CN109456996B - 构建abca1基因敲除仓鼠模型的试剂盒和方法 - Google Patents
构建abca1基因敲除仓鼠模型的试剂盒和方法 Download PDFInfo
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Abstract
本发明公开了构建ABCA1基因敲除仓鼠模型的试剂盒和方法,所述试剂盒包括sgRNA,所述sgRNA由如SEQ ID NO:2和SEQ ID NO:3所示人工序列经PCR扩增、体外转录所得。所述方法包括:设计仓鼠ABCA1基因特异性打靶序列、制备cas9 mRNA和sgRNA;收集和培养仓鼠受精卵;显微注射:将sgRNA和cas9 mRNA共同注射至仓鼠受精卵的细胞质中;将显微注射后的受精卵植入代孕仓鼠体内,F0代仓鼠出生。经鉴定该方法成功构建了ABCA1基因敲除仓鼠模型,能够为研究ABCA1与冠心病的关系提供研究基础。
Description
技术领域
本发明涉及疾病动物模型及其制备技术领域,具体地,涉及一种心血管疾 病动物模型及其构建方法,尤其是构建ABCA1基因敲除仓鼠模型的试剂盒和 方法。
背景技术
冠状动脉粥样硬化性心脏病(Coronary atherosclerotic heart disease,CHD,简称冠心病)的患病率和死亡率呈逐年上升趋势。动脉粥样硬化 (Atherosclerosis,As)作为冠心病的主要病理学基础,其发生是一个多因素参 与的复杂过程,其中血管壁胆固醇过量蓄积和炎症反应是两个相互促进的关键 环节,因此,抑制血管炎症和减少巨噬细胞内胆固醇蓄积是防治As的重要措 施。三磷酸腺苷结合盒转运体A1(ABCA1)在胆固醇逆转运和高密度脂蛋白 (HDL)生成的起始步骤中起重要作用,它是介导胆固醇流出的关键蛋白,也是As防治的重要靶点。ABCA1以ATP为能源,促进细胞内游离胆固醇和磷脂流 出,并结合到细胞表面乏脂的载脂蛋白AI(Apolipoprotein AI,apoAI),形成 新生的高密度脂蛋白(High-density lipoprotein,HDL),对于减少血管壁脂质 蓄积泡沫细胞形成和血管壁炎症反应均具有重要意义。
人类ABCA1基因敲除可导致血浆HDL水平降低,增加冠心病风险。多个临 床研究发现,ABCA1基因至少存在50多种突变,这些突变导致的最常见疾病是 丹吉尔病和家族性HDL缺乏症(Familial HDL deficiency,FHA),该类疾病表 现出低HDL水平、胆固醇蓄积于外周和早发性As,其冠心病发病率是正常人群 的6倍。然而,临床研究和动物实验的许多结果并不一致,如,以小鼠为模型的 研究发现,ABCA1突变虽能降低血浆HDL,但并不形成As;且ABCA1突变模 型小鼠与As模型小鼠杂交的双基因敲除动物在喂饲高脂饲料后,亦不促进As病 变,更没有冠心病发生,与人类疾病明显不符。而大动物模型存在经济成本、 时间成本、伦理审查要求过高,实验操作便利性差的问题。上述情况导致以 ABCA1为靶点进行As防治的研究没有重要进展。
发明内容
针对现有技术中缺乏与人类脂代谢接近的ABCA1基因敲除动物模型、小 鼠模型与人类临床表现明显不符、大动物模型存在经济成本和、时间成本、伦 理审查要求过高以及实验操作便利性差的技术问题,本发明提供构建ABCA1 基因敲除仓鼠模型的试剂盒。
以及,本发明还提供采用上述试剂盒构建ABCA1基因敲除仓鼠模型的方 法。
为实现上述目的,本发明实施例提供了构建ABCA1基因敲除仓鼠模型的 试剂盒,该试剂盒中包括sgRNA,所述sgRNA由如SEQ ID NO:2和SEQ ID NO:3所示人工序列经PCR扩增、体外转录所得。
