CN108410873A - 构建lcat基因敲除仓鼠模型的系统和方法 - Google Patents
构建lcat基因敲除仓鼠模型的系统和方法 Download PDFInfo
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- CN108410873A CN108410873A CN201810201435.0A CN201810201435A CN108410873A CN 108410873 A CN108410873 A CN 108410873A CN 201810201435 A CN201810201435 A CN 201810201435A CN 108410873 A CN108410873 A CN 108410873A
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- sgrna
- lcat
- cas9mrna
- gene knockout
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Abstract
本发明公开了一种构建卵磷脂胆固醇酰基转移酶(LCAT)基因敲除仓鼠模型的系统和方法,所述系统包括仓鼠LCAT基因特异性打靶序列sgRNA和放大sgRNA的PCR引物。所述方法包括:设计所述仓鼠LCAT基因特异性打靶序列、制备所述cas9mRNA和sgRNA;收集和培养仓鼠受精卵;显微注射:将所述sgRNA和cas9 mRNA共同注射至仓鼠受精卵的细胞质中;最后将显微注射后的受精卵植入代孕仓鼠体内,F1代仓鼠出生,经鉴定构建了LCAT基因敲除的仓鼠模型。
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,尤其是一种卵磷脂胆固醇酰基转移酶(LCAT)基因敲除仓鼠的构建方法和系统。
背景技术
卵磷脂胆固醇酰基转移酶(Lecithin—cholesterol acyltransferase,LCAT)是催化高密度脂蛋白(High-density lipoprotein,HDL)中游离胆固醇的酯化酶,并接受外周组织包括动脉壁的胆固醇,转运到肝脏降解,是胆固醇逆转运的关键因子之一。在脂蛋白代谢途径中,LCAT对胆固醇、甘油三酯、磷脂的代谢都起到重要调节作用。人群调查研究发现,LCAT增高对冠心病有保护作用;而以小鼠为模型的研究则发现LCAT转基因过表达可促进动脉硬化(atherosclerosis,缩写As),敲除LCAT反而减轻As,与LCAT在胆固醇逆转运中的作用以及人群中的研究结果明显相悖。因此,必须应用拟人化动物模型,才能阐明LCAT究竟是延缓还是促进冠心病的发生发展这样一个重要科学问题。
在基因工程小鼠技术建立之后,虽然小鼠成为疾病动物模型的主要模式动物,然而科研人员越来越认识到小鼠疾病和人类疾病的区别。因此,研发和构建人源化、类人化动物模型开展转化医学研究,也是近年来生物医学领域发展的一个方向。随着近年来基因工程仓鼠的研究不断进展,其在代谢性心血管病研究中的优势逐步被广泛地重新认识。和小鼠、家兔相比,仓鼠的糖脂代谢机制和人类更加相似,包括对高脂饮食的反应性,血浆脂质谱有较高的低密度脂蛋白(low-density lipoprotein,LDL)组分,表达胆固醇酯转运蛋白(CETP)、HL、ApoAII,肝脏编辑ApoB100,高果糖饲料能诱导2型糖尿病,因此在代谢性心血管病研究上优势非常明确。
基于仓鼠的类人化特点,对仓鼠的LCAT基因敲除(LCAT-/-)和转基因,有可能为争议中的LCAT对As的影响提供比较明确的答案,为进一步的干预方法研究提供理论基础。
发明内容
本发明要解决的技术问题是提供一种构建LCAT基因敲除仓鼠模型的系统和方法。本发明实施例应用高效基因组编辑CRISPR/Cas9技术,构建出LCAT基因敲除的拟人化仓鼠动物模型,用于解决临床和基础研究中一直争议的LCAT和动脉粥样硬化的关系问题。
为实现上述目的,本发明实施例采用的技术方案是:
一种构建LCAT基因敲除仓鼠模型的系统,所述系统包括仓鼠LCAT基因特异性打靶序列和sgRNA:
所述仓鼠LCAT基因特异性打靶序列如SEQ ID NO:1所示;
所述sgRNA的制备:采用如SEQ ID NO:2和SEQ ID NO:3所示人工序列,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,所述系统还包括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cas9mRNA的DNA模板,所得cas9mRNA的DNA模板经体外转录得cas9 mRNA粗品。
可选的,所述cas9mRNA的制备中:
cas9mRNA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RNA酶水溶解,得浓度为200-800ng/μl的cas9 mRNA。
可选的,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。
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,sgRNA的DNA模板转录的条件为:采用Megascript T7 Kit试剂盒,在37℃反应3-5h。
