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首页> 外文期刊>Biochemistry >Double-reciprocal crossover mediated by FLP-recombinase: a concept and an assay.
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Double-reciprocal crossover mediated by FLP-recombinase: a concept and an assay.

机译：FLP重组酶介导的双向交换：概念和分析。

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摘要

FLP recombinase induces a double-reciprocal crossover event between sets of different FLP recognition target (FRT) sites. Therefore, if these sites flank an expression cassette at a given genomic locus, it can be exchanged for another cassette that has been constructed in the analogous was [Schlake & Bode (1994) Biochemistry 33, 12746-12751]. Here we demonstrate that an integrated expression cassette, flanked by a wild type and a mutated site, remains completely stable in the presence of constitutive FLP activity, obviating the need for a timing of this parameter. Therefore the only variable left for optimization is the initial concentration of the exchange plasmid. Since the exchange plasmid lacks a promoter, random integration is not expected to confer resistance to the selection marker, the expression of which requires the acquisition of the SV40 promoter provided at the predetermined integration site. Due to the presence of a luciferase reporter in a specific bicistronic expression cassette, recombination generates bioluminescence upon recombination, indicating the extent of the exchange reaction. This principle is utilized to compare the potential of various cell lines to support the exchange reaction and to adjust the optimum parameters.
机译：FLP重组酶诱导一组不同的FLP识别目标（FRT）位点之间的双向交换事件。因此，如果这些位点在给定基因组基因座的表达盒的侧翼，则可以将其交换为已经类似构建的另一盒[Schlake＆Bode（1994）Biochemistry 33，12746-12751]。在这里，我们证明了侧翼为野生型和突变位点的整合表达盒在组成型FLP活性存在的情况下仍保持完全稳定，从而消除了对该参数进行计时的需要。因此，剩下的用于优化的唯一变量是交换质粒的初始浓度。由于交换质粒缺少启动子，因此预期不会随机整合赋予选择标记抗性，选择标记的表达需要获得在预定整合位点提供的SV40启动子。由于特定双顺反子表达盒中荧光素酶报道分子的存在，重组后重组产生生物发光，表明交换反应的程度。利用该原理来比较各种细胞系支持交换反应和调整最佳参数的潜力。

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