application of a novel strategy of engineering conditional alleles to a single exon gene, sox2应用程序的一个新策略的工程条件单外显子基因的等位基因,sox2.pdfVIP

Application of a Novel Strategy of Engineering Conditional Alleles to a Single Exon Gene, Sox2 1 1¤a 1,2 1¤b 2 Nikolaos Mandalos , Marannia Saridaki , Jessica Lea Harper , Anastasia Kotsoni , Peter Yang , 2 1 Aris N. Economides *, Eumorphia Remboutsika * 1 Stem Cell Biology Laboratory, Institute of Molecular Biology and Genetics, Biomedical Sciences Research Centre ‘‘Alexander Fleming’’, Vari, Greece, 2 Regeneron Pharmaceuticals Inc., Tarrytown, New York, United States of America Abstract Background: The Conditional by Inversion (COIN) method for engineering conditional alleles relies on an invertible optimized gene trap-like element, the COIN module, for imparting conditionality. The COIN module contains an optimized 39 splice site-polyadenylation signal pair, but is inserted antisense to the target gene and therefore does not alter transcription, until it is inverted by Cre recombinase. In order to make COIN applicable to all protein-coding genes, the COIN module has been engineered within an artificial intron, enabling insertion into an exon. Methodology/Principal Findings: Therefore, theoretically, the COIN method should be applicable to single exon genes, and to test this idea we engineered a COIN allele of Sox2. This single exon gene presents additional design challenges, in that its proximal promoter and coding region are entirely contained within a CpG island, and are also spanned by an overlapping transcript, Sox2Ot, which contains mmu-miR1897. Here, we show that despite disruption of the CpG island by th