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。
本发明实施例还提供了构建上述LCAT基因敲除仓鼠模型的方法,包括以下步骤:
步骤一、设计仓鼠LCAT受体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、显微注射: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;
步骤四、将显微注射后的受精卵植入代孕仓鼠体内,F1代仓鼠出生,鉴定。
采用上述技术方案产生的有益效果在于:本发明实施例采用sgRNA序列高效,不易脱靶;操作简单,高效率,低致死率;采用本发明实施例提供的拟人化的LCAT-/-仓鼠模型,对明确LCAT与动脉粥样硬化尤其是冠状动脉硬化的关系具有重要的意义。
附图说明
图1为仓鼠基因序列,其中斜体字体是sgRNA在LCAT基因第二外显子上识别位点示意图,斜体加粗字体是PAM序列,下划线为突变部分;
图2A-2C分别为仓鼠模型构建成功后的LCAT基因型,并与正常仓鼠的基因型进行比较,其中斜体字体为sgRNA位置,斜体加粗字体为PAM序列,---为缺失部分;
图3为LCAT缺失Δ12bp仓鼠组织经PCR扩增后的DNA凝胶电泳图,缺失片段DNA长度为108bp,野生型(wt)、杂合子(+/-)、纯合子(-/-)三种基因型;
图4分别显示了LCAT基因缺失碱基Δ6bp、Δ12bp、Δ56bp的纯合子及野生型对照仓鼠的血浆总胆固醇图及甘油三酯含量图、HDL-C的含量图;
图5A-C为缺失Δ56纯合子仓鼠进行表型分析,检测血浆胆固醇的含量;
图6A和图6B分别为LCAT基因缺失仓鼠和野生型仓鼠TC、TG中脂蛋白图谱,其中,LCAT敲除仓鼠为(-/-),野生型仓鼠为(WT);
图7A-C分别为仓鼠血浆载脂蛋白ApoB100、ApoE、ApoAI的western blot分析及LCAT基因缺失仓鼠和野生型仓鼠血浆中LCAT活性分布图;
图8为动脉粥样硬化病变图,其中A为表示动脉硬化病变的代表图;B为.动脉硬化病变面积的定量检测。
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。
本发明实施例的基本构思:根据仓鼠LCAT基因信息(Gene ID:101823034),设计向导RNA靶向其第二外显子中atctcaatatgtttctacccctgg序列为sgRNA序列,基于此分别构建cas9 mRNA和sgRNA。
实施例1
用于构建LCAT基因敲除仓鼠模型的系统,包括仓鼠LCAT基因特异性打靶序列、cas9mRNA和sgRNA:
所述仓鼠LCAT基因特异性打靶序列如SEQ ID NO:1所示;
所述cas9mRNA的制备:
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cas9mRNA的DNA模板,所述cas9mRNA的DNA模板经体外转录得cas9mRNA粗品;
所述sgRNA的制备: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,取扩增产物中124bp的DNA片段作为sgRNA的DNA模板,所述sgRNA的DNA模板经体外转录得sgRNA粗品。
在NCBI(National Center for Biotechnology Information,美国国立生物技术信息中心)中,仓鼠全基因组测序和拼接工作已经完成度非常高了,数据库已经建立,本实施例的基因修饰模型制备和鉴定工作基本依据该数据库完成。找到数据库中的LCAT基因信息(Gene ID:101823034)在其第二外显子中设计sgRNA的靶序列位置,所述靶序列的位置,参见图1和SEQ ID NO:1所示。
本实施例中,cas9 mRNA的制备中,采用的cas9DNA模板是含有人源化cas9 cDNA的PXT7质粒。转录前,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cas9mRNA的DNA模板。
所得cas9 mRNA的DNA模板经体外转录得cas9 mRNA。其中,体外转录所用试剂盒为mMESSAGE mMACHINE T7 kit(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RNA酶水溶解,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。
SEQ ID NO:2和SEQ ID NO:3所示引物,互为模板,进行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。PCR产物经过琼脂糖凝胶电泳,用胶回收试剂盒,比如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。进一步,酚-氯仿抽提,乙醇沉淀,得sgRNA的DNA模板。
所得sgRNA的DNA模板用体外转录试剂盒,比如Megascript T7 Kit(Ambion)试剂盒,进行转录。转录体系于37℃充分反应4小时。反应所得sgRNAmRNA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。