该试剂盒以仓鼠为动物模型,其原因在于:仓鼠脂蛋白组成、载脂蛋白含 量及分布、脂代谢特点、As易感性等与人类高度相似,血浆中有较高的低密度 脂蛋白,与小鼠相比,仓鼠对高脂饮食的高反应性更接近人类,高脂饲料可使 仓鼠血TG和胆固醇皆明显升高,易诱导发生肥胖、胰岛素抵抗、动脉粥样硬 化病变,且病变斑块性质与人类相似。同时仓鼠还具有易于操作、成本低、易 繁殖的特点。因此,我们以仓鼠为对象构建ABCA1基因敲除仓鼠模型,用于 研究ABCA1对脂代谢、As、冠心病等的作用。仓鼠基因序列与小鼠等其他动 物不同,因此我们对人工序列进行设计,用如SEQ ID NO:2和SEQ ID NO:3所 示人工序列经PCR扩增、体外转录制成sgRNA,使含有该sgRNA的试剂盒可 用于构建ABCA1基因敲除仓鼠模型。
可选的,所述sgRNA的制备步骤为:采用所述SEQ ID NO:2和SEQ ID NO:3 人工序列经PCR扩增,取扩增产物中124bp的DNA片段作为sgRNA的DNA 模板,所得的sgRNA的DNA模板经体外转录得sgRNA粗品。
可选的,所述试剂盒还包括cas9 mRNA,其制备步骤为:采用含有人源化 cas9cDNA的PXT7质粒,经XbaI限制性内切酶作用,纯化得cas9 mRNA的 DNA模板,所得cas9 mRNA的DNA模板经体外转录得cas9 mRNA粗品。
可选的,所述cas9 mRNA的制备中:
cas9 mRNA的DNA模板纯化的方法为:PXT7质粒经XbaI限制性内切酶 处理后,用蛋白酶K处理20~50min,经酚-氯仿抽提,乙醇沉淀;和/或
cas9mRNA的DNA模板转录的条件为:采用mMESSAGE mMACHINE T7 kit试剂盒,在37℃反应1~3小时。
所得cas9 mRNA粗品的纯化方法为:cas9 mRNA粗品经酚-氯仿抽提、异 丙醇沉淀、无RNase水溶解,得浓度为200~800ng/μL的cas9 mRNA。
可选的,所述PCR扩增的反应条件为:
98℃30s;
98℃10s,56℃30s,72℃15s,共35个循环;
72℃10min。
可选的,在sgRNA的制备中:
所得sgRNA的DNA模板的选取和纯化方法为:所述PCR产物经过琼脂糖 凝胶电泳,用胶回收试剂盒回收124bp的DNA条带,再经蛋白酶K处理 20~50min,酚-氯仿抽提,乙醇沉淀,得sgRNA的DNA模板;和/或
所得sgRNA粗品纯化的方法为:用MEGAclear Kit试剂盒进行纯化,无 RNase水溶解为浓度100~500ng/μL的sgRNA。
可选的,sgRNA的DNA模板转录的条件为:采用Megascript T7Kit试剂 盒,在37℃反应3~5h。
本发明实施例还提供了采用上述试剂盒构建ABCA1基因敲除仓鼠模型的 方法,包括以下步骤:
步骤一、设计仓鼠ABCA1基因特异性打靶序列,如SEQ ID NO:1所示; 以含有人源化cas9 cDNA的PXT7质粒制备cas9 mRNA,采用如SEQ ID NO:2 和SEQ ID NO:3所示人工序列经PCR反应和转录制得sgRNA;
步骤二、收集和培养仓鼠受精卵;
步骤三、将步骤一中所制得的sgRNA和cas9 mRNA注射至仓鼠受精卵的 细胞质中,得到ABCA1基因敲除的受精卵;
步骤四、将ABCA1基因敲除的受精卵,植入代孕仓鼠体内,F0代仓鼠出 生,鉴定。
可选的,上述步骤三中,所述sgRNA和cas9 mRNA的注射浓度分别为 10ng/μL和20ng/μL。
采用上述技术方案产生的有益效果在于:本发明实施例所提供的构建 ABCA1基因敲除仓鼠模型的试剂盒中采用的sgRNA序列具有高效、不易脱靶 的特点,采用该试剂盒构建ABCA1基因敲除仓鼠模型的方法操作简单,效率 高,致死率低;采用本发明实施例提供的ABCA1基因敲除仓鼠模型具有高度 的拟人性,对明确ABCA1对脂代谢、As、冠心病的作用具有重要的意义。
附图说明
图1检验例中野生型仓鼠及ABCA1基因敲除仓鼠的DNA测序峰及敲除序 列示意图;
图2为检验例中ABCA1基因敲除仓鼠血浆中总胆固醇与甘油三酯浓度;
