CN113969264B - Cog5基因敲除的人胚胎干细胞系、构建方法及应用 - Google Patents
Cog5基因敲除的人胚胎干细胞系、构建方法及应用 Download PDFInfo
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Abstract
本发明公开了COG5基因敲除的人胚胎干细胞系、构建方法及应用，通过CRISPR/Cas9基因编辑技术在人胚胎干细胞系chHES‑90中敲除COG5，并将其诱导为中脑神经元，为该基因病的研究提供了便利、可靠的实验模型；同时利用hESC的多向分化能力，也为研究COG5缺陷造成的多系统病变提供平台。本发明通过CRISPR/Cas9基因编辑技术构建了COG5缺陷的人胚胎干细胞系，并将其诱导为中脑神经元作为细胞模型来进行研究，为该基因病提供了便利、可靠的实验模型。
Description
技术领域
本发明涉及一种人胚胎干细胞系的构建，具体涉及COG5基因敲除的人胚胎干细胞系、构建方法及应用。属于基因工程技术领域。
背景技术
保守寡聚高尔基体(conserved oligomeric golgi，COG)复合物是高尔基体逆行运输的核心，负责将完成上一次功能的糖基转移酶重新定位到高尔基体扁平囊中。COG复合物由八个不同的亚基组成，COG1-4组成叶A，COG5-8组成叶B。叶A的缺失对细胞生存构成威胁，而叶B的障碍通常造成糖基化过程的异常。COG5和COG7相互作用的缺陷，不会扰乱叶B的稳定性，但对小泡转运和扁平囊中糖基的后期加工产生影响。
COG5缺陷的糖基化病，是一种常染色体隐性遗传病，基因突变致使蛋白的完全缺失导致疾病的发生。2009年Paesold-Burda报道了第一例COG5基因突变的病例。至今，共 7篇文章进行了报道。病人的临床症状主要包括发育迟缓、智力障碍、大/小脑萎缩、小头畸形、肌张力低下等。目前国际上暂无针对该基因病患者的有效治疗手段。由于人的神经细胞体外几乎无法获取，也不清楚糖基化异常对神经细胞造成了什么改变。利用COG5突变的动物模型或者细胞模型进行研究，是可能的研究手段。目前，国内外未查到COG5突变小鼠模型的报道，并且介于脑发育以及神经系统在小鼠和人类中的差异，小鼠脑细胞毕竟不能真实反映人脑细胞的情况，利用小鼠模型进行研究存在局限性。
近年来出现了许多基因组编辑技术，包括锌指核酸酶(ZFNs)、转录激活物样效应核酸酶(TALENs)和RNA引导的CRISPR/Cas9核酸酶系统。前两种技术使用内切酶催化域的拴系策略来模块化DNA结合蛋白，从而在特定的基因组位点诱导靶向DNA双链断裂(DSB)。相比之下，Cas9是一种由小RNA引导的核酸酶，通过PAM序列与靶DNA配对，代表了一种更容易设计、高度特异、高效的系统，非常适合于各种细胞类型的基因编辑。
CRISPR/Cas9是一种基因编辑系统。由引导RNA(sgRNA)引导Cas9核酸内切酶识别外源遗传元件上的PAM(NGG)序列并在其上游几个碱基处进行切割，由此刺激目标基因组位点上的双链断裂(DSB)来促进基因组编辑。一旦被Cas9切割，目标位点通常经历两条主要的DNA损伤修复途径：非同源末端连接(NHEJ)或同源修复(HDR)途径。在没有修复模板的情况下，DSB通过NHEJ过程重新连接，这会以插入/缺失(INDEL)突变的形式留下印记。发生在编码外显子内的INDELs可能导致移码突变和蛋白翻译提前终止，从而介导基因敲除。相同的CPRSPR/Cas9质粒，成功转染胚胎干细胞的效率远低于肿瘤细胞，同时位点的剪切效率不高，成功基因缺陷的细胞少；药物筛选、单克隆培养等都增加了编辑细胞的生长难度，同时需考虑这些过程对干细胞干性，核型等的影响。
