CN114480508A - Tecrl敲除小鼠模型的构建方法及其应用 - Google Patents
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Abstract
本发明提供一种Tecrl敲除小鼠模型的构建方法及其应用,所述Tecrl敲除小鼠模型的构建方法为:A、根据基因敲除的靶点,设计针对Tecrl基因的sgRNA;B、将Cas9mRNA及sgRNA同时注射入实验小鼠的受精卵中,得到Tecrl基因成功敲除的F0代小鼠。本发明首次构建了一种Tecrl敲除小鼠模型,并发现该先天的小鼠基因缺陷导致小鼠在早期的时候就会出现心脏收缩和舒张功能的受损,为研究左心功能提供了模型。且该Tecrl敲除小鼠在肾上腺素和咖啡因的诱导下出现了多形性及双向性室速,模拟了临床上CPVT患者在运动或压力状态下出现的临床表现,为儿茶酚胺敏感性室速的病理生理机制研究提供有力的工具。
Description
技术领域
本发明涉及一种小鼠模型构建技术领域,尤其是涉及一种Tecrl敲除小鼠模型的构建方法及其应用。
背景技术
由心律失常引起的心源性猝死是无心脏器质性改变的患者死亡的主要原因。儿茶酚胺敏感性室速(CPVT)是一种遗传性的离子通道病,主要发生于7-12岁的儿童,发病率约为1/10000。其主要的临床特征为运动或情绪激动所诱发的多形性或双向性室速。部分儿茶酚胺敏感性室速患者的首发症状即为晕厥或猝死。但目前具体的发病机制仍未明确。
儿茶酚胺敏感性室速的诊断主要依靠具有特征性的心电图及基因检测确诊。对于典型CPVT患者,心电图运动负荷试验或注射儿茶酚胺类药物可以有效诱发出多形性或双向性室速。并且在未经任何治疗的情况下,发生心律失常的阈值及心律失常的形态可重复出现。
目前CPVT主要分为三型,分别为CPVT1型、CPVT2型及CPVT3型。CPVT1型是由RyR2基因突变所引起常染色体显性遗传病,约占CPVT患者总数的50-60%。CPVT2型是由CaSQ2基因突变引起的常染色体隐性遗传病,约占CPVT患者总数的5%。CPVT3型由TECRL基因缺陷引起,由法国的Devalla教授在2016年首次发并报道。然而目前缺乏对于TECRL基因缺陷患者在未出现心脏器质性改变前就发生CPVT的发病机制清晰的阐述。
TECRL主要位于内质网,与TECR基因高度同源。TECRL基因的缺陷会伴随有RyR2和CASQ2的减少。而在hiPSC-CM细胞上的研究发现TECRL基因缺陷还会导致细胞内外钙离子失衡,从而导致DADs发生的增加。
近年来,越来越多研究CPVT发病机制的小鼠模型得以构建,但主要包括RyR2基因突变的小鼠模型,如RyR2(R4496C,N2386I,A165D)等表现出CPVT的典型临床表现。CASQ2位点突变的小鼠模型CASQ2(Casq2D307H,Casq2DeltaE9/DeltaE9)可体外模拟CPVT。具有这些突变的小鼠均表现出了运动或压力诱发的心律失常,体内钙环境的紊乱及线粒体超微结构的受损。但目前为止,仍没有Tecrl突变的CPVT小鼠模型。因此构建Tecrl基因敲除的小鼠模型对于研究Tecrl基因缺陷对心脏功能改变的影响有着至关重要的作用。
发明内容
针对现有技术中的缺陷,本发明的目的是提供一种Tecrl敲除小鼠模型的构建方法及其应用。
第一方面,本发明提供了一种Tecrl基因缺陷小鼠模型的构建方法,包括以下步骤:
A、根据基因敲除的靶点,设计针对Tecrl基因的sgRNA,所述sgRNA的序列如SEQ IDNO.1和SEQ ID NO.2所示;
B、将Cas9 mRNA及sgRNA同时注射入体外培养的实验小鼠受精卵中,再将受精卵移植到超排的母鼠内,得到Tecrl基因成功敲除的F0代小鼠。
优选地,所述方法还包括将Tecrl基因成功敲除的F0代小鼠进行回交得到稳定基因型的F1代小鼠,将F1代小鼠与野生型小鼠杂交得到F2代小鼠,再将F2代小鼠相互交配得到F3代Tecrl敲除小鼠的步骤。