图3为检验例中ABCA1基因敲除仓鼠血浆中游离胆固醇浓度;
图4为检验例中ABCA1基因敲除仓鼠血浆中HDL浓度;
图5为检验例中ABCA1基因敲除仓鼠血浆的快速蛋白液相色谱图;
图6为检验例中ABCA1基因敲除仓鼠血浆的变性梯度凝胶电泳结果;
图7为检验例中ABCA1基因敲除仓鼠血浆的聚丙烯酰胺凝胶电泳结果;
图8为检验例中喂饲正常饲料的5周龄的野生型仓鼠及ABCA1基因敲除 仓鼠的主动脉粥样硬化病变结果比较。
具体实施方式
为了使本发明所要解决的技术问题、技术方案及有益效果更加清楚明白, 以下结合附图及实施例,对本发明进行进一步详细说明。应当理解,所述实施 例只用于解释本发明,并非用于限定本发明的范围。下述实施例中所使用的试 验方法如无特殊说明,均为常规方法;所使用的材料、试剂等,如无特殊说明, 为可从商业途径得到的试剂和材料。
实施例1
用于构建ABCA1基因敲除仓鼠模型的试剂盒,包括sgRNA和cas9mRNA, 所述sgRNA由如SEQ ID NO:2和SEQ ID NO:3所示人工序列经PCR扩增、体 外转录所得。
所述sgRNA的制备步骤为:
采用如SEQ ID NO:2和SEQ ID NO:3所示人工序列,互为引物和模板,经 PCR扩增,PCR扩增的方法为:于50μl体系中进行,反应条件为98℃,30s; 98℃,10s,56℃,30s,72℃,15s,共35个循环;72℃10min。该PCR产物 经过琼脂糖凝胶电泳,用胶回收试剂盒(TAKARA试剂盒)回收124bp的DNA 条带,然后经蛋白酶K处理30min,尽可能除去样品中的RNA酶,再经酚-氯 仿抽提,乙醇沉淀,得纯化的sgRNA的DNA模板。将该模板经体外转录得 sgRNA粗品。其中体外转录所用试剂盒为Megascript T7Kit(Ambion)试剂盒, 转录体系于37℃充分反应4小时。反应所得sgRNA mRNA用MEGAclear Kit (Ambion)进行纯化,无RNase水溶解使其浓度为200ng/μL。储存于-80℃冰 箱,备用,用于构建模型中的显微注射。
所述cas9mRNA的制备步骤为:
采用含有人源化cas9 cDNA的PXT7质粒,在XbaI限制性内切酶的作用下 充分线性化,反应终止后,用蛋白酶K处理30min,尽可能除去样品中的RNA 酶,进一步酚-氯仿抽提,乙醇沉淀,得纯化的cas9 mRNA的DNA模板,用该 模板经体外转录得cas9 mRNA粗品。其中,体外转录所用试剂盒为mMESSAGE mMACHINE T7kit(Ambion)试剂盒,转录体系于37℃充分反应2小时。反 应所得cas9 mRNA用酚-氯仿抽提的方法进行纯化,异丙醇沉淀,用无RNase水溶解,使其浓度为500ng/μL。储存于-80℃冰箱,备用,用于构建模型中的显 微注射
采用本实施例的试剂盒可用于构建ABCA1基因敲除的仓鼠模型。
实施例2
本实施例以从北京维通利华实验动物技术公司购得的野生型仓鼠为例,说 明采用实施例1的试剂盒,构建ABCA1基因敲除仓鼠模型的方法。本实施例 中仓鼠模型的制备、分析等实验计划和过程均通过伦理委员会实验动物伦理审 查。
所述野生型仓鼠按清洁级标准饲养。保持湿度50-60%、温度22-24℃。光 照周期为7:00-19:00光照,19:00-7:00黑暗。
用野生型仓鼠制备仓鼠模型的具体方法包括以下步骤:
步骤一、在NCBI(National Center for Biotechnology Information,美国国 立生物技术信息中心)中确定数据库中的ABCA1基因信息(Gene ID: 101838333),在其第二外显子中设计仓鼠ABCA1基因特异性靶序列(sgRNA 的靶序列)位置,参见SEQ ID NO:1所示;用实施例1试剂盒中sgRNA和cas9 mRNA的制备步骤,采用如SEQ ID NO:2和SEQ ID NO:3所示人工序列经PCR 反应和体外转录制得sgRNA,以含有人源化cas9 cDNA的PXT7质粒制备cas9 mRNA;
