CN116171942A - 一种six1基因干扰构建涡虫视神经系统缺失模型的方法 - Google Patents
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Abstract
本发明涉及基因干扰技术领域,具体公开了一种SIX1基因干扰构建涡虫视神经系统缺失模型的方法,包括以下步骤:步骤一:扩增SIX1基因,作为dsRNA体外合成的模板;步骤二:dsRNA体外合成与纯化;步骤三:干扰涡虫,完成涡虫视神经系统(眼点)缺失模型构建。本发明构建的涡虫视神经系统(眼点)缺失模型为筛选独立于脑部缺陷的视神经系统(眼点)调控基因的研究奠定基础。
Description
技术领域
本发明涉及基因干扰技术领域,尤其是涉及一种SIX1基因干扰构建涡虫视神经系统缺失模型的方法。
背景技术
日本三角涡虫(Dugesia japonica),隶属扁形动物门,涡虫纲,三肠目,一般生活在温度较低、水流缓慢的溪流中。日本三角涡虫身体似扁平叶状,呈淡褐色,背面着色较深,头部呈三角形,故因此而得名。涡虫具有脑、眼、神经、肌肉、肠、肾、卵巢、睾丸等器官,是三胚层生物中唯一能完全再生的模式生物。涡虫作为一种简单的后生动物,又兼具体型小、饲养条件简单、实验操作简便等特点,已经成为研究再生的理想生物模型。
涡虫是具有明确的双边对称性、背腹极性、中枢神经系统(CNS)和简单的大脑结构的早期生物群。涡虫中枢神经系统位于身体的腹侧,由三部分结构组成:前脑、光感受器(眼点)和腹神经索。涡虫眼点位于头部神经节的背侧,由两种细胞类型组成:杯状色素细胞和光敏神经元组成。尽管涡虫眼点结构比人眼简单得多,但仍有许多相似之处,涡虫的眼点表达许多与脊椎动物感光细胞和视网膜色素上皮细胞同源的基因,因此可用涡虫作为研究眼睛再生的模型,为人类视觉功能恢复提供新的思路。
视神经系统是中枢神经系统的组成部分,其内在或自发再生能力受其他CNS组织再生影响因素的限制,因此独立于其他CNS组织损伤研究视神经系统再生机制是十分必要的。然而现有研究一般是将涡虫头尾切割后靶向目的基因进行RNA干扰,观察涡虫脑部及眼点再生情况,但脑畸形会导致涡虫眼点出现继发性缺陷,不能说明目的基因对涡虫眼点发育具有直接的调控作用。因此,通过SIX1基因干扰构建涡虫视神经系统(眼点)缺失模型具有较好的适用性,为筛选独立于脑部缺陷的视神经系统(眼点)调控基因的研究奠定基础。
发明内容
本发明要解决的技术问题是克服现有的缺陷,提供SIX1基因干扰构建涡虫视神经系统(眼点)缺失模型的方法及应用,以解决上述背景技术中提出的眼点缺陷可能继发于脑畸形的问题。
为实现上述目的,本发明提供如下技术方案:SIX1基因干扰构建涡虫视神经系统(眼点)缺失模型的方法及应用,包括以下步骤:
步骤一:扩增SIX1基因,作为dsRNA体外合成的模板。从现存的涡虫转录组测序结果中获得SIX1基因的cDNA序列。利用Primer Primer5软件进行引物设计,随后,配置SIX1扩增体系,均匀混合后放入PCR仪中,程序结束后,将PCR扩增产物进行DNA凝胶电泳点样并胶回收纯化PCR产物;将纯化产物与pMD 19-T载体4℃连接过夜,然后转化感受态DH5α细胞;37℃培养箱培养10-12h,挑取10个单克隆菌落摇菌和菌落PCR鉴定。将PCR扩增产物进行琼脂糖凝胶电泳,选取PCR产物大小和目的基因大小相对应的样品,送生物公司测序,剩余菌液保存于4℃;待测序结果返回后,使用SnapGene软件进行测序结果比对,将测序结果正确的菌液扩大培养。需要扩大培养的菌液接种到30mL氨苄抗性LB培养基中,放入恒温震荡培养箱,37℃过夜培养;待菌液浑浊时,取700μL菌液保种,剩余菌液用于提取质粒。