优选地,步骤B中,所述Cas9 mRNA和sgRNA注射量分别为20ng/μL,5ng/μL。
优选地,步骤B中,还包括通过鉴定获得Tecrl基因成功敲除的F0代小鼠,所述鉴定的方法包括以下步骤:
提取F0代小鼠的尾部DNA,并进行PCR扩增,扩增产物进行凝胶电泳。
更优选地,所述PCR扩增采用的WT引物序列如SEQ ID NO.3所示,Mutant引物序列如SEQ ID NO.4和SEQ ID NO.5所示;
所述PCR扩增的反应体系以20μL体积计,包括:2×TaqMaster Mix 10μL、上游引物0.8μL、下游引物0.8μL、ddH2O 6.4μL、模版DNA 2μL;
所述PCR扩增反应程序为:
第二方面,本发明提供了一种Tecrl基因缺陷导致左心功能下降的小鼠模型的构建方法,其特征在于,包括将前述的F3代Tecrl敲除小鼠进行正常饲养6-8周后,表现为心脏收缩和舒张功能受损,即构建得到左心功能下降的小鼠模型。
第三方面,本发明提供了一种前述方法构建的小鼠模型在筛选治疗左心功能下降的药物中的应用。
第四方面,本发明提供了一种Tecrl基因缺陷介导的室性心动过速小鼠模型的构建方法,包括将前述的F3代Tecrl敲除小鼠进行正常饲养6-8周后,对F3代Tecrl敲除小鼠使用腹腔注射肾上腺素和咖啡因,得到室性心动过速的小鼠模型。
优选地,所述肾上腺素和咖啡因的注射剂量分别为1.6-3mg/kg、120mg/kg。
优选地,所述室性心动过速包括儿茶酚胺敏感性室速。
第五方面,本发明提供了一种根据前述方法构建的小鼠模型在筛选治疗室性心动过速的药物中的应用。
第六方面,本发明提供了一种用于构建Tecrl基因缺陷小鼠模型的sgRNA,所述sgRNA的序列如SEQ ID NO.1和SEQ ID NO.2所示。
与现有技术相比,本发明具有如下的有益效果:
本发明首次构建了一种Tecrl敲除小鼠模型,并发现该先天的小鼠基因缺陷导致小鼠在早期的时候就会出现心脏收缩和舒张功能的受损,为研究左心功能提供了模型。
本发明构建的Tecrl敲除小鼠在不加任何干预的情况下出现了收缩和舒张功能的受损并且在肾上腺素和咖啡因的诱导下出现了多形性及双向性室速,模拟了临床上CPVT患者在运动或压力状态下出现的临床表现。该小鼠为儿茶酚胺敏感性室速的病理生理机制研究提供有力的工具。
附图说明
通过阅读参照以下附图对非限制性实施例所作的详细描述,本发明的其它特征、目的和优点将会变得更明显:
图1为实施例1中对FO小鼠进行琼脂糖凝胶电泳的鉴定结果;
图2为实施例2的Tecrl敲除小鼠(KO)和野生型C57BL/6小鼠(WT)的M型超声心动图像;
图3为实施例2的Tecrl敲除小鼠(KO)和野生型C57BL/6小鼠(WT)的心脏功能相关指标的检测结果;
图4为实施例3中野生型C57BL/6小鼠(WT)注射ISO及咖啡因前的心电图;
图5为实施例3中野生型C57BL/6小鼠(WT)注射ISO及咖啡因后的心电图;
图6为实施例3中Tecrl敲除小鼠(KO)注射ISO及咖啡因前的心电图;
图7为实施例3中Tecrl敲除小鼠(KO)注射ISO及咖啡因后的心电图;
图8为实施例1中设计的各sgRNA的活性结果。
具体实施方式
下面结合具体实施例对本发明进行详细说明。以下实施例将有助于本领域的技术人员进一步理解本发明,但不以任何形式限制本发明。应当指出的是,对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。
实施例1
本实施例提供一种Tecrl基因敲除小鼠的构建方法,包括以下步骤:
1.1sgRNA的设计及鉴定
