Schübeler D, Maass K, Bode J
GBF, National Research Center for Biotechnology, Braunschweig, Germany.
Biochemistry. 1998 Aug 25;37(34):11907-14. doi: 10.1021/bi9807052.
The transcriptional activity of transgenes in eukaryotic host cells critically depends on the sites of their integration where it is modulated by interactions between the promoter and surrounding chromatin structures. Retroviruses integrate their genome into chromosomal loci favoring expression from one long-terminal repeat (LTR). We have therefore developed a strategy in which retroviral vectors are provided with "tags", that is, targets for a site-specific recombinase (Flp). Presence of two 48 bp Flp recognition target (FRT) sequences permits the excision of a selection marker whereby the reading frame of a reporter gene (lacZ) is restored and beta-galactosidase activity can be monitored to characterize the integration site regarding the level and stability of expression. The location of the remaining FRT tag permits the subsequent Flp-mediated insertion of a truncated selection marker which is then expressed from the LTR. This step represents an authentic site-specific recombination event which can be demonstrated by a number of criteria, among these its reversibility in the presence of Flp activity. Thereby the "expression trap" principle permits the efficient recovery of stable insertion events, and if a gene of interest is linked to the truncated marker, the established properties of a given genomic site can be utilized for transcription studies or for the generation of highly expressing clones, even for biotechnological purposes.
转基因在真核宿主细胞中的转录活性关键取决于其整合位点，在该位点，启动子与周围染色质结构之间的相互作用会对转录活性进行调节。逆转录病毒将其基因组整合到有利于从一个长末端重复序列（LTR）进行表达的染色体位点。因此，我们开发了一种策略，为逆转录病毒载体提供“标签”，即位点特异性重组酶（Flp）的靶标。两个48 bp的Flp识别靶标（FRT）序列的存在允许切除选择标记，从而恢复报告基因（lacZ）的阅读框，并可监测β-半乳糖苷酶活性，以根据表达水平和稳定性来表征整合位点。剩余FRT标签的位置允许随后通过Flp介导插入一个截短的选择标记，该标记随后从LTR进行表达。这一步代表了一个真实的位点特异性重组事件，可通过多种标准来证明，其中包括在Flp活性存在下其可逆性。因此，“表达陷阱”原理允许高效回收稳定的插入事件，如果将感兴趣的基因与截短的标记相连，给定基因组位点的既定特性可用于转录研究或用于生成高表达克隆，甚至用于生物技术目的。
