Nagarkar-Jaiswal Sonal, Lee Pei-Tseng, Campbell Megan E, Chen Kuchuan, Anguiano-Zarate Stephanie, Gutierrez Manuel Cantu, Busby Theodore, Lin Wen-Wen, He Yuchun, Schulze Karen L, Booth Benjamin W, Evans-Holm Martha, Venken Koen J T, Levis Robert W, Spradling Allan C, Hoskins Roger A, Bellen Hugo J
Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, United States.
Program in Developmental Biology, Baylor College of Medicine, Houston, United States.
Elife. 2015 Mar 31;4:e05338. doi: 10.7554/eLife.05338.
Here, we document a collection of ∼7434 MiMIC (Minos Mediated Integration Cassette) insertions of which 2854 are inserted in coding introns. They allowed us to create a library of 400 GFP-tagged genes. We show that 72% of internally tagged proteins are functional, and that more than 90% can be imaged in unfixed tissues. Moreover, the tagged mRNAs can be knocked down by RNAi against GFP (iGFPi), and the tagged proteins can be efficiently knocked down by deGradFP technology. The phenotypes associated with RNA and protein knockdown typically correspond to severe loss of function or null mutant phenotypes. Finally, we demonstrate reversible, spatial, and temporal knockdown of tagged proteins in larvae and adult flies. This new strategy and collection of strains allows unprecedented in vivo manipulations in flies for many genes. These strategies will likely extend to vertebrates.
在此，我们记录了约7434个Minos介导的整合盒（Minos Mediated Integration Cassette，MiMIC）插入事件，其中2854个插入到编码内含子中。这些插入事件使我们能够创建一个包含400个绿色荧光蛋白（GFP）标记基因的文库。我们发现，72%的内部标记蛋白具有功能，并且超过90%的标记蛋白可以在未固定的组织中成像。此外，标记的mRNA可以通过针对GFP的RNA干扰（RNAi against GFP，iGFPi）被敲低，标记的蛋白可以通过降解荧光蛋白技术（deGradFP technology）被有效敲低。与RNA和蛋白敲低相关的表型通常对应于功能的严重丧失或无效突变体表型。最后，我们展示了在幼虫和成年果蝇中对标记蛋白进行可逆、空间和时间上的敲低。这种新策略和菌株集合允许对果蝇中的许多基因进行前所未有的体内操作。这些策略可能会扩展到脊椎动物。