步骤二、收集和培养仓鼠受精卵:
选取91-150日龄,90-130g雌鼠诱导其超数排卵:仓鼠动情周期为四天, 在周期第二天,即发情症状消失后,先后腹腔注射马血清促性腺激素(PMSG) 和人绒毛膜促性腺激素(HCG),以促进排卵,然后与雄性仓鼠交配,交配是 否成功通过阴道分泌物检查判断。HCG注射24小时后,选取交配成功的雌性 仓鼠取受精卵,置于37℃预热的M2培养基中(Sigma-Aldrich,St.Louis,MO, USA)。
仓鼠受精卵的体外培养基是HECM-10(配方为:NaCl 113.8mM,KCl 3mM, NaHCO325mM,乳酸钠4.5mM,CaCl2 1mM,MgCl2 2mM,谷氨酸0.01mM, 谷氨酰胺0.2mM,甘氨酸0.01mM,组氨酸0.01mM,赖氨酸0.01mM,脯氨酸 0.01mM,丝氨酸0.01mM,天冬酰胺0.01mM,天门冬氨酸0.01mM,半胱氨酸 0.01mM,牛磺酸0.5mM,泛酸盐0.003mM,聚乙烯醇0.1mg/ml)。用石蜡油将HECM-10培养液滴覆盖。受精卵培养温度为37.5℃,CO2浓度为10%培养。
步骤三、显微注射:
在注射皿中加入一滴(100μl)M2培养基,矿物油液封,作为注射液滴, 将受精卵置于该注射液滴内,将步骤一中所得sgRNA和cas9 mRNA共同注射 至仓鼠受精卵的细胞质中,其注射浓度为10ng/μL和20ng/μL。该操作自受精 卵离开孵箱后在15分钟内完成,注射后将受精卵放回孵箱中继续培养,培养2 小时左右选择健康受精卵回输至代孕母鼠体内。
步骤四、将显微注射后的受精卵植入代孕仓鼠体内,F0代仓鼠出生,鉴定。
挑选与供卵仓鼠年龄相同、动情周期一致的雌鼠作为代孕仓鼠。代孕鼠动 情第一天晚19点与雄鼠合笼过夜交配。次日,将代孕鼠麻醉后,打开腹腔找出 卵巢输卵管,通过输卵管伞口,将步骤三得到的显微注射后受精卵吹入输卵管, 每只吹入30枚。缝合苏醒后,正常饲养,产仔获得F0代ABCA1基因敲除仓 鼠。
检验例
经过基因组DNA PCR产物测序分析,确认了ABCA1基因的敲除,证明 ABCA1基因敲除仓鼠构建成功。如图1所示,野生型仓鼠的DNA测序峰中两 条竖线之间为敲除片段,即野生型仓鼠的DNA序列中标有下划线的片段。
对该仓鼠进行初步表型分析,发现ABCA1基因敲除仓鼠表现为:
总胆固醇不变,甘油三酯升高(如图2所示);
游离胆固醇增高(如图3所示);
HDL消失(如图4~图6所示);
VLDL增高,LDL不变,HDL消失(如图5所示);
ApoA1减小(如图7所示)。
上述表现与ABCA1突变病人相同。而现有技术中可知,ABCA1基因敲除 小鼠则表现为总胆固醇降低,甘油三酯不变,VLFL不变,LDL消失,与ABCA1 突变病人表现不同。
正常饲料喂饲5个月时,检测到ABCA1敲除仓鼠出现自发性动脉粥样硬 化,如图8所示,该表现与ABCA1突变病人相同。
以上结果说明,本发明实施例所提供的ABCA1基因敲除仓鼠具有高度的 拟人性。
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发 明的精神和原则之内所作的任何修改、等同替换和改进等,均应包含在本发明 的保护范围之内。
SEQUENCE LISTING
<110> 河北伊维沃生物科技有限公司
<120> 构建ABCA1基因敲除仓鼠模型的试剂盒和方法
<130> 2018.10.11
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 54
<212> DNA
<213> 靶序列
<400> 1
cctgatcctg attgccgtcc gcctgagcta cccgccctat gaacaacatg agtg 54
<210> 2
<211> 18