基因敲除的关键在于靶点选择,作用于正确靶点可通过序列突变使基因功能缺失。本实施例在确定基因敲除的靶点后,利用CRISPR Design软件(http://crispr.mit.edu/)设计针对Tecrl基因的8对sgRNA(依次命名为sgRNA1~sgRNA16),按照已设计sgRNA序列合成相应引物,通过退火聚合的方式连入pCS载体,连接产物转化后送样测序验证正确。随后利用百奥斯图公司自主研发的CRISPR/Cas9活性检测方法-UCATM方式检测sgRNA的活性,结果如图8所示,筛选确定其中活性较高的两个sgRNA。具体为sgRNA8和sgRNA14,其序列分别为SgRNA8(SEQ ID NO.1):5’-TTAGGATATGCCCTAGATAAAGG-3’和SgRNA14(SEQ ID NO.2):5’-GATCGAAATACCTACGATCGAGG-3’。
将SgRNA8(SEQ ID NO.1)和SgRNA14(SEQ ID NO.2)分别连入带T7启动子质粒载体上并进行体外转录,得到进行显微注射的RNA。
1.2 Tecrl基因敲除小鼠的构建与鉴定
将sgRNA8、sgRNA14与Cas9 mRNA(三者的注入量分别为:5ng/μL,5ng/μL,20ng/μL)同时注入体外培养的C57BL/6小鼠的受精卵,将受精卵移植到超排的母鼠(通过常规的超排方法处理得到的母鼠)内,得到F0代小鼠。提取F0代小鼠尾部DNA,并且进行PCR扩增技术,所得扩增产物利用琼脂糖凝胶电泳进行鉴定。
采用的PCR扩增的引物序列如下:
WT引物:
Tecrl-WT-F(SEQ ID NO.3):5’-TGTATTGCACAGTTTTGGGCTCATGG-3’
Mutant引物:
Tecrl-Mutant-F(SEQ ID NO.4):5’-CATAGGGACTCGATTGTTGTCCGTG-3’
Tecrl-Mutant-R(SEQ ID NO.5):5’-TGCACTTGAATGGAAAAAGACTGGA-3’
扩增反应体系如下:
PCR反应组分 | 20μL反应体系(μL) |
2×TaqMaster Mix | 10 |
上游引物(浓度10μM) | 0.8 |
下游引物(浓度10μM) | 0.8 |
ddH<sub>2</sub>O | 6.4 |
模版DNA(浓度7.5-15ng/μL) | 2 |
PCR反应程序如下:
鉴定结果如图1所示。鉴定为阳性的F0代小鼠回交得到F1代Tecrl基因敲除小鼠。将F1代Tecrl基因敲除小鼠与野生型C57BL/6小鼠杂交,得到F2代Tecrl基因敲除小鼠并进行鉴定,选取F2代杂合子小鼠相互交配,最终得到F3代Tecrl敲除小鼠模型用于后续实验。F1、F2、F3代Tecrl基因敲除小鼠的鉴定方法与F0代小鼠的鉴定方法相同。
实施例2
将实施例1得到的F3代Tecrl敲除(Tecrl-/-)小鼠模型正常饲养6-8周后,将小鼠于3.5ml/min异氟烷下进行麻醉,应用Vevo2100小动物超声影像系统对6-8周的Tecrl敲除小鼠进行超声检查小鼠心脏获得M型超声心动图像及心脏功能相关指标。结果如图2和图3所示,与野生型C57BL/6小鼠(WT)相比,Tecrl敲除小鼠(KO)手所求心脏射血分数及短轴短轴率显著降低。舒张期时,相对野生型小鼠(WT)而言,Tecrl-/-小鼠(KO)等容舒张时间明显延长,左室舒张末期内径明显增加。这一结果表明,Tecrl敲除小鼠在6-8周时就出现了心脏收缩和舒张功能受损的左心功能下降表现,该小鼠模型可用于研究左心功能下降,以及筛选用于治疗左心功能下降的药物。
实施例3
将实施例1得到的F3代Tecrl敲除(Tecrl-/-)小鼠模型正常饲养6-8周后,使用腹腔注射方法同时注入肾上腺素(ISO)及咖啡因,咖啡因剂量为120mg/kg,肾上腺素剂量为2mg/kg(肾上腺素剂量可以在1.6-3mg/kg之间选择,都能够使Tecl-/-小鼠出现明显的双向性的室性心动过速)。在注射前和注射后分别采集小鼠的心电数据,以对比小鼠在注射肾上腺素和咖啡因前后的心电图变化。具体采集小鼠的心电数据的方法为:将小鼠于3.5ml/min异氟烷下进行麻醉,仰卧位固定于心电图记录板上,四肢分别使用胶带固定,接近心电图表面导联,使用心电图软件采集小鼠心电数据。以野生型C57BL/6小鼠作为对照。