发明内容
本发明的目的是为克服上述现有技术的不足，针对COG缺陷病缺少研究模型，提供COG5基因敲除的人胚胎干细胞系、构建方法及应用。
为实现上述目的，本发明采用下述技术方案：
1、COG5基因敲除的人胚胎干细胞系的构建方法，具体步骤如下：
(1)先将SEQ ID NO.1和SEQ ID NO.2所示的两条sgRNA退火成单链，克隆到含有嘌呤霉素抗性和BsmBI内切酶的改良Lenti-CRISPR质粒中，得到含有sgRNA的载体 Lenti-CRISPR-COG5质粒；
(2)接着将质粒N75、质粒VSV-G和Lenti-CRISPR-COG5质粒共转染80％会合度293T细胞，离心取上清，再将上清感染chHES-90细胞，筛选，得到存活细胞；
(3)然后将存活细胞进行单克隆扩增，测序，保留COG5基因缺陷的细胞，即得COG5基因敲除的人胚胎干细胞系。
优选的，步骤(2)中，上清应先通过0.45μm过滤器过滤后，再进行细胞感染。
优选的，步骤(2)中，细胞感染的具体方法为：先将上清与chHES-90细胞作用24 小时，然后更换新鲜培养基，继续进行感染，直至总体作用时间为48小时。
优选的，步骤(2)中，利用3μg/mL嘌呤进行细胞筛选。
2、利用上述构建方法得到的COG5基因敲除的人胚胎干细胞系。
3、COG5基因敲除的人胚胎干细胞系在制备COG5缺陷病模型中的应用。
优选的，上述人胚胎干细胞系先形成拟胚体，再经历5天悬浮和35天的贴壁培养，形成中脑神经元，即可实现COG5缺陷病细胞模型的制备。
本发明的有益效果：
本发明通过CRISPR/Cas9基因编辑技术在人胚胎干细胞系chHES-90中敲除COG5，并将其诱导为神经元，为该基因病的研究提供了便利、可靠的实验模型；同时利用hESC 的多向分化能力，也为研究COG5缺陷造成的多系统病变提供平台。
本发明通过CRISPR/Cas9基因编辑技术构建了COG5缺陷的人胚胎干细胞系，并将其诱导为神经元作为细胞模型来进行研究，为该基因病提供了便利、可靠的实验模型。
本发明的主要优势在于，以往的研究通过小鼠等实验动物研究人神经系统疾病，具有物种间的差异，而本发明通过人胚胎干细胞诱导成为中脑神经元，提供了人源的神经细胞，跨越了物种障碍，实验结果更全面真实。
附图说明
图1是利用慢病毒载体介导COG5基因在人胚胎干细胞系中的敲除流程图。
图2是COG5缺陷的异常人胚胎干细胞DNA的Sanger测序图，结果显示一条链缺失4bp，另一条链缺失21bp。
图3是Western Blot检测缺陷型hESC的蛋白表达图。野生型作为正常对照，β-actin作为内参蛋白。缺陷型无COG5蛋白表达。
图4是Western Blot检测缺陷型hESC的LAMP2蛋白迁移图。野生型作为正常对照，β-actin作为内参蛋白。缺陷型的LAMP2蛋白在SDS-PAGE上发生迁移，暗示糖基化的缺陷。
图5是BFA处理前后野生型及缺陷型hESC的高尔基体情况图。BFA可导致高尔基体的崩塌并向内质网迁移，而COG5的缺失可使这一过程滞后。野生型及缺陷型在BFA(2ug/ml)环境下11分钟，后用Giantin标记高尔基体。野生型的Giantin荧光标记弥散，表明高尔基体正向内质网融合；而缺陷型的荧光标记未发生明显弥散。
图6是透射电镜检测野生型和缺陷型hESC的高尔基体形态图。
图7是免疫荧光检测敲缺陷型的多能性标记NANOG，TRA-1-81，OCT4，SOX2图。
图8是流式细胞术检测缺陷型的多能性标记图，其中，A为NANOG，B为OCT4。
