述方法构建得到。
第三方面,本发明提供上述动物模型在Lesch-Nyhan综合症研究中的应用。
第四方面,本发明提供一种基于CRISPR-Cas9基因敲除技术的HPRT基因敲除试剂盒,所述HPRT基因敲除试剂盒包括HPRT-gRNA体外转录载体,所述HPRT-gRNA体外转录载体以pT7-gRNA载体为出发载体,含针对HPRT基因的gRNA;所述gRNA由SEQ ID NO.6和SEQ IDNO.7所示的引物退火得到。
与现有技术相比,本发明的有益效果如下:
(1)本发明以家兔作为构建HPRT基因敲除的动物模型,由于家兔相较于比大鼠、小鼠,其神经系统更为丰富,在模拟人类神经系统疾病上更有优势。基于此,本发明利用高效的CRISPR/Cas9基因编辑技术和显微注射技术构建了HPRT敲除的家兔模型,以期模拟LNS神经方面的表型,为LNS疾病发病机制及治疗方式探索提供理想的动物模型。
(2)本发明针对HPRT基因第3外显子设计一条sgRNA,sgRNA的靶位点碱基长度为18-20bp左右,将sgRNA序列与T7启动子相连,获得体外转录载体,利用体外转录试剂盒,以构建的体外转录载体为模板,制备显微注射使用的Cas9 mRNA和gRNA,接着将Cas9 mRNA和gRNA混和后,注射到单细胞期兔受精卵中,通过PCR扩增及Sanger测序获得HPRT基因敲除的家兔模型,本发明构建HPRT基因敲除的动物模型的方法简单、高效。
附图说明
图1为家兔HPRT位点体外sanger测序;
图2为四只新生兔照片;
图3为新生兔HPRT位点基因型鉴定;
图4为2#、3#、4#小兔耳组织成纤维细胞的细胞增殖率;
图5为2#、3#、4#小兔耳组织成纤维细胞的CCK-8细胞增长率。
具体实施方式
为更好地说明本发明的目的、技术方案和优点,下面将结合具体实施例对本发明作进一步说明。本领域技术人员应当理解,此处所描述的具体实施例仅用以解释本发明,并不用于限定本发明。
实施例中所用的试验方法如无特殊说明,均为常规方法;所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
实施例1
本实施例构建HPRT基因敲除的动物模型流程为:针对HPRT基因第3外显子设计一条sgRNA,sgRNA的靶位点碱基长度为18-20bp左右,将sgRNA序列与T7启动子相连,获得体外转录载体,利用体外转录试剂盒,以构建的体外转录载体为模板,制备显微注射使用的Cas9mRNA和gRNA,接着将将Cas9 mRNA和gRNA混和后,注射到单细胞期兔受精卵中,通过PCR扩增及Sanger测序获得HPRT基因敲除的家兔模型。具体方法如下:
1.1 Cas9靶位点的选择与确认
Cas9靶位点包含20个碱基,其中5’端应为GG;紧邻靶点3’端的3个碱基构成PAM区,PAM区要求序列为NGG(N为任意碱基),其余序列为任意碱基。5’端选择GG并非Cas9靶点本身的要求,而是由于本实验所用的gRNA体外转录载体采用了T7启动子。T7启动子要求转录起始位点的前两位为GG,并且第三位最好为G或A;如果采用其他的启动子,可以随之更改。可参考如下的Cas9靶位点预测网站:(http://crispr.mit.edu),根据该原则设计的针对兔HPRT基因第3外显子的sgRNA的靶向识别序列为GGGCTTTAATATAATCCAGCAGG(SEQ ID NO.1)。靶位点选择完毕后,需在NCBI上与家兔全基因组序列进行比对,确认靶序列的唯一性,避免脱靶。
1.2根据确定好的靶点合成相应的引物
针对HPRT基因第3外显子的sgRNA的靶向识别序列的两条引物分别为:
HPRT-F1:CACACTTCATGGGCACAAAGGA(SEQ ID NO.2);
HPRT-R1:CCGCCTCTCTCTCTGTGAAC(SEQ ID NO.3)。
1.3构建HPRT-gRNA体外转录载体
根据设计好的靶位点合成相应引物分别为:
1F:5’-ataGGGCTTTAATATAATCCAGCgt-3’(SEQ ID NO.4);
1R:5’-taaaacGCTGGATTATATTAAAGCC-3’(SEQ ID NO.5)。
用ddH2O分别将引物溶解为10μM的溶液,退火,98℃加热10min,自然冷却至室温。退火程序如表1所示。
表1
1F | 10μL(10μM) |
1R | 10μL(10μM) |
buffer | 5μL |
H2O | 25μL |
Total | 50μL |
然后将引物退火后连入至BbsI酶切后的gRNA克隆骨架载体pT7-gRNA中,连接、转化、挑取克隆并测序鉴定得到正确的HPRT-gRNA体外转录载体。
1.4制备Cas9 mRNA和gRNA
(1)制备Cas9 mRNA
1)制备Cas9 mRNA的体外转录模板