采用本实施例的系统可用于构建LCAT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的系统,构建LCAT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6:00-20:00光照,20:00-6:00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、按照实施例1的系统,设计所述仓鼠LCAT基因特异性靶序列、制备所述cas9mRNA和sgRNA。
步骤二、收集和培养仓鼠受精卵
选取8-12周龄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,腹腔注射孕马血清促性腺激素(PMSG)浓度为125U/kg。下一动情周期第一天晚上19点与雄鼠合笼过夜进行交配。合笼后第二天下午14点雌鼠腹腔注射过量的戊巴比妥钠处死。将其输卵管剪下,置于37℃预热的M2培养基中(Sigma-Aldrich,St.Louis,MO,USA)。在解剖显微镜下,将输卵管膨大的壶腹部撕破,含有受精卵的堆积物流出,收集受精卵。
仓鼠受精卵的体外培养基是HECM-10(NaCl 113.8mM,KCl 3mM,NaHCO3 25mM,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(aspartate)0.01mM,半胱氨酸(cysteine)0.01mM,牛磺酸(taurine)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:将步骤一中所述sgRNA和cas9 mRNA共同注射至仓鼠受精卵的细胞质中。
制备注射液滴,注射皿中,加入一滴100μl M2培养基,矿物油液封。所有的受精卵离开孵箱时间少于15分钟。sgRNA和cas9 mRNA共同注射至细胞质中,其注射浓度为10ng/μl和20ng/μl。注射后受精卵在孵箱中培养1-2小时后,用于回输代孕母鼠体内。
步骤四、将显微注射后的受精卵植入代孕仓鼠体内,F1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,将注射过的受精卵从输卵管伞口植入代孕鼠体内。每侧输卵管植入15个受精卵。
出生仓鼠一周龄后,取其皮肤组织、脚趾组织或血细胞提取的基因组DNA,PCR扩增目的片段。引物:上游5’-CAGACGCCAGTCATAGGGTG-3’(SEQ ID NO:4),下游5’-CCAGAACAAAAGCTGGTGCC-3’(SEQ ID NO:5),PCR后电泳检测得到特异性条带后送测序,分序列及色谱峰图,确定突变位置。发生突变的仓鼠,进一步序列验证,将PCR产物链接至T载体上,转化至感受态细胞,接种至加有氨苄西林的LB培养基平板上,37℃孵育12h,随机挑取20个单克隆菌落,接种至LB液体培养基中,37℃摇床孵育12h后菌液送测序,分析序列及色谱峰图,确定碱基突变数目及位置以及可能产生的突变类型。由于cas9内切酶在特异性位点酶切后,受损DNA由末端直接连接或同源重组进行修复,因此产生体内有多种不同基因型的LCAT基因突变嵌合体仓鼠,将founder和野生型杂交后,F1代多个突变位点出现分离,后代(F1代)提取尾巴全基因组DNA进行测序,共获得A(△12bp)、B(△6bp)、C(△56bp)分别缺失12、6、56个碱基的三个类型的基因突变的品系(如图2所示)。都通过上述方法鉴定并繁殖纯合子和杂合子,鉴定的琼脂糖凝胶电泳结果(如图3所示)。经过表型分析确定留一到两个品系繁殖并用于实验。
游离胆固醇检测试剂盒(普利莱生物技术有限公司)检测血浆中游离胆固醇含量。以表型最明显的△56bp品系纯合子仓鼠继续进行表型分析,检测血浆发现在HDL-C(高密度脂蛋白胆固醇)(图5A)显著下降的情况下,HDL/TC比值(图5B)也显示血浆总胆固醇中几乎没有HDL组分。在总胆固醇没有显著变化下,血浆游离胆固醇(free cholesterol)(图5C)显著升高,因此胆固醇酯肯定显著降低。***表示t-test统计学检验p<0.001。
血浆脂蛋白中脂质组分检测,采用快速蛋白液相色谱(fast protein liquidchromatography,FPLC)仪,superose 6HR10/30色谱柱首先分离血浆脂蛋白组分。血浆200μl通过0.22μm针筒式过滤器后上样,以每分钟0.5mL的速度自动收集分离组分,每个组分500μl,共40个组分。将收集的40管组分分别测定胆固醇(TC)和甘油三酯(TG)浓度,绘制成脂蛋白谱图,参见图6A和图6B,图6A显示的各个组分的峰分别为极低密度脂蛋白(VLDL)、低密度脂蛋白(LDL)、高密度脂蛋白(HDL),图6B中的各个组分峰和A中位置相同。结果显示LCAT敲除仓鼠VLDL组分显著升高,HDL组分显著降低。