步骤二:dsRNA体外合成与纯化。从质粒上扩增带有T7启动子序列的目的基因,PCR程序结束后,胶回收PCR产物;配置dsRNA体外合成体系于37℃金属浴过夜孵育,第二天利用乙酸铵纯化dsRNA。
步骤三:干扰涡虫,完成涡虫视神经系统(眼点)缺失模型构建。选取饥饿7天、体长相近的涡虫进行头尾切割,浸泡在含有一定浓度dsRNA的水中,每天换水,第7天检测干扰效果及涡虫眼点发育情况。
进一步地,步骤一中,所述SIX1基因的引物序列前加上T7启动子序列:TAATACGACTCACTATAGG。
进一步地,步骤二中,胶回收PCR扩增产物时,用DEPC水洗脱,作为SIX1-dsRNA体外合成的模板。
进一步地,体外合成dsRNA,为确保双链RNA正确配对,需进行变性与退火:95℃变性3min,75℃孵育3min,50℃孵育3min,室温静置5min即可。
进一步地,步骤三中,从实验室已有的pEGFP-N1质粒上扩增涡虫中不表达的EGFP基因,作为对照检测SIX1-dsRNA处理后的干扰效果及涡虫眼点发育情况。
进一步地,检测干扰效果及涡虫眼点发育情况的具体方法为:分别提取实验组和对照组的RNA,并进行反转录,以此cDNA为模板进行RT-qPCR分析SIX1基因表达变化;体式显微镜观察涡虫眼点发育情况。
与现有技术相比,本发明提供了一种SIX1基因干扰构建涡虫视神经系统(眼点)缺失模型模型的方法及应用,具备以下有益效果:
1、本发明使用涡虫进行实验,涡虫与人类基因同源性高达80%。此外,涡虫具有强大的组织再生能力,作为一种简单的后生动物,是研究再生的理想生物模型。因此,涡虫作为模式生物的优势很突出;
2、本发明提供了一种涡虫视神经系统(眼点)缺失模型的构建方法,构建方法简单易行、稳定可重复,能够为筛选独立于脑畸形的眼睛发育基因提供优良的动物模型。
附图说明
图1为构建涡虫视神经系统(眼点)缺失模型的技术路线图;
图2为体外合成dsRNA凝胶电泳图;
图3为dsRNA干扰后SIX1基因表达变化;
图4为dsRNA干扰后涡虫产生的形态学变化。
具体实施方式
为更好的说明本发明的目的、技术方案和优点,下面结合附图和具体实例对本发明作进一步说明。本发明的技术路线见图1。
以下实施例中所使用的实验方法如无特殊说明,均为常规方法。
以下实施例中所用的材料、试剂等,如无特殊说明,均为商业途径获得。
实施例1、涡虫SIX1基因克隆
引物设计:选取饥饿7天以上并未做其他处理涡虫的cDNA作为基因扩增模板。根据实验室所存日本三角涡虫转录组数据获得目的基因cDNA序列,利用软件设计引物并将T7启动子序列添加到设计好的引物序列前,引物序列如表1所示。
表1本发明中所用SIX1基因扩增引物
PCR、琼脂糖凝胶电泳与胶回收:
50μL体系如下:
2×Rapid Taq Master Mix 5.0μL
正向引物(10μM) 2.0μL
反向引物(10μM) 2.0μL
cDNA 5.0μL
ddH2O 16.0μL
加样完成后,吹打混匀液体,放入PCR仪中,设置程序为95℃,3min;
95℃,15s;54℃,15s;72℃,15s;72℃,5min;4℃,∞;
将PCR扩增产物预留5μL,剩余溶液全部用于琼脂糖凝胶电泳,设置电泳条件为140V,15min。电泳结束后,根据DNA maker的标识,选择与预期DNA大小片段一致的单一条带,将其切下放入1.5mL EP管中,做好标记,随后按照DNA凝胶回收试剂盒说明书进行胶回收。
