title: Application of a novel strategy of engineering conditional alleles to a single exon gene, Sox2.
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 3' 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.
  
  
  RESULTS
  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 the COIN module intron, the COIN allele of Sox2 (Sox2(COIN)) is phenotypically wild type, and also does not interfere with expression of Sox2Ot and miR1897. Furthermore, the inverted COIN allele of Sox2, Sox2(INV) is functionally null, as homozygotes recapitulate the phenotype of Sox2(βgeo/βgeo) mice, a well-characterized Sox2 null. Lastly, the benefit of the eGFP marker embedded in the COIN allele is demonstrated as it mirrors the expression pattern of Sox2.
  
  
  CONCLUSIONS
  Our results demonstrate the applicability of the COIN technology as a method of choice for targeting single exon genes.
authors.affiliation: Stem Cell Biology Laboratory, Institute of Molecular Biology and Genetics, Biomedical Sciences Research Centre Alexander Fleming, Vari, Greece.
authors.forename: Nikolaos
authors.initials: N
authors.lastname: Mandalos
authors.forename: Marannia
authors.initials: M
authors.lastname: Saridaki
authors.forename: Jessica Lea
authors.initials: JL
authors.lastname: Harper
authors.forename: Anastasia
authors.initials: A
authors.lastname: Kotsoni
authors.forename: Peter
authors.initials: P
authors.lastname: Yang
authors.forename: Aris N
authors.initials: AN
authors.lastname: Economides
authors.forename: Eumorphia
authors.initials: E
authors.lastname: Remboutsika
mesh_terms: D000483:Alleles; D000818:Animals; D001483:Base Sequence; D004622:Embryo, Mammalian / Q000378:metabolism; D005091:Exons; D005786:Gene Expression Regulation; D005818:Genetic Engineering / Q000379:methods*; D006824:Hybridization, Genetic; D019426:Integrases / Q000096:biosynthesis / Q000235:genetics; D007438:Introns; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D055748:SOXB1 Transcription Factors / Q000235:genetics* / Q000378:metabolism; D057345:Sequence Inversion
chemical_list: D055748:SOXB1 Transcription Factors; C527783:Sox2 protein, mouse; C045073:Cre recombinase; D019426:Integrases