通过pmeI单酶切线性化MLM3613载体(Amp抗性),线性化条件为:37℃,4h以上;取少量电泳确认线性化完全后,直接回收线性化产物。
2)体外转录Cas9 mRNA
Cas9 mRNA 体外转录体系如表2所示,其中,T7 Enzyme Mix购自T7 mMESSAGEKit,Life:
表2
2×NTP/CAP | 10μL |
10×Reaction Buffer | 2μL |
Linearized template DNA | 1μg(<6μL) |
T7 Enzyme Mix | 2μL |
DEPC H2O | up to 20μL |
37℃,2h;加入1μL TURBO DNase,37℃、15min以去除DNA模板。
3)添加polyA聚合酶、回收得到可用于显微注射的Cas9 mRNA。mRNA加polyA的反应体系如表3所示。
表3
ATP(10mM) | 5μL |
10×Reaction Buffer | 5μL |
E.coli Poly(A)聚合酶 | 1μL |
DEPC H2O | up to 50μL |
向体系中添加polyA聚合酶可增强mRNA的稳定性和翻译效率,于37℃、孵育1h;取0.3μL跑电泳查看转录效果。
(2)制备gRNA
1)制备gRNA的PCR体外转录模板
用T7-cr fwd(SEQ ID NO.6)和tracr rev(SEQ ID NO.7)引物对,以构建好的gRNA体外转录载体HPRT-gRNA质粒为模板,使用高保真酶PCR,得到gRNA体外转录模板(58℃退火,延伸30sec,40cycle,40μL体系)。取1μL PCR产物电泳,确认为单一条带(125bp)后直接回收PCR产物,用于后续的步骤。
T7-cr fwd序列:5’-GAAATTAATACGACTCACTATA-3’(SEQ ID NO.6);
tracr rev序列:5’-AAAAAAAGCACCGACTCGGTGCCAC-3’(SEQ ID NO.7)。
2)体外转录gRNA
gRNA体外转录体系(AmbionT7 Kit)如表4所示。
表4
2.5mmol/L NTP | 4μL |
10×Reaction Buffer | 2μL |
Template DNA | 1μg(<6μL) |
T7 Enzyme Mix | 2μL |
DEPC H2O | up to 20μL |
37℃,孵育1h;加入1μL TURBO DNase,37℃、15min以去除DNA模板;然后取0.3μL,跑电泳查看转录效果,产物100bp左右。
3)回收gRNA
建议使用Ambion的mirVanaTM miRNA Isolation Kit进行回收。也可采用LiCl沉淀法,但效率较低。
用mirVanaTM miRNA Isolation Kit回收小片段RNA,步骤如下:
用RNase-free water将gRNA转录体系稀释到300μL,加入330μL无水乙醇;
将溶液加到回收柱中,10000g离心15s(转速太高会损伤滤膜);
加入700μL的miRNA Wash Solution I,离心5~10s;
加入500μL的Wash Solution II,离心5~10s;重复一次;
弃去收集管中的液体,离心1min,去除残余的液体;
加入适量95℃预热的RNase-free water或Elution Solution,最大转速离心20~30s,收集得到gRNA溶液。
1.5靶点突变效率检测
根据实验室已有的经验,将Cas9 mRNA和gRNA以最终浓度分别为200ng/μL和20ng/μL混和后,注射到单细胞期兔受精卵胞核中。取3-5天囊胚或桑葚胚期的胚胎(1枚/管),裂解获取基因组DNA,PCR并测序检测靶位点突变效率。加5μl裂解液(含0.45%NP40和2mg/mlproteinase K)于56℃处理1h;95℃处理10min后取样进行PCR。
初步结果如表5和图1所示,共注射了10枚胚胎,有8枚发育至囊胚,PCR及Sanger测序结果显示有7枚发生了基因敲除,基因敲除效率高达87.5%。靶点突变效率检测结果显示:我们选用的靶点和构建的HPRT-gRNA可以高效的在靶点进行切割,因此可以进行下一步实验。
表5家兔HPRT位点体外突变效率检测
1.6制备基因打靶兔构建HPRT基因敲除的动物模型
将Cas9 mRNA和gRNA混和,注射到单细胞期兔受精卵胞质中。注射后的兔受精卵在培养箱静置2-3小时后,按每只受体移植6-14枚胚胎的数量手术植入发情母兔子宫。移植后15天可检查母兔是否受孕。生产后取小兔少量耳缘组织提取DNA,PCR,测序鉴定打靶情况。PCR须扩增打靶位点序列,由于模版较少,因此尽量选取较短片段扩增(小于500bp为好),并且20bp打靶序列居中。本次实验的鉴定引物为:鉴定-F1:CACACTTCATGGGCACAAAGGA(SEQ IDNO.8);鉴定-R1:CCGCCTCTCTCTCTGTGAAC(SEQ ID NO.9)。PCR扩增条件为95℃5min;(95℃30s,55℃30s,72℃15s)×38cycle;72℃5min。