仓鼠血浆载脂蛋白ApoB100、ApoE、ApoAI的western blot分析,图7A为血浆脂蛋白超速离心分离并脱脂后,SDS-PAGE电泳后用相应载脂蛋白抗体杂交后,化学发光法显示的蛋白条带,图7B为扫描定量后柱状图。结果显示LCAT敲除仓鼠(-/-)血浆ApoB100显著增多,ApoAI检测不到,ApoE没有显著变化。LCAT活性的测定采用SIGMA公司胆固醇酰基转移酶活性检测试剂盒(MAK107),结果显示基因敲除的纯合子(LCK)仓鼠的血浆中LCAT活性相对野生型(WT)仓鼠,显著降低。***表示t-test统计学检验p<0.001。
以上结果表明:采用本实施例的方法成功制备了LCAT基因敲除仓鼠模型。利用这个LCAT基因敲除仓鼠模型,给予12周的高脂饲料,LCAT突变仓鼠的主动脉动脉硬化病变明显较正常对照组仓鼠的增多,表明LCAT基因缺陷可以促进动脉粥样硬化病变,如图8所示:A为表示动脉硬化病变的代表图;B为.动脉硬化病变面积的定量检测。
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。
<110> 河北伊维沃生物科技有限公司
<120> 构建LCAT基因敲除仓鼠模型的系统和方法
<130> 2018.02.27
<160> 5
<170> PatentIn version 3.5
<210> 1
<211> 24
<212> DNA
<213> 靶序列
<400> 1
atctcaatat gtttctaccc ctgg 24
<210> 2
<211> 64
<212> DNA
<213> CRISPR-Forward
<400> 2
gaaattaata cgactcacta tagatctcaa tatgtttcta ccccgtttta gagctagaaa 60
tagc 64
<210> 3
<211> 80
<212> DNA
<213> sgRNA-Reverse
<400> 3
aaaagcaccg actcggtgcc actttttcaa gttgataacg gactagcctt attttaactt 60
gctatttcta gctctaaaac 80
<210> 4
<211> 20
<212> DNA
<213> 人工序列
<400> 4
cagacgccag tcatagggtg 20
<210> 5
<211> 20
<212> DNA
<213> 人工序列
<400> 5
ccagaacaaa agctggtgcc 20
Claims (9)
1.构建LCAT基因敲除仓鼠模型的系统,其特征在于,所述系统包括仓鼠LCAT基因特异性打靶序列和sgRNA:
所述仓鼠LCAT基因特异性打靶序列如SEQ ID NO:1所示;
所述sgRNA的制备:采用如SEQ ID NO:2和SEQ ID NO:3所示人工序列,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。
2.如权利要求1所述的构建LCAT基因敲除仓鼠模型的系统,其特征在于,所述系统还包括cas9mRNA,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cas9mRNA的DNA模板,所得cas9mRNA的DNA模板经体外转录得cas9mRNA粗品。
3.如权利要求2所述的构建LCAT基因敲除仓鼠模型的系统,其特征在于,所述cas9mRNA的制备中:
cas9mRNA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/或所得cas9mRNA粗品的纯化方法为:cas9mRNA粗品经酚-氯仿抽提、异丙醇沉淀、无RNA酶水溶解,得浓度为200-800ng/μl的cas9mRNA。
4.如权利要求2所述的构建LCAT基因敲除仓鼠模型的系统,其特征在于,cas9mRNA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。
5.如权利要求1所述的构建LCAT基因敲除仓鼠模型的系统,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1所述的构建LCAT基因敲除仓鼠模型的系统,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。
7.如权利要求1所述的构建LCAT基因敲除仓鼠模型的系统,其特征在于,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。
8.采用权利要求1-7任一项所述的系统构建LCAT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LCAT受体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cas9mRNA,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、显微注射:将步骤一中所制得的sgRNA和cas9mRNA注射至仓鼠受精卵的细胞质中;
步骤四、将显微注射后的受精卵植入代孕仓鼠体内,F1代仓鼠出生,鉴定。
9.如权利要求8所述的构建LCAT基因敲除仓鼠模型的方法,其特征在于:步骤三中,所述sgRNA和cas9mRNA的注射浓度分别为10ng/μl和20ng/μl。
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