CN114934074A - 一种ApoC3基因敲除仓鼠模型的构建方法 - Google Patents
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Abstract
本发明属于疾病动物模型构建技术领域,具体的说是一种ApoC3基因敲除仓鼠模型的构建方法,包括仓鼠受精卵的收集和培养,然后显微注射,最后受精卵回输三个步骤,本发明通过构建ApoC3敲除动物模型,将CRISPR/Cas9技术应用于叙利亚金黄仓鼠,这是一种在脂代谢特征方面与人类相似的小型啮齿类动物模型,通过CRISPR/Cas9产生ApoC3敲除(ApoC3‑/‑)仓鼠,可以更好的理解ApoC3与脂质代谢和动脉粥样硬化之间的关系,使其成为基因修饰动物模型是研究基因功能及其与疾病关系的重要工具。
Description
技术领域
本发明属于疾病动物模型构建技术领域,具体的说是一种ApoC3基因敲除仓鼠模型的构建方法。
背景技术
载脂蛋白C3(ApoC3)是一种分子量为8.8kDa的小分泌糖蛋白,主要由肝脏和小肠合成,主要定位于循环中的富含甘油三酯的脂蛋白(TRL),如乳糜微粒(CM)和极低密度脂蛋白(VLDL)。ApoC3的关键功能是抑制脂蛋白脂肪酶(LPL)介导的TRL上TG的水解,损害肝脏中脂蛋白受体清除TRL残余物。同时,ApoC3还刺激肝脏的VLDL分泌,扰乱高密度脂蛋白(HDL)的正常功能。因此,载脂蛋白C3是血浆甘油三酯的重要调节因子,载脂蛋白C3与高甘油三酯血症之间的关系已被充分证实。
所以,为了更好地理解ApoC3与脂质代谢和动脉粥样硬化之间的关系,提出了一种ApoC3基因敲除仓鼠模型的构建方法。
发明内容
为了弥补现有技术的不足,为了更好地理解ApoC3与脂质代谢和动脉粥样硬化之间的关系,本发明提出了一种ApoC3基因敲除仓鼠模型的构建方法。
本发明解决其技术问题所采用的技术方案是:一种ApoC3基因敲除仓鼠模型的构建方法,包括如下步骤:
S100:仓鼠受精卵的收集和培养;
S200:显微注射;
S300:受精卵回输。
优选的,所述S100步骤中包括如下子步骤:
S101:确定雌鼠动情周期;
S102:供卵鼠超排;
S103:假孕鼠的准备;
S104:取卵及受精卵培养。
优选的,所述S102步骤中,供卵鼠超排时,优选体重130-150g,91-150日龄的雌鼠。
优选的,所述S102步骤中,供卵鼠超排后,进行为期5天的转基因制备。
优选的,所述S103子步骤中,选用6-8周龄的雄性仓鼠做输精管结扎。
优选的,所述S104子步骤中,采用如下分步骤进行:
S1041:首先用HECM-10培养基70μL/滴,覆盖石蜡油,放于培养箱中,用于培养受精卵,37℃预热M2及HECM-10培养基,用于取卵;
S1042:雌鼠做***涂片,取***涂片中观察到***的雌鼠作供卵鼠;
S1043:预热一小时培养基后,用3%戊巴比妥钠(3.5μL/g)麻醉供卵鼠,打开腹腔,取出输卵管,置于预热的M2培养基中,在体式显微镜下,从输卵管壶腹部取出卵团,反复吹打卵团,得到单个分散的受精卵,将受精卵移到HECM-10培养基中,置于培养箱中培养,将显微操作后的受精卵在体外培养1-2小时。
优选的,所述S104子步骤进行时间为转基因制备第五天。
优选的,所述S200步骤中显微注射选用Cas9mRNA和gRNA。
优选的,所述S300步骤中受精卵回输是指将假孕雌鼠麻醉后,从背部开口,拉出输卵管,做双侧输卵管伞口回输,每侧输卵管回输15-20枚显微注射后的受精卵,且仓鼠孕期为17天。
本发明的有益效果如下:
通过构建ApoC3敲除动物模型,将CRISPR/Cas9技术应用于叙利亚金黄仓鼠,这是一种在脂代谢特征方面与人类相似的小型啮齿类动物模型,通过CRISPR/Cas9产生ApoC3敲除(ApoC3-/-)仓鼠,可以更好的理解ApoC3与脂质代谢和动脉粥样硬化之间的关系,使其成为基因修饰动物模型是研究基因功能及其与疾病关系的重要工具。