<212> DNA
<213> ABCA1-X-F1
<400> 2
tcagagccca gcagcagt 18
<210> 3
<211> 18
<212> DNA
<213> sgRNA-Reverse
<400> 3
agccagccat caccgagt 18
Claims (9)
1.构建ABCA1基因敲除仓鼠模型的试剂盒,其特征在于,包括sgRNA,所述sgRNA由如SEQID NO:2和SEQ ID NO:3所示人工序列经PCR扩增、体外转录所得。
2.如权利要求1所述的构建ABCA1基因敲除仓鼠模型的试剂盒,其特征在于,所述sgRNA的制备步骤为:采用所述SEQ ID NO:2和SEQ ID NO:3人工序列经PCR扩增,取扩增产物中124bp的DNA片段作为sgRNA的DNA模板,所得的sgRNA的DNA模板经体外转录得sgRNA粗品。
3.如权利要求1或2所述的构建ABCA1基因敲除仓鼠模型的试剂盒,其特征在于,所述试剂盒还包括cas9 mRNA,其制备步骤为:采用含有人源化cas9 cDNA的PXT7质粒,经XbaI限制性内切酶作用,纯化得cas9 mRNA的DNA模板,所得cas9 mRNA的DNA模板经体外转录得cas9mRNA粗品。
4.如权利要求3所述的构建ABCA1基因敲除仓鼠模型的试剂盒,其特征在于,所述cas9mRNA的制备中:
cas9 mRNA的DNA模板纯化的方法为:PXT7质粒经XbaI限制性内切酶处理后,用蛋白酶K处理20~50min,经酚-氯仿抽提,乙醇沉淀;和/或
cas9 mRNA的DNA模板体外转录的条件为:采用mMESSAGE mMACHINE T7kit试剂盒,在37℃反应1~3小时;和/或
所得cas9 mRNA粗品的纯化方法为:cas9 mRNA粗品经酚-氯仿抽提、异丙醇沉淀、无RNase水溶解,得浓度为200~800ng/μL的cas9 mRNA。
5.如权利要求2所述的构建ABCA1基因敲除仓鼠模型的试剂盒,其特征在于,所述PCR扩增的反应条件为:
98℃30s;
98℃10s,56℃30s,72℃15s,共35个循环;
72℃10min。
6.如权利要求2所述的构建ABCA1基因敲除仓鼠模型的试剂盒,其特征在于,所述sgRNA的制备中:
所得sgRNA的DNA模板的选取和纯化方法为:所述PCR产物经过琼脂糖凝胶电泳,用胶回收试剂盒回收124bp的DNA条带,再经蛋白酶K处理20~50min,酚-氯仿抽提,乙醇沉淀,得sgRNA的DNA模板;和/或
所得sgRNA粗品纯化的方法为:用MEGAclear Kit试剂盒进行纯化,无RNase水溶解为浓度100~500ng/μL的sgRNA。
7.如权利要求2所述的构建ABCA1基因敲除仓鼠模型的试剂盒,其特征在于,sgRNA的DNA模板体外转录的条件为:采用Megascript T7Kit试剂盒,在37℃反应3~5h。
8.采用权利要求1~7任一项所述的试剂盒构建ABCA1基因敲除仓鼠模型的方法,其特征在于,包括以下步骤:
步骤一、设计仓鼠ABCA1基因特异性打靶序列,如SEQ ID NO:1所示;以含有人源化cas9cDNA的PXT7质粒制备cas9 mRNA,采用如SEQ ID NO:2和SEQ ID NO:3所示人工序列经PCR反应和体外转录制得sgRNA;
步骤二、收集和培养仓鼠受精卵;
步骤三、将步骤一中所制得的sgRNA和cas9 mRNA注射至仓鼠受精卵的细胞质中,得到ABCA1基因敲除的受精卵;
步骤四、将ABCA1基因敲除的受精卵,植入代孕仓鼠体内,F0代仓鼠出生,鉴定。
9.如权利要求8所述的构建ABCA1基因敲除仓鼠模型的方法,其特征在于:步骤三中,所述sgRNA和cas9 mRNA的注射浓度分别为10ng/μL和20ng/μL。
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