图9是缺陷型的三胚层分化能力检测图。在无bFGF的完全培养基(DMEM，敲除血清替代物，非必需氨基酸，β巯基乙醇，L-谷氨酰胺)悬浮培养7天形成拟胚体，再贴璧14 天自由分化。后免疫荧光分别检测内(AFP)、中(α-SMA)、外(TUBB3)胚层标记。
图10是缺陷型的核型分析图。结果显示无染色体异常。
图11是缺陷型支原体检测结果图。使用Lonza的支原体检测试剂盒检测缺陷型是否存在支原体污染。B/A<1时，表示无支原体污染。结果显示无污染。
图12是STEMCELL公司提供的由人胚胎干细胞系向多巴胺能神经元分化流程图。
图13是缺陷型诱导40天的人多巴胺能神经元亮视野图。
图14是免疫荧光检测缺陷型诱导40天的人多巴胺能神经元成熟标记。MAP2，神经元标记；TH，多巴胺能神经元标记。
图15是野生型与缺陷型的N-糖基化位点差异分析统计图。
图16是缺陷型糖基化位点上下游氨基酸出现频率分布图。缺陷型的N-糖基化位点遵循N-X-S/T氨基酸序列。
图17是缺陷型相比野生型的N-糖基化下降蛋白质的功能聚类图。
图18是野生型与缺陷型的N-糖基化位点差异情况分布图。
图19是野生型与缺陷型的N-糖基化差异位点在KEGG通路的富集热图。缺陷型的N-糖基化位点显著下降的情况集中分布于亨廷顿病，海马体的长效抑制，多巴胺突触等。
具体实施方式
下面结合附图和实施例对本发明进行进一步的阐述，应该说明的是，下述说明仅是为了解释本发明，并不对其内容进行限定。
实施例：
1、COG5缺陷病的人胚胎干细胞模型的制备方法流程图如图1，具体步骤如下：
(1)由引导RNA(sgRNA)引导Cas9核酸内切酶识别外源遗传元件上的PAM(NGG) 序列并在其上游几个碱基处进行切割，由此刺激目标基因组位点上的双链断裂来促进 COG5基因组编辑。靶向的人COG5的PAM位点设计如下：
TACAGTCCGGGAACTTCTGC(+)AGG
(2)并针对适当的限制位点设计了sgRNA：
F5’-3’：CACCGTACAGTCCGGGAACTTCTGC，如SEQ ID NO.1所示；
R5’-3’：AAACGCAGAAGTTCCCGGACTGTAC，如SEQ ID NO.2所示；
(3)将两条sgRNA退火成双链，克隆到含有嘌呤霉素抗性和BsmBI内切酶的改良Lenti-CRISPR质粒中，形成Lenti-CRISPR-COG5质粒。
(4)使用lipofectamine 2000(INVITROGEN公司购买)将两种包装质粒(N75、VSV-G，帮助CRISPR质粒更好进入宿主的辅助质粒)和含有sgRNA的Lenti-CRISPR-COG5质粒共转染至80％会合度的293T细胞中。
(5)24小时后收集含有sgRNA的病毒颗粒上清液，并通过0.45μm过滤器过滤。
(6)慢病毒上清液与人胚胎干细胞(chHES-90)作用24h。24h后更换新鲜培养基mTeSRTM(STEMCELL公司购买)。
(7)感染48时后，用3μg/mL嘌呤霉素筛选细胞4天。
(8)存活细胞在Matrigel(CORNING)上进行单克隆扩增。
(9)进行Sanger测序(图2)，保留COG5基因缺陷(纯合突变，复合杂合突变)的细胞，将其中一个单克隆命名为chHES-90-Cog5-/--C2C2。Western Blot检测COG5蛋白含量(图3)，确定COG5的纯合突变导致其蛋白缺失。