附图说明
下面结合附图对本发明作进一步说明。
图1为仓鼠ApoC3第二外显子特异性sgRNA的设计;
图2为ApoC3基因敲除仓鼠DNA PCR电泳基因型鉴定结果;
图3为ApoC3基因敲除仓鼠肝脏的RT-PCT结果;
图4为ApoC3基因敲除仓鼠血浆SDS-PAGE;
图5为ApoC3基因敲除仓鼠血浆TG\TC\HDL-C;
图6为ApoC3基因敲除纯合子仓鼠Oral Fat Load;
图7为ApoC3基因敲除纯合子VLDL分泌;
图8-9为ApoC3基因敲除纯合子仓鼠脂蛋白圆盘电泳;
图10为ApoC3基因敲除纯合子仓鼠血浆western blot;
图11-13为喂饲HCHF后ApoC3基因敲除纯合子仓鼠血浆TG\TC\HDL-C;
图14为喂饲HCHF后ApoC3基因敲除纯合子仓鼠脂蛋白圆盘电泳;
图15为喂饲HCHF后ApoC3基因敲除纯合子仓鼠血浆western blot;
图16-17为喂饲HCHF条件下动脉粥样硬化情况。
具体实施方式
为了使本发明实现的技术手段、创作特征、达成目的与功效易于明白了解,下面结合具体实施方式,进一步阐述本发明。
如图1至图17所示,本发明所述的一种ApoC3基因敲除仓鼠模型的构建方法,采用如下步骤进行:
S100:仓鼠受精卵的收集和培养,其包括如下子步骤:
S101:确定雌鼠动情周期:
雌性仓鼠一般在8周龄时性成熟,此后出现稳定的动情周期。动情周期一般为4天,动情周期的第一天为发情期。在动情周期的第二天,***口会出现白色粘稠状分泌物,此分泌物可以作为判断动情周期时程的标志。
S102:供卵鼠超排:
在适宜条件下,雌性仓鼠的发情期一年四季都可以出现,全年都能进行超***。仓鼠体重和超***效果有关,130-150g区间体重的雌鼠超***最合适。从年龄上看,91-150日龄的雌鼠超***效果最好。超***的时间和动情周期相吻合十分重要。
转基因制备第一天:选择动情周期第二天的雌鼠(***口有白色粘稠状分泌物)于中午12:00-13:00腹腔注射PMSG20IU;
转基因制备第五天:取卵前对雌鼠做***涂片,如果发现***,证明交配成功,可以用这样的雌鼠作供卵鼠。
S103:假孕鼠的准备:将6-8周龄的雄性仓鼠做输精管结扎。手术前禁食6h,3%戊巴比妥钠(3.5μL/g)麻醉。固定后,手术区剃毛、消毒,双侧下腹部作0.5cm长的切口,然后依次剪开皮肤、皮下组织。将组织钝性分离,形成约1cm长的切口,夹住附睾脂肪将输精管拉出,用烧红的镊子夹断输精管,去掉1cm长度。然后将输精管放回腹腔。分层缝合创口。对侧输精管手术同上述操作。术后四周左右伤口愈合,与雌鼠能顺利交配且***中未检测到***即说明手术成功,这样的雄鼠可用于***母鼠交配。转基因制备第四天20:00-22:00,将动情周期第一天的正常雌鼠与结扎成功的雄鼠合笼,观察交配。如果有多于10次的交配动作,认为对雌鼠假孕诱导成功,这样的雌鼠可以作为***鼠。
S104:取卵及受精卵培养:
在转基因制备第五天时,进行如下分步骤:
S1041:首先用HECM-10培养基70μL/滴,覆盖石蜡油,放于培养箱中,用于培养受精卵。37℃预热M2及HECM-10培养基,用于取卵。
S1042:雌鼠做***涂片,取***涂片中观察到***的雌鼠作供卵鼠。
S1043:预热一小时培养基后,用3%戊巴比妥钠(3.5μL/g)麻醉供卵鼠,打开腹腔,取出输卵管,置于预热的M2培养基中。在体式显微镜下,从输卵管壶腹部取出卵团,反复吹打卵团,得到单个分散的受精卵。将受精卵移到HECM-10培养基中,置于培养箱中培养。
S200:显微注射;
受精卵转移至M2培养基中,培养基液滴上用石蜡油覆盖,做显微注射。将Cas9mRNA与带有目的基因靶序列的gRNA混合,稀释浓度至:Cas9mRNA,100ng/μL;gRNA,25ng/μL。然后注射至受精卵胞浆,镜下见到有液体进入胞浆表明注射成功。
将显微操作后的受精卵在体外培养1-2小时。
S300:受精卵回输;