本实验共获得四只新生兔如图2所示,取四只小兔耳组织测序鉴定后,Sanger测序结果如图3所示,其中三只小兔为杂合子(编号依次为1#、2#、3#),一只小兔为纯合子(编号为4#),所得纯合子小兔即为本发明HPRT基因敲除的动物模型(HPRT-/-兔模型)。
1.7HPRT-/-兔模型鉴定
取编号依次为2#、3#、4#小兔耳组织,得到兔耳成纤维细胞。利用6-硫鸟嘌呤(6-Thioguanine,6-TG),是一种基于鸟嘌呤结构的硫类似物。6-TG通过HPRT转化为6-硫鸟嘌呤单磷酸盐,抑制嘌呤的合成和利用,并且抑制细胞生长,而HPRT敲除后则不会出现该现象。
本实验通过对兔耳成纤维细胞的细胞增殖率和细胞增殖-毒性检测试剂盒(CellCounting Kit-8,CCK-8)两种方法进行检测,观察细胞生长情况。首先通过细胞计数的方法计算细胞增殖率,结果如图4所示,可以看出2#、3#小兔的耳组织成纤维细胞加入药物6-TG后,细胞增殖率降低;而4#HPRT-/-兔的耳组织成纤维细胞加入药物6-TG后,细胞增殖速率没有明显变化。接下来,我们又做了CCK-8实验,通过测量细胞的OD值计算细胞增长速率,结果如图5所示。CCK-8所得结果基本与细胞计数法所得结果相同,2#、3#兔耳组织成纤维细胞加入药物6-TG后,细胞增长率降低;而4#HPRT-/-兔耳组织成纤维细胞加入药物6-TG后,细胞增长速率没有明显变化,因此可以判断我们成功得到了HPRT-/-4#兔,即本发明通过上述方法成功构建出了HPRT基因敲除的模型兔。
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。
SEQUENCE LISTING
<110> 五邑大学
<120> HPRT基因敲除的动物模型的构建方法和应用
<130> H220806381
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<170> PatentIn version 3.5
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<213> sgRNA的靶向识别序列
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Claims (4)
1.一种HPRT基因敲除的动物模型的构建方法,其特征在于,包括如下步骤:
(1)靶向家兔HPRT基因的Cas9靶位点的选择;
(2)构建HPRT-gRNA体外转录载体;
(3)制备gRNA和cas9mRNA;
(4)将gRNA和cas9mRNA混合注射到单细胞期兔受精卵胞质中,体外培养后植入母兔子宫;
(5)F0代家兔出生与鉴定,所得纯合子即为HPRT基因敲除的动物模型;
步骤(1)中所述Cas9靶位点为针对兔HPRT基因第3外显子的sgRNA,sgRNA的靶向识别序列如SEQ ID NO.1所示;
步骤(2)构建HPRT-gRNA体外转录载体的方法如下:
依据所述Cas9靶位点合成相应引物,其序列分别如SEQ ID NO.4和SEQ ID NO.5所示,将引物退火后连入至BbsI酶切后的gRNA克隆骨架载体pT7-gRNA中,连接、转化、挑取克隆并测序鉴定得到正确的HPRT-gRNA 体外转录载体。
2.根据权利要求1所述构建方法,其特征在于,在步骤(3)制备cas9mRNA的体系中添加有polyA聚合酶。
3.根据权利要求1所述构建方法,其特征在于,步骤(3)制备sgRNA包括如下步骤:
用如SEQ ID NO.6和SEQ ID NO.7所示引物对,以步骤(2)构建好的HPRT-gRNA体外转录载体为模板,使用高保真酶PCR,得到gRNA体外转录模板;经PCR电泳确认为单一条带后回收PCR产物,收集gRNA。
4.一种基于CRISPR-Cas9基因敲除技术的HPRT基因敲除试剂盒,其特征在于,所述HPRT基因敲除试剂盒包括HPRT-gRNA体外转录载体,所述HPRT-gRNA体外转录载体以pT7-gRNA载体为出发载体,含针对HPRT基因的gRNA;所述gRNA由SEQ ID NO.6和SEQ ID NO.7所示的引物退火得到;所述gRNA的靶向识别序列如SEQ ID NO.1所示。
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