Expression and characterization of ribozymes and short hairpin RNA in mammalian cells
Expression and characterization of ribozymes and short hairpin RNA in mammalian cells
Functional genomic research using R N A interference techniques (R N A i ) has been carried out extensively in recent years.R N A i is the newest and most successful of a variety of techniques for regulating gene expression, which are generally based on antisense effects. To introduce antisense effectors into cells, there are two main approaches: introduction of already synthesized effector molecules from outside the cell or intracellular expression of the effector molecules by means of appropriate vectors. Down-regulation of gene expression induced by exogenous introduction is transient and unsuitable for studies on proteins with long half-lives, and other potential problems arise from low or frequently changing transfection efficiencies. Written by T. Friedman et al, translated by W. Qin et al. This experiment is from "Gene Transfer".
Operation method
Construction and expression of oligonucleotide-based antisense cassettes Move Construction and expression of oligonucleotide-based antisense cassettes Materials Reagents 26. Aspirate the culture medium and add 150ul of passive lysis buffer (provided in the Dual Fluorokinase Reporter System kit) to each well and incubate for 15 min at room temperature with gentle shaking. 27. Measure the expression of the reporter gene on a fluorescence/chemiluminescence detector (refer to the operation manual of the Dual Fluorokinase Reporter System kit). 28 The internal control firefly fluorokinase was used as a standard to specificate all samples target-specific Renilla fluoresceinase expression. The expression efficiency of the control (cells transfected with irrelevant effectors) was set to 1 00%, and the relative expression levels of the other samples were calculated against the control. At least two independent parallel experiments were performed, and two samples that were constructed and demonstrated efficient expression of the effector were selected for further study.
![: 中 表 有 福 个 型 二 二 A = ^丄包白特异性®序列可以减少形成目的基因错误二级结构的机会。前引物和后引物分 占从S X A 〇1和 驗 1酶切位点，用于将1^产物定向克隆进psiCheck2 载体。确保酶切位 点外面还包含另外4〜6个核苷酸以提髙对P C R 产物的酶切效率。 18. 要克隆目的c D N A ，参照步骤4〜1 5 , 但某些步骤有所修改。在步骤9,插入序列的 摩尔数应是线性载体的2〜3 倍。在步骤1 5 , 酣 x / w I + JVGrt酶切释放出插入序列 来分析转化菌D N A 。 19. 通过酶切鉴定的阳性克隆，用 RLuc_F〇1^ TK_Rev引物扩增。 双萤光素酶报道系统与效应因子载体共转染 20. 在转染前16〜24h ，将细胞接种在2 4 孔 板 里 （每 孔 0.5m l )，当开始转染时，细胞 汇合度应达到60% 〜80% 。 2 1 . 每种细胞接种到3 个孔，为 所 有 的 样 品 （]V个）准 备 Lip/med (Lipofectamine 2000/培养液）混合液： Lip/m e d : 50 X JVjul 的基础培养液+1. 5X JVpl 的 Lipofectamine 2000 室温下孵育5〜l O m i n。 22. 混合报道D N A 和效应因子D N A (包括一个无关的效应因子作为对照）用于转染 (条件： IOOng 报道基因、 IOOng 效应因子、每孔 0 •5；xl Lipofectamine 2000): lOOng/f J 的报道基因 3今 1 lOOng/p l的效应因子 3. Oyl 基础培养液 50fxl Lip/med (见步骤 2〇) 50m1 混 匀 ，室 温 孵 育30min (见步骤21)。 23. 在孵育混合物的过程中，吸出24孔板中的培养液，向每孔加人300I山新鲜的全培养 液。或者只从每孔吸出200f J 旧培养液。 24. 将°5〇4步骤2 2 的混合物溶解在500W 全培养液中，向 3 个孔中每个孔加人200m 1。 • 528 •](http://img.dxycdn.com/trademd/upload/userfiles/image/2016/08/A1470641622508sb8f6tsrpzpng_small.jpg)
25. Co-incubate the mixture with the cells (without changing the culture medium during this period) until 24 h after transfection.
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