将假孕雌鼠麻醉后,从背部开口,拉出输卵管,做双侧输卵管伞口回输。每侧输卵管回输15-20枚显微注射后的受精卵。仓鼠孕期为17天。
基因修饰动物模型是研究基因功能及其与疾病关系的重要工具,构建ApoC3敲除动物模型的意义:
载脂蛋白C3(ApoC3)是一种分子量为8.8kDa的小分泌糖蛋白,主要由肝脏和小肠合成,主要定位于循环中的富含甘油三酯的脂蛋白(TRL),如乳糜微粒(CM)和极低密度脂蛋白(VLDL)。ApoC3的关键功能是抑制脂蛋白脂肪酶(LPL)介导的TRL上TG的水解,损害肝脏中脂蛋白受体清除TRL残余物。同时,ApoC3还刺激肝脏的VLDL分泌,扰乱高密度脂蛋白(HDL)的正常功能。因此,载脂蛋白C3是血浆甘油三酯的重要调节因子,载脂蛋白C3与高甘油三酯血症之间的关系已被充分证实。
所以,为了更好地理解ApoC3与脂质代谢和动脉粥样硬化之间的关系,将CRISPR/Cas9技术应用于叙利亚金黄仓鼠,这是一种在脂代谢特征方面与人类相似的小型啮齿类动物模型。通过CRISPR/Cas9产生ApoC3敲除(ApoC3-/-)仓鼠。
以上显示和描述了本发明的基本原理、主要特征和优点。本行业的技术人员应该了解,本发明不受上述实施例的限制,上述实施例和说明书中描述的只是说明本发明的原理,在不脱离本发明精神和范围的前提下,本发明还会有各种变化和改进,这些变化和改进都落入要求保护的本发明范围内。本发明要求保护范围由所附的权利要求书及其等效物界定。
Claims (9)
1.一种ApoC3基因敲除仓鼠模型的构建方法,其特征在于:包括如下步骤:
S100:仓鼠受精卵的收集和培养;
S200:显微注射;
S300:受精卵回输。
2.根据权利要求书1所述的一种ApoC3基因敲除仓鼠模型的构建方法,其特征在于:所述S100步骤中包括如下子步骤:
S101:确定雌鼠动情周期;
S102:供卵鼠超排;
S103:假孕鼠的准备;
S104:取卵及受精卵培养。
3.根据权利要求书2所述的一种ApoC3基因敲除仓鼠模型的构建方法,其特征在于:
所述S102步骤中,供卵鼠超排时,优选体重130-150g,91-150日龄的雌鼠。
4.根据权利要求书3所述的一种ApoC3基因敲除仓鼠模型的构建方法,其特征在于:
所述S102步骤中,供卵鼠超排后,进行为期5天的转基因制备。
5.根据权利要求书2所述的一种ApoC3基因敲除仓鼠模型的构建方法,其特征在于:所述S103子步骤中,选用6-8周龄的雄性仓鼠做输精管结扎。
6.根据权利要求书2所述的一种ApoC3基因敲除仓鼠模型的构建方法,其特征在于:所述S104子步骤中,采用如下分步骤进行:
S1041:首先用HECM-10培养基70μL/滴,覆盖石蜡油,放于培养箱中,用于培养受精卵,37℃预热M2及HECM-10培养基,用于取卵;
S1042:雌鼠做***涂片,取***涂片中观察到***的雌鼠作供卵鼠;
S1043:预热一小时培养基后,用3%戊巴比妥钠(3.5μL/g)麻醉供卵鼠,打开腹腔,取出输卵管,置于预热的M2培养基中,在体式显微镜下,从输卵管壶腹部取出卵团,反复吹打卵团,得到单个分散的受精卵,将受精卵移到HECM-10培养基中,置于培养箱中培养,将显微操作后的受精卵在体外培养1-2小时。
7.根据权利要求书6所述的一种ApoC3基因敲除仓鼠模型的构建方法,其特征在于:所述S104子步骤进行时间为转基因制备第五天。
8.根据权利要求书1所述的一种ApoC3基因敲除仓鼠模型的构建方法,其特征在于:所述S200步骤中显微注射选用Cas9mRNA和gRNA。
9.根据权利要求书1所述的一种ApoC3基因敲除仓鼠模型的构建方法,其特征在于:所述S300步骤中受精卵回输是指将假孕雌鼠麻醉后,从背部开口,拉出输卵管,做双侧输卵管伞口回输,每侧输卵管回输15-20枚显微注射后的受精卵,且仓鼠孕期为17天。
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