(10)对挑拣出的人胚胎干细胞单克隆进行细胞特性鉴定。免疫荧光检测多能性标记 NANOG、SOX-2、OCT4、TRA-1-81(图7)及多向分化潜能标记AFP、α-SMA、TUBB3 (图9)；证明基因编辑后的细胞仍具备hESC的特性。流式细胞术检测多能性标记NANOG (图8中A)和OCT4(图8中B)，确定COG5基因缺陷的单克隆细胞仍然是高纯度的人胚胎干细胞。同时，对单克隆进行核型检测，确定其遗传稳定性(图10)。对单克隆进行有无支原体检测，证明其无常见微生物污染(图11)。
2、COG5缺陷病的人胚胎干细胞及其诱导的多巴胺能神经元细胞模型的表型结果如下：
(1)按照本实验室确定的诱导流程对COG5缺陷hESC向人中脑多巴胺能神经元进行诱导(图12)。COG5缺陷细胞在aggrewellTM(STEMCELL公司购买)容器中形成拟胚体，经历5天悬浮和35天的贴壁培养，形成中脑神经元，诱导过程依次使用STEMCELL公司的神经诱导试剂盒/中脑神经元分化试剂盒(+SHH)/中脑神经元成熟试剂盒。诱导后多巴胺能神经元形态如图13。通过免疫荧光检测诱导的成熟人多巴胺能神经元标记，可见MAP2 (成熟神经元微管蛋白的标记)和TH(成熟多巴胺能神经元神经递质标记)呈阳性(图 14)。
(2)人胚胎干细胞显示出COG5缺陷造成的细胞表型，如图4所示，COG5缺陷的人胚胎干细胞表现出LAMP2蛋白迁移的拖尾现象，与COG5基因突变病人的样本蛋白电泳表现出一致的现象；蛋白转运抑制剂BFA处理可导致高尔基体的崩塌并向内质网迁移，而 COG5的缺失可使这一过程滞后。正常hESC及COG5缺陷hESC在2ug/ml BFA处理11 分钟，再用Giantin标记高尔基体。如图5所示，BFA处理后正常hESC出现迁移现象，而 COG5缺陷的人胚胎干细胞的高尔基体未见发生迁移现象。COG5缺陷的hESC电镜结果显示出高尔基体的肿胀，空泡增多的现象，也与COG5基因突变病人的细胞电镜表现出一致的现象(图6)；以上结果说明敲除的hESC已初步具备cog5缺陷病人细胞相同的现象。
(3)为了证明hESC诱导的中脑多巴胺能神经元是研究COG5缺陷糖基化病引起的神经系统病变的良好细胞模型，申请人应用LC-MS/MS定量N-糖蛋白组学，比较了正常人胚胎干细胞和COG5缺陷人胚胎干细胞中蛋白质和糖蛋白的表达水平。文库搜索和质谱数据分析的结果给出了不同样品中每个肽的信号强度。基于此信息，计算蛋白质的相对数量。比较样品对中每个修饰位点的所有生物重复定量值的平均值的比率被视为差异倍数变化 (FC)。显著上调的变化阈值大于1.5，显著下调的变化阈值小于1/1.5。结果显示上调修饰位点426个，下调修饰位点223个。由于可能有多个肽对应于一种蛋白质，因此鉴定出289种上调蛋白质和155种下调蛋白质(图15)。
(4)大规模的修饰组学实验可以在一次实验中鉴定出数千个蛋白质翻译后修饰位点，了解引起这些修饰的潜在生物学过程是蛋白质组学的重要研究方面。由于酶对给定底物的部分生化偏好可能由修饰位点周围的残基决定，因此使用由所有已识别修饰位点的上游和下游10个氨基酸组成的肽序列作为分析对象。N-糖基化序列基序热图显示，最显著富集的基序是N-糖基化位点：丙氨酸(A)、半胱氨酸(C)、组氨酸(H)、甘氨酸(G)、丝氨酸(S)、苏氨酸(T)、缬氨酸(V)和酪氨酸Y。值得注意的是，丝氨酸与苏氨酸都频繁出现在第二位(图16)。
(5)由于COG5的缺陷主要可能导致糖基化酶回收和定位异常，也就意味着可能神经细胞相关的突触、神经递质、通道蛋白的糖基化程度降低，所以申请人的分析策略主要先关注在糖基化降低的糖蛋白分析上。检测结果也证明，如图17所示，糖基化程度降低的差异糖蛋白主要集中在受体活性、通道活性、突触和膜等方面(P<0.005)。