构建含有SIX1基因T载体:
10μL连接体系如下:
基因片段 0.5μL
载体 1.5μL
SolutionⅠ 3.0μL
ddH2O 5.0μL
加样完成后,吹打混匀,2000g离心将溶液离心至PCR管底部,4℃接过夜。
转化:取出超低温保存的DH5α感受态细胞,待其融化后取30μl于1.5mL EP管中,加入10μL上述连接产物,冰上静置30min;将转化体系放入42℃水浴锅中热激50s,50s后平稳的将转化体系取出置于冰上静置2min;向转化体系中加入200μL无抗LB液体培养基,放入恒温摇床中,37℃培养50min;取出菌液,用涂布棒将菌液均匀涂布于氨苄抗性的平板上,正置平板于37℃恒温培养箱中,待菌液干后,将平板倒置过夜培养。
菌落PCR检测阳性克隆:待平板上菌落长至合适大小时,分别挑取10个单菌落于500μL氨苄抗性培养基中混匀;放入恒温培养震荡箱中,37℃、200r/min,培养约3~5h(视菌液浑浊情况而定),待菌液稍浑浊时,取1μL菌液进行菌液PCR鉴定,该加样体系扩增模板为菌液DNA,其余反应体系、程序同SIX1基因扩增体系。PCR扩增反应结束后,取全部PCR反应溶液点样,140V电泳15min;根据电泳结果选取正确PCR条带所对应的样品,送生物公司测序,剩余菌液于4℃保存。
质粒提取:待测序结果返回后,使用SnapGene软件进行结果比对,将测序结果正确的菌液扩大培养。将需要扩大培养的菌液接种到30mL氨苄抗性LB培养基中,放入恒温震荡培养箱,37℃、200r/min,过夜培养;待菌液浑浊时,取700μL用于保种(1.5mL Ep管中加入700μL菌液与300μL甘油),剩余菌液用于提取质粒;菌液7500g离心5min;弃上清,可将50mL离心管倒置于吸水纸上弃去多余菌液;加入1mL P1,重悬细菌;向离心管中加入1mL P2温和翻转离心管,裂解菌体使其变得清亮粘稠;按照1:1.4比例加入P3,温和翻转离心管,出现白色絮状物;12000g离心10min。将上清转移到新的离心管内,加入0.3倍上清体积的异丙醇,混匀后将不超过700μL的溶液转移至吸预先活化好的附柱,12000g离心2min,多次过柱;加入500μL PD,12000g离心1min,弃废液;加入500μL PW,静置2min,12000g离心1min,,该步骤进行两次;室温晾干5min以便去除乙醇;然后将吸附柱置于干净1.5mL EP管中向滤膜中部加入双蒸水洗脱质粒,静置2min,12000g离心2min;该步骤进行两次,最后得到提取的质粒。
实施例二、dsRNA合成与纯化
dsRNA模板扩增:对照组以涡虫中不表达的EGFP基因作为对照,EGFP基因从实验室已有的pEGFP-N1质粒上进行扩增;实验组可直接利用上述构建的SIX1-T载质粒作为SIX1基因扩增模板。目的基因扩增反应结束后,进行琼脂糖凝胶电泳并对扩增产物进行胶回收,需要注意的是,PCR产物用20μl DEPC处理水洗脱并测定浓度。
表2本发明中dsRNA模板扩增引物
dsRNA体外合成:为防止RNase对实验的干扰,在进行dsRNA体外合成之前需要提前准备好冰盒与RNase-free离心管,提前打开37℃金属浴,用75%酒精喷洒实验台面,所用枪头需用DEPC水处理并灭菌,加样应尽可能迅速。
50μldsRNA体外扩增体系:
5×Transcription Buffer 10μL
rNTP(10mM) 10μL
