前言
转染是将遗传物质如DNA和双链RNA导入到哺乳动物细胞内的过程。将DNA导入细胞使得它可利用细胞自身的细胞机制表达和合成蛋白质，将RNA导入细胞则是用来通过终止翻译来降低某特定蛋白的合成。转染的RNA在细胞质中发挥作用，DNA则需被转运到细胞核中从而达到有效的转染。在核内，DNA会被短暂表达或整合到基因组DNA而将该遗传改变传代到下一代细胞。
本短片讲述了化学转染的基本原理，介绍了一些最常用的试剂，包括带电荷脂质体，聚合物和磷酸钙。我们将讲解从准备转染的细胞开始到最后分析转染效率过程中的每个步骤。另外，本视频文章的应用部分还讲述了导入核酸到哺乳动物细胞的其他方法，电击转染和基因枪转染。它还介绍了转染的更进一步的应用，同时转染DNA和干扰RNA来下调某天然存在的蛋白并同时引入该蛋白的突变体到同一细胞。
转染的介绍
转染是将遗传物质如DNA和双链RNA导入到哺乳动物细胞内的过程。将DNA导入细胞使得它可利用细胞自身的细胞机制表达和合成蛋白质，将RNA导入细胞则是用来通过终止翻译来降低某特定蛋白的合成。该功能强大的操作工具使得研究人员可以来更好地研究基因表达和功能，蛋白质功能以及基因突变。
没有一种转染试剂或方法可以适用于所有细胞类型。所幸的是在过去几十年里发展出了许多方法和试剂用于各种各样细胞类型的转染。这些方法主要可分为两大类：化学转染和物理转染。
本短片中我们将重点讲述不同的化学转染系统，它们的应用在最近几年更为普遍。其中包括使用脂质体的方法，磷酸钙介导的转染，和使用带正电荷的聚合物的方法。
转化的原理
所有化学转染方法的基本原理类似。它们都是使用带正电荷的承载分子和核酸结合并包裹核酸从而转运到细胞内。这些带正电荷的分子可以中和细胞膜表面的负电荷从而穿过膜来转运其包裹的核酸。
脂转染中，带正电荷的脂类形成一个脂质体，然后与核酸结合一起形成一个转染复合物。而磷酸钙只是简单地将DNA浓缩使其产生正电荷。此外，带正电荷的聚合物如聚乙烯亚胺可将DNA浓缩成为带正电荷的颗粒。
化学介导转染的下一步是这些带正电荷的复合物通过简单的静电吸引结合到带负电荷的细胞膜。
然后复合物利用细胞内吞作用进入细胞内部 –细胞内吞是一种分子通过称之为包涵体的包膜颗粒而进入细胞的过程。
一旦进入细胞，核酸以一种目前还未知的机制从内涵体释放到细胞质，并最终到达细胞核，在那里它被细胞机制转录生成mRNA，并进一步翻译产生蛋白质。细胞质是小分子干扰RNA或称siRNA起作用的地方，它们通过干扰蛋白质合成机制的一部分而降低蛋白质的生成。
转染进入的DNA可以稳定或瞬时存在。稳定转染是指转染的DNA整合到基因组中并在细胞分裂后仍然保留。瞬时转染时，DNA并不整合到基因组，其编码的蛋白会在转染后24到96小时后丢失。
通常将插入基因与报告系统连在一起来检测DNA转染的效率。这些系统可简单地通过直接观察报告分子，如绿色荧光蛋白，或用比色分析测量酶活性，如荧光素酶报告分子，来进行检测。
siRNA沉默是否成功可通过免疫杂交目的蛋白的表达水平来判断。成功转染siRNA应该会降低细胞内目的蛋白的表达而不改变管家基因GAPDH的表达量。
转染过程
为达到最高转染效率，细胞应在转染时处于对数生长期，并处于40%到80%生长密度之间。这需要在转染前一天收集细胞，计数，然后重新接种到多孔板中，接种浓度要使得转染时能达到正确的细胞密度。
接下来，混合化学试剂和核酸，并孵育一段时间使它们形成核酸-试剂复合物。每种化学转染系统中用到的各组分的浓度必须优化达到最佳。
然后将核酸-试剂复合物加入到平板生长的细胞中并孵育过夜使得复合物有足够的时间结合到细胞并介导转染。24小时后，需将培养液吸去换成新鲜培养液。
应用
转染技术可以有很多的改动和应用。共转染使得研究人员能够研究错义突变对细胞蛋白功能的影响。这里，将RNAi转染到Hela细胞来降低内源BRCA1蛋白，它会导致GFP阳性细胞数目的减少。同时BRCA1突变蛋白也转染进入细胞并得到表达。若突变蛋白的功能完整，就会使GFP阳性细胞数目得到恢复，但若突变对功能有负面影响，则GFP阳性细胞数目仍然会少。
您这里看到的是研究人员在用化学转染的一种替代方法，使用基因枪将吸附了DNA的金颗粒轰击培养的细胞。最后细胞质中含了附着DNA颗粒的细胞很大可能会被转染。
另一个转染的替代方法是电击转染。电击转染是使用电流来破坏细胞膜从而使得DNA或RNA在细胞得到修复前的短时间内进入到细胞内。这里电极丝被放置在小鼠脑的周围，让电子短脉冲通过脑组织来进行离体转染含RNAi的蓝色溶液。然后观察基因沉默对发育皮质结构的影响。
结语
您刚观看的是JoVE关于细胞转染的短片。我们一如既往地感谢您的观看！
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