Nagarkar-Jaiswal Sonal, DeLuca Steven Z, Lee Pei-Tseng, Lin Wen-Wen, Pan Hongling, Zuo Zhongyuan, Lv Jiangxing, Spradling Allan C, Bellen Hugo J
Howard Hughes Medical Institute, Baylor College of Medicine, Houston, United States.
Department of Embryology, Howard Hughes Medical Institute, Carnegie Institution for Science, Baltimore, United States.
Elife. 2015 Jun 23;4:e08469. doi: 10.7554/eLife.08469.
Previously, we described a large collection of Minos-Mediated Integration Cassettes (MiMICs) that contain two phiC31 recombinase target sites and allow the generation of a new exon that encodes a protein tag when the MiMIC is inserted in a codon intron (Nagarkar-Jaiswal et al., 2015). These modified genes permit numerous applications including assessment of protein expression pattern, identification of protein interaction partners by immunoprecipitation followed by mass spec, and reversible removal of the tagged protein in any tissue. At present, these conversions remain time and labor-intensive as they require embryos to be injected with plasmid DNA containing the exon tag. In this study, we describe a simple and reliable genetic strategy to tag genes/proteins that contain MiMIC insertions using an integrated exon encoding GFP flanked by FRT sequences. We document the efficiency and tag 60 mostly uncharacterized genes.
此前，我们描述了大量的Minos介导的整合盒（MiMICs），其包含两个phiC31重组酶靶位点，并且当MiMIC插入密码子内含子时，能够产生一个编码蛋白质标签的新外显子（Nagarkar-Jaiswal等人，2015年）。这些修饰基因有多种应用，包括评估蛋白质表达模式、通过免疫沉淀继以质谱法鉴定蛋白质相互作用伙伴，以及在任何组织中可逆地去除标记蛋白。目前，这些转化仍然耗时且费力，因为它们需要向胚胎注射含有外显子标签的质粒DNA。在本研究中，我们描述了一种简单可靠的遗传策略，利用一个编码绿色荧光蛋白（GFP）且两侧带有FRT序列的整合外显子来标记含有MiMIC插入的基因/蛋白质。我们记录了该方法的效率，并标记了60个大多未被表征的基因。