模板(1ug) XμL
RNase inhibitor 1.25μL
T7 RNA聚合酶 1.5μL
DTT 5μL
DEPC处理水 补足50μL
加样完成后,用手弹匀并瞬时离心至EP管底,置于37℃金属浴反应1h,补加1.25μLT7 RNA聚合酶,37℃金属浴反应过夜。反应完成后每管各加2μL RNase-free DNase I,37℃反应30min,以消化模板。
dsRNA纯化:dsRNA利用乙酸铵进行纯化,具体步骤如下:在离心管中加入等体积DEPC处理水,充分混匀;加入等体积5mM的乙酸铵,充分混匀;加入2倍体积无水乙醇,充分混匀后放入冰盒中,-20℃静置1h。18000g、4℃离心15min,离心后可见白色沉淀即为dsRNA;避开白色沉淀,弃尽上清;加入500μL预冷的70%乙醇洗涤,再次18000g、4℃离心15min;弃上清于室温下晾干;根据白色沉淀的量加入50-100μL DEPC处理水溶解白色沉淀,并瞬时离心至管底。
为确保双链RNA正确配对,进行变性与退火:95℃变性3min后75℃孵育3min,然后于室温静置5min即可。
取3μL进行琼脂糖凝胶电泳检测dsRNA的合成质量并测定浓度,电泳结果见图1。剩余部分则存放于-80℃或随即干扰涡虫。
实施例三、涡虫视神经系统(眼点)缺失模型的构建
干扰涡虫:取饥饿一周、体型大小相近的涡虫数只,将涡虫头尾切割后随机分为两组并做好标记,放入干净的24孔板内,每个孔2只涡虫。对照组加入20ng/μL EGFP dsRNA,实验组加入20ng/μL SIX1 dsRNA。再生过程中,检测干扰效果及眼点发育情况。
干扰效果检测:RNA提取。每组样本各3只涡虫,吸到1.5ml RNase-free离心管中,做好标记,尽量除去多余水分;每管加入500μL TRIzol,用枪头使劲吹打,使涡虫样品充分裂解;每管加入100μL三氯甲烷,晃动20s混匀,冰盒内静置3min;12000g、4℃离心15min;慢慢吸取含有总RNA的上层无色水相至一新的标记好的RNase-free离心管中并记住所吸液体体积,注意不要吸到中间的蛋白与下层的有机相;加入与RNA水相等体积的异丙醇,上下轻轻颠倒混匀,冰上静置10min;12000g、4℃离心15min,此时可以看到RNA沉淀;小心的弃去上清,每管加入500μL 75%乙醇,吹打混匀;7500g、4℃离心15min;小心的弃去上清,室温下晾干;根据RNA沉淀加入适量的DEPC处理水(20-50μL)溶解RNA,取1μL检测RNA浓度,并进行琼脂糖凝胶电泳检测RNA提取质量。
反转录。对提取的RNA进行反转录反应,反应分为基因组去除和反转录两部分,按照反转录试剂盒说明书于冰上配置混合液,于PCR仪进行相应反应程序。反应结束后,将获得的cDNA于-20℃或-80℃进行保存,或随即进行下一步实验。
RT-qPCR。以上述反转录合成的cDNA为模板,进行RT-qPCR检测SIX1基因相对表达量。RT-qPCR反应体系按照试剂盒说明书进行加样,引物序列如表3所示。完成加样后将溶液混匀,离心至管底。设置反应程序为95℃、5min;95℃、10s;60℃、30s;39个循环,65℃、5s;95℃、0.5s。反应结束后,按2-ΔΔCt计算基因相对差异表达量,实验结果见图2。
表3本发明中RT-qPCR引物
眼点发育情况统计:选取3个不同时间点(2d、4d、7d)对涡虫眼点发育情况进行拍照统计。拍照时,将涡虫置于冰上,待涡虫完全伸展躯体时进行拍照记录,涡虫眼点发育情况见图3。