(6)此外，根据差异表达倍数将差异表达糖蛋白进一步分为四部分，称为Q1至Q4(图 18)。Q1和Q2为糖基化下调差异蛋白，Q3和Q4为上调差异蛋白。申请人对每个Q组进行GO分类、KEGG和蛋白质结构域富集，并进行聚类分析，以发现蛋白质功能与不同差异表达倍数的相关性。KEGG富集结果表明，N-糖基化修饰下调的神经递质与突触和细胞外基质的相互作用聚集在COG5缺陷的人胚胎干细胞衍生的多巴胺能神经元中，可能与精神和神经相关疾病有关(图19)。在那些通过糖基化修饰的显著不同的蛋白质中，AMPAR 和NMDAR在这些途径中最常下调。这一发现可能表明谷氨酰胺及其受体的切断可能是神经系统异常的一个重要原因。
(7)在COG5缺陷的多巴胺能神经元糖基化水平显著下降的蛋白中，申请人重点关注到α-L-岩藻糖苷酶(FUCA1)。岩藻糖沉积症是一种常染色体隐性溶酶体贮存病，由α-L-岩藻糖苷酶缺陷引起，并在组织中积累岩藻糖。临床特征包括血管角化瘤、进行性精神运动迟缓、神经症状、面部特征粗糙和多发性骨发育不良。这些症状与COG5缺陷病例的症状高度重合，提示COG5缺陷可能引起了FUCA1的糖基化异常而发生功能紊乱，使患者体内岩藻糖蓄积而患病。除此，申请人还发现几种酶、受体和神经递质的糖基化显著降低，可作为后续研究的靶标蛋白。
(8)综上所述，通过构基因编辑质粒对hESC进行COG5的敲除，获得了COG5纯合缺失的hESC。再向神经元诱导，可获得遗传背景完全一致的正常与缺陷细胞样本对，克服了病人神经细胞不可取的不足，也弥补了老鼠在脑发育和神经元功能上与人不同的不足，建立了COG缺陷病研究的细胞模型，提供了COG5基因敲除的人胚胎干细胞系、构建方法及应用的实例。
上述虽然结合附图对本发明的具体实施方式进行了描述，但并非对本发明保护范围的限制，在本发明的技术方案的基础上，本领域技术人员不需要付出创造性劳动即可做出的各种修改或变形仍在本发明的保护范围以内。
序列表
<110> 中南大学
<120> COG5基因敲除的人胚胎干细胞系、构建方法及应用
<130> 2021
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 1
caccgtacag tccgggaact tctgc 25
<210> 2
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 2
aaacgcagaa gttcccggac tgtac 25
Claims (3)
1. COG5基因敲除的人胚胎干细胞系，是通过以下方法构建得到的：
（1）先将SEQIDNO.1和SEQIDNO.2所示的两条sgRNA退火成单链，克隆到含有嘌呤霉素抗性和BsmBI内切酶的改良Lenti-CRISPR 质粒中，得到含有sgRNA的载体Lenti-CRISPR-COG5质粒；
（2）接着将质粒N75、质粒VSV-G和Lenti-CR ISPR-COG5质粒共转染80%会合度293T细胞，离心取上清，再将上清感染chHES-90细胞，筛选，得到存活细胞；
（3）然后将存活细胞进行单克隆扩增，测序，保留COG5基因纯合突变的细胞，即得COG5基因敲除的人胚胎干细胞系。
2.权利要求1所述COG5基因敲除的人胚胎干细胞系在制备COG缺陷病模型中的应用。
3.根据权利要求2所述的应用，其特征在于，权利要求1所述人胚胎干细胞系先形成拟胚体，再经历5天悬浮和35天的贴壁培养，形成中脑神经元，即可实现COG5缺陷病模型的制备。
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