结果如图4-图7所示,在注射咖啡因及肾上腺素前,野生型C57BL/6小鼠(WT)及Tecrl-/-小鼠(KO)均未出现室性心动过速的表现(图4和图6);在注射入咖啡因及肾上腺素后,野生型C57BL/6小鼠(WT)仍未出现明显室速的表现(图5和图7),而Tecl-/-小鼠(KO)出现了明显的双向性的室性心动过速。这一结果表明,肾上腺素和咖啡因可以诱发Tecrl-/-小鼠出现双向型室速,这一结果与临床上CPVT患者在注射肾上腺素后可以诱发双向性或多形性室速的表现相一致。由此可见,该小鼠模型为儿茶酚胺敏感性室速的病理生理机制研究提供有力的工具,可以用于筛选治疗CPVT患者的药物。
上述的对实施例的描述是为便于该技术领域的普通技术人员能理解和使用发明。熟悉本领域技术的人员显然可以容易地对这些实施例做出各种修改,并把在此说明的一般原理应用到其他实施例中而不必经过创造性的劳动。因此,本发明不限于上述实施例,本领域技术人员根据本发明的揭示,不脱离本发明范畴所做出的改进和修改都应该在本发明的保护范围之内。
序列表
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<120> Tecrl敲除小鼠模型的构建方法及其应用
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Claims (10)
1.一种Tecrl基因缺陷小鼠模型的构建方法,其特征在于,包括以下步骤:
A、根据基因敲除的靶点,设计针对Tecrl基因的sgRNA,所述sgRNA的序列如SEQ IDNO.1和SEQ ID NO.2所示;
B、将Cas9 mRNA及sgRNA同时注射入体外培养的实验小鼠受精卵中,再将受精卵移植到超排的母鼠内,得到Tecrl基因成功敲除的F0代小鼠。
2.根据权利要求1所述的Tecrl基因缺陷小鼠模型的构建方法,其特征在于,所述方法还包括将Tecrl基因成功敲除的F0代小鼠进行回交得到稳定基因型的F1代小鼠,将F1代小鼠与野生型小鼠杂交得到F2代小鼠,再将F2代小鼠相互交配得到F3代Tecrl敲除小鼠的步骤。
3.根据权利要求1所述的Tecrl基因缺陷小鼠模型的构建方法,其特征在于,步骤B中,所述Cas9 mRNA和sgRNA注射量分别为20ng/μL,5ng/μL。
4.一种Tecrl基因缺陷导致左心功能下降的小鼠模型的构建方法,其特征在于,包括将权利要求2所述的F3代Tecrl敲除小鼠进行正常饲养6-8周后,表现为心脏收缩和舒张功能受损,即构建得到左心功能下降的小鼠模型。
5.一种根据权利要求4所述方法构建的小鼠模型在筛选治疗左心功能下降的药物中的应用。
6.一种Tecrl基因缺陷介导的室性心动过速小鼠模型的构建方法,其特征在于,包括将权利要求2所述的F3代Tecrl敲除小鼠进行正常饲养6-8周后,对F3代Tecrl敲除小鼠使用腹腔注射肾上腺素和咖啡因,得到室性心动过速的小鼠模型。
7.根据权利要求6所述的Tecrl基因缺陷介导的室性心动过速小鼠模型的构建方法,其特征在于,所述肾上腺素和咖啡因的注射剂量分别为1.6-3mg/kg、120mg/kg。
8.根据权利要求6所述的Tecrl基因缺陷介导的室性心动过速小鼠模型的构建方法,其特征在于,所述室性心动过速包括儿茶酚胺敏感性室速。
9.一种根据权利要求6-8任一项所述方法构建的小鼠模型在筛选治疗室性心动过速的药物中的应用。
10.一种用于构建Tecrl基因缺陷小鼠模型的sgRNA,其特征在于,所述sgRNA的序列如SEQ ID NO.1和SEQ ID NO.2所示。
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