CN106811483B - 一种高效转染真核细胞的方法 - Google Patents
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Abstract
本发明公开了一种高效转染真核细胞的方法，该方法包括以下步骤：（1）受体细胞的培养；（2）转染液的制备：A液：用培养基MEM稀释1~10μg浓度为10g/L的供体DNA，定量至100μL；B液：将培养基MEM定量至100μL，然后吸取2~15μg的LR加入到MEM中，室温静置15分钟；A/B复合物：将A液滴加入B液，轻轻弹下离心管壁，室温静置20min；（3）转染：将配置好的Ca2+载体工作液加入培养好的细胞培养液中，摇匀，将所述A/B复合物缓缓加入步骤（1）所述的细胞培养液中，摇匀，37℃温箱置6～24小时，观察转染效率。本发明快速的将质粒转入真核细胞内，减少脂质体对细胞的毒性作用时间，更高效的将目的基因整合入真核细胞基因组中，减少了后期药物筛选作用时间。
Description
技术领域
本发明属于生物技术领域，尤其涉及一种高效转染真核细胞的方法。
背景技术
细胞转染的方法主要包括：电穿孔法、显微注射、基因枪、磷酸钙共沉淀法、脂质体转染法、多种阳离子物质介导、病毒介导的转染等，理想的细胞转染方法是具有高转染效率、对细胞的毒性作用小等。
脂质体（lipofectin regeant，LR）试剂是阳离子脂质体N-[1-2，3-Dioleyoxy，Propyl]-n，n，n-Trimethylammonium Chloride（DOTMA）和Dioleoyl photidye-thanolamine（DOPE）的混合物[1：1（w/w）]。它适用于把DNA转染入悬浮或贴壁培养细胞中，是目前条件下最方便的转染方法之一。
用LR进行转染时，首先需优化转染条件，应找出该批LR对转染某一特定细胞适合的用量、作用时间等，对每批LR而言，均先要固定一个DNA的量和DNA/LR混合物与细胞相互作用的时间，DNA可从1~5μg和孵育时间6小时开始，按这两个参数绘出相应LR需用量的曲线，再选用LR和DNA两者最佳的剂量，确定出转染时间（2~24小时）。
因LR对细胞有一定的毒性，转染时间以不超过24小时为宜。细胞种类：COS-7、BHK、NIH3T3、Hela和Jurkat等任何一种细胞均可作为受体细胞。阳离子脂质体对细胞的毒性相对较高，为了防止其毒性，需要对脂质体与质粒的比例、细胞转染时间等要素准确掌握。
发明内容
本发明的目的在于提供一种高效转染真核细胞的方法。
本发明是这样实现的，一种高效转染真核细胞的方法，该方法包括以下步骤：
（1）受体细胞的培养
在转染前一天接种待转染细胞，接种密度为2×105/cm2，用含10%胎牛血清的DMEM液，37℃、5%CO2培养，待细胞占50～70%瓶底面积时，用于转染试验；
（2）转染液的制备
A液：用培养基MEM稀释1~10μg 浓度为10g/L的供体DNA，定量至100μL；
B液：将培养基MEM定量至100μL，然后吸取2~15μg的LR加入到MEM中，室温静置15分钟；
A/B复合物：将A液滴加入B液，轻轻弹下离心管壁，室温静置20min；
（3）转染
将配置好的Ca2+载体工作液加入培养好的细胞培养液中，摇匀，将所述A/B复合物缓缓加入步骤（1）所述的细胞培养液中，摇匀，37℃温箱置6～24小时，观察转染效率；其中，所述Ca2+载体工作液配置为：MEM、15mmol/L Hepes、0.168mg/ml NaHCO3 以及5μmol/LA23187。
优选地，在步骤（3）中，所述Ca2+载体工作液的配置包括以下步骤：取1mg A23187在室温情况下，用1.9ml DMSO或DMSO/乙醇混合液溶解为1mmol/L A23187浓缩液；将所述A23187浓缩液、MEM、Hepes、NaHCO3比例混合，得到所述Ca2+载体工作液。
优选地，在步骤（2）中，所述供体DNA利用去内毒素质粒大提试剂盒提取得到。
本发明克服现有技术的不足，提供一种高效转染真核细胞的方法。在本发明中，A23187（ IA）载体是一种移动性离子载体，但它的功能是运输钙离子、镁离子等二价阳离子。钙离子载体A23187通过Ca2+的跨膜转运，能迅速诱导细胞内Ca2+浓度升高，利于细胞吞入摄取，或通过细胞膜脂相收缩时裂开的空隙进入细胞内，Ca2+能促进膜对DNA-脂质体的吸收，Haberland的体外转染实验表明，Ca2+之所以能促进转染是由于形成了磷酸钙微小沉淀，能促进DNA-复合体的运输和从包涵体膜内的释放。
