今天向大家介绍一种在哺乳动物细胞蛋白表达领域被广泛应用的表达技术-Flp-In™系统。传统的哺乳动物细胞基因重组技术通常用电转化或阳离子脂质体将目的基因导入细胞，进而把目的基因随机插入到宿主基因组中。由于目的基因插入到宿主细胞基因组的位置具有随机性，因而我们得到的是包含了不同表达量，不同折叠方式及糖基化形式的细胞库。完成基因重组后我们需要通过加压筛选从上千个细胞克隆里筛选得到表达量高及目的蛋白活性高的细胞系。该方式具有不可预知性且耗时耗力，尤其在前期药物筛选阶段会浪费较多时间。Flp-In™系统是为了克服传统技术的缺陷而发展起来的定点基因插入技术，该技术十分符合药物生产中流行的Quality By Design理念并已在药物蛋白表达领域获得广泛应用。
Flp-In™系统是将目的基因插入到哺乳动物细胞基因组中特定位置的一项基因表达技术。利用Flp-In™系统构建重组细胞株需要两步：首先需要将Flp Recombination Target (FRT) 位点导入到宿主细胞基因组中；然后通过Flp重组酶介导的DNA重组将目的基因插入到FLP位点。使用的Flp-In™系统构建的细胞系有诸多优点：1）将FRT位点插入基因组得到Flp-In™宿主细胞后，将来插入多种目的基因将会快速高效；2）可得到基因组完全相同的稳表达细胞库；3）可得到可进行多克隆筛选的稳表达细胞系。
FRT位点，最初分离自酿酒酵母，是Flp重组酶的特异性结合位点。最小FRT位点由34个碱基序列组成，该序列包含两个反向重复的13bp的序列及一个Xba1酶切位点。Flp重组酶与13bp的重复序列结合后会在下图中C8位置完成切割。
Flp-In™系统包括了三个载体：载体一为含有pFRT/lacZeo位点的质粒，它用于获得Flp-In™宿主细胞系；第二个载体为包含了目的蛋白基因及FRT位点的pcDNA5/FRT表达载体；第三个载体为可以表达Flp重组酶的pOG44质粒。构建目的基因插入到特定位点的细胞系，您只需要完成以下几步：
- 将pFRT / lacZeo导入到您所选择的哺乳动物细胞系得到FLP-In™宿主细胞系。
- 将目的基因克隆到pcDNA5 / FRT表达载体。
- 将pcDNA5 / FRT表达载体及Flp重组酶表达载体pOG44一起倒入到FLP-In™宿主细胞系中。
- 分析目的蛋白的表达情况。
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问一下，这次我构建好了之后，下次我需要其他的目的基因转染细胞，我还需要整个过程再来一遍吗
这个系统不需要筛选吗？请问质粒在哪里购买？
也是需要筛选的啊，筛选基因是潮霉素。 查一下应该很多公司再卖，比如http://www.biovector.net/product/43717.html，
再比如Thermo Fisher， https://www.thermofisher.com/uk/en/home/life-science/protein-biology/protein-expression/mammalian-protein-expression/stable-cell-line-development/flp-in-system.html