综上所述,在涡虫再生过程中,与野生型涡虫(SIX1+)相比,SIX1-涡虫没有发育出眼点,并且未表现出大脑发育异常或任何其他形态缺陷,即涡虫视神经系统(眼点)缺失模型构建成功,该涡虫视神经系统(眼点)缺失模型可用于评估目的基因在其他解剖结构正常的情况下是否可以调节视神经系统(眼点)再生。
最后应当说明的是,上述实施例仅为本发明的具体实施方式,但本发明不受上述实施例的限制,在不脱离本发明原理前提下,做出的改进和润饰,都涵盖在本发明的保护范围之内。因此,本发明的保护范围应该以权利要求书所限定的保护范围为准。
Claims (6)
1.一种SIX1基因干扰构建涡虫视神经系统缺失模型的方法,其特征在于,包括以下步骤:
步骤一:扩增SIX1基因,作为dsRNA体外合成的模板。从现存的涡虫转录组测序结果中获得SIX1基因的cDNA序列。利用Primer Primer5软件进行引物设计,随后,配置SIX1扩增体系,均匀混合后放入PCR仪中,程序结束后,将PCR扩增产物进行DNA凝胶电泳点样并胶回收以纯化PCR产物;将纯化产物与pMD 19-T载体4℃连接过夜,然后转化感受态DH5α细胞;37℃培养箱培养10-12h,挑取10个单克隆菌落摇菌和菌落PCR鉴定。将PCR扩增产物进行琼脂糖凝胶电泳,选取PCR产物大小和目的基因大小相对应的样品,送生物公司测序,剩余菌液保存于4℃;待测序结果返回后,使用SnapGene软件进行测序结果比对,将测序结果正确的菌液扩大培养。需要扩大培养的菌液接种到30mL氨苄抗性LB培养基中,放入恒温震荡培养箱,37℃过夜培养;待菌液浑浊时,取700μL菌液保种,剩余菌液用于提取质粒。
步骤二:dsRNA体外合成与纯化。从质粒上扩增带有T7启动子序列的目的基因,PCR程序结束后,胶回收PCR产物;配置dsRNA体外合成体系于37℃金属浴过夜孵育,第二天利用乙酸铵纯化dsRNA。
步骤三:干扰涡虫,完成涡虫视神经系统(眼点)缺失模型构建。选取饥饿7天、体长相近的涡虫进行头尾切割,浸泡在含有一定浓度dsRNA的水中,每天换水,第7天检测干扰效果及涡虫眼点发育情况。
2.根据权利要求1所述的SIX1基因干扰构建涡虫视神经系统缺失模型的方法,其特征在于步骤一中,所述SIX1基因的引物序列前加上T7启动子序列:TAATACGACTCACTATAGG。
3.根据权利要求1所述的SIX1基因干扰构建涡虫视神经系统缺失模型的方法,其特征在于步骤二中,胶回收PCR扩增产物时,用DEPC水洗脱,作为SIX1-dsRNA体外合成的模板。
4.根据权利要求3所述的SIX1基因干扰构建涡虫视神经系统缺失模型的方法,其特征在于体外合成dsRNA,为确保双链RNA正确配对,需要进行变性与退火:95℃变性3min,75℃孵育3min,50℃孵育3min,室温静置5min即可。
5.根据权利要求1所述的SIX1基因干扰构建涡虫视神经系统缺失模型的方法,其特征在于步骤三中,从实验室已有的pEGFP-N1质粒上扩增涡虫不表达的EGFP基因,作为对照检测SIX1-dsRNA处理后的干扰效果及涡虫眼点发育情况。
6.根据权利要求5所述的SIX1基因干扰构建涡虫视神经系统缺失模型的方法,其特征在于检测干扰效果及涡虫眼点发育情况的具体方法为:分别提取实验组和对照组的RNA,并进行反转录,以此cDNA为模板进行RT-qPCR分析SIX1基因表达变化;体式显微镜观察涡虫眼点发育情况。
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