相比于现有技术的缺点和不足，本发明具有以下有益效果：本发明快速的将质粒转入真核细胞内，减少脂质体对细胞的毒性作用时间，更高效的将目的基因整合入真核细胞基因组中，减少了后期药物筛选作用时间。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。
实施例
（1）Ca2+载体工作液的制备
取1mg IA（A23187）在室温情况下，用1.9ml DMSO 或DMSO/乙醇混合液溶解为1mmol/L A23187浓缩液。应用时，用去离子水或细胞培养液（无血清MEM）稀释后使用。
室温下，配置Ca2+载体工作液（A23187工作液），MEM+ 15mmol/L Hepes + 0.168mg/ml NaHCO3 + 5μmol/L A23187；
（2）供体DNA制备
利用去内毒素质粒大提试剂盒提取高浓度质粒（10g/L）。
（3）受体细胞的培养
一般在转染前一天接种细胞，接种密度为2×105/cm2，用含10%胎牛血清的DMEM液，37℃、5% CO2培养，待细胞占50～70%瓶底面积时，用于转染试验。以六孔板为例，向每孔中加入2mL含1～2×105个细胞培养液，37℃ CO2培养至50%～70%汇合时。
（4）转染液制备
在聚苯乙稀管中制备以下两液，A液：用不含血清培养基（MEM）稀释1~10μg DNA，最终定量至100μL；B液：用MEM稀释2~15μg LR，首先将需要的MEM最终定量至100μL，然后吸取LR小心滴加入MEM中，室温静置15分钟；
A/B复合物（转染液）配置，将A液滴加入B液，轻轻弹下离心管壁，室温静置20分钟，如出现沉淀可能因LR或DNA浓度过高所致。
（5）转染：在混合液静置期间，室温下，提前5分钟将配置好的Ca2+载体工作液加入培养液中，摇匀，然后把A/B复合物缓缓加入细胞培养液中，摇匀，37℃温箱置6～24小时，可以在显微镜下观察转染效率。
本发明实施例中，应用未加A23187试剂的脂质体进行转染，转染后12小时的细胞转染率为50%~55%，而利用A123187和脂质体联合作用后转染效率可达到65%~75%。
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。
Claims (3)
1.一种高效转染真核细胞的方法，其特征在于，该方法包括以下步骤：
（1）受体细胞的培养
在转染前一天接种待转染细胞，接种密度为2×105/cm2，用含10%胎牛血清的DMEM液，37℃、5%CO2培养，待细胞占50～70%瓶底面积时，用于转染试验；
（2）转染液的制备
A液：用培养基MEM稀释1~10μg 浓度为10g/L的供体DNA，定量至100μL；
B液：将培养基MEM定量至100μL，然后吸取2~15μg的LR加入到MEM中，室温静置15分钟；
A/B复合物：将A液滴加入B液，轻轻弹下离心管壁，室温静置20min；
（3）转染
将配置好的Ca2+载体工作液加入培养好的细胞培养液中，摇匀，将所述A/B复合物缓缓加入步骤（1）中的细胞培养液中，摇匀，37℃温箱置6～24小时，观察转染效率；其中，所述Ca2+载体工作液配置为：MEM、15mmol/L Hepes、0.168mg/ml NaHCO3 以及5μmol/L A23187。
2.如权利要求1所述的高效转染真核细胞的方法，其特征在于，在步骤（3）中，所述Ca2+载体工作液的配置包括以下步骤：取1mg A23187在室温情况下，用1.9ml DMSO或DMSO/乙醇混合液溶解为1mmol/L A23187浓缩液；将所述A23187浓缩液、MEM、Hepes、NaHCO3比例混合，得到所述Ca2+载体工作液。
3.如权利要求1所述的高效转染真核细胞的方法，其特征在于，在步骤（2）中，所述供体DNA利用去内毒素质粒大提试剂盒提取得到。
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| CN103071161A (zh) * | 2013-01-09 | 2013-05-01 | 浙江大学 | 聚阳离子脂质体/磷酸钙纳米粒给药载体的制备方法 |
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