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. 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 the COIN module intron, the COIN allele of Sox2 (Sox2COIN) is phenotypically wild type, and also does not interfere with expression of Sox2Ot and miR1897. Furthermore, the inverted COIN allele of Sox2, Sox2INV is functionally null, as homozygotes recapitulate the phenotype of Sox2？geo/？geo 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/Significance Our results demonstrate the applicability of the COIN technology as a method of choice for targeting single exon genes.
References
[1]
Kamachi Y, Iwafuchi M, Okuda Y, Takemoto T, Uchikawa M, et al. (2009) Evolution of non-coding regulatory sequences involved in the developmental process: reflection of differential employment of paralogous genes as highlighted by Sox2 and group B1 Sox genes. Proceedings of the Japan Academy Series B, Physical and biological sciences 85: 55–68.
[2]
Brosius J, Gould SJ (1992) On “genomenclature”: a comprehensive (and respectful) taxonomy for pseudogenes and other “junk DNA”. Proceedings of the National Academy of Sciences of the United States of America 89: 10706–10710.
[3]
Gentles AJ, Karlin S (1999) Why are human G-protein-coupled receptors predominantly intronless? Trends in genetics : TIG 15: 47–49.
[4]
Venter JC, Adams MD, Myers EW, Li PW, Mural RJ, et al. (2001) The sequence of the human genome. Science 291: 1304–1351.
[5]
Sakharkar KR, Sakharkar MK, Culiat CT, Chow VT, Pervaiz S (2006) Functional and evolutionary analyses on expressed intronless genes in the mouse genome. FEBS letters 580: 1472–1478.
[6]
Sakharkar MK, Perumal BS, Lim YP, Chern LP, Yu Y, et al. (2005) Alternatively spliced human genes by exon skipping–a database (ASHESdb). In silico biology 5: 221–225.
[7]
Hill AE, Sorscher EJ (2006) The non-random distribution of intronless human genes across molecular function categories. FEBS letters 580: 4303–4305.
[8]
Friend K, Lovejoy AF, Steitz JA (2007) U2 snRNP binds intronless histone pre-mRNAs to facilitate U7-snRNP-dependent 3' end formation. Molecular cell 28: 240–252.
[9]
Medlin J, Scurry A, Taylor A, Zhang F, Peterlin BM, et al. (2005) P-TEFb is not an essential elongation factor for the intronless human U2 snRNA and histone H2b genes. The EMBO journal 24: 4154–4165.
[10]
Maquat LE, Li X (2001) Mammalian heat shock p70 and histone H4 transcripts, which derive from naturally intronless genes, are immune to nonsense-mediated decay. RNA 7: 445–456.
[11]
Huang Y, Carmichael GG (1997) The mouse histone H2a gene contains a small element that facilitates cytoplasmic accumulation of intronless gene transcripts and of unspliced HIV-1-related mRNAs. Proceedings of the National Academy of Sciences of the United States of America 94: 10104–10109.
[12]
Schilham MW, van Eijk M, van de Wetering M, Clevers HC (1993) The murine Sox-4 protein is encoded on a single exon. Nucleic acids research 21: 2009.
[13]
Elkouris M, Balaskas N, Poulou M, Politis PK, Panayiotou E, et al. (2011) Sox1 maintains the undifferentiated state of cortical neural progenitor cells via the suppression of Prox1-mediated cell cycle exit and neurogenesis. Stem Cells 29: 89–98.
[14]
Remboutsika E, Elkouris M, Iulianella A, Andoniadou CL, Poulou M, et al. (2011) Flexibility of neural stem cells. Front Physiol 2: 16.
[15]
Kirby PJ, Waters PD, Delbridge M, Svartman M, Stewart AN, et al. (2002) Cloning and mapping of platypus SOX2 and SOX14: insights into SOX group B evolution. Cytogenetic and genome research 98: 96–100.
[16]
Behbahaninia M, Ramey WL, Sindhwani MK, Kalani MY (2011) Differential expression of pluripotency factors Sox2 and Oct4 regulate neuronal and mesenchymal lineages. Neurosurgery 69: N19.
[17]
Yoon DS, Kim YH, Jung HS, Paik S, Lee JW (2011) Importance of Sox2 in maintenance of cell proliferation and multipotency of mesenchymal stem cells in low-density culture. Cell proliferation 44: 428–440.
[18]
Parrinello S, Napoli I, Ribeiro S, Digby PW, Fedorova M, et al. (2010) EphB signaling directs peripheral nerve regeneration through Sox2-dependent Schwann cell sorting. Cell 143: 145–155.
[19]
Avilion AA, Nicolis SK, Pevny LH, Perez L, Vivian N, et al. (2003) Multipotent cell lineages in early mouse development depend on SOX2 function. Genes & development 17: 126–140.
[20]
Cavallaro M, Mariani J, Lancini C, Latorre E, Caccia R, et al. (2008) Impaired generation of mature neurons by neural stem cells from hypomorphic Sox2 mutants. Development 135: 541–557.
[21]
Taranova OV, Magness ST, Fagan BM, Wu Y, Surzenko N, et al. (2006) SOX2 is a dose-dependent regulator of retinal neural progenitor competence. Genes & development 20: 1187–1202.
[22]
Donner AL, Episkopou V, Maas RL (2007) Sox2 and Pou2f1 interact to control lens and olfactory placode development. Developmental biology 303: 784–799.
[23]
Okubo T, Pevny LH, Hogan BL (2006) Sox2 is required for development of taste bud sensory cells. Genes & development 20: 2654–2659.
[24]
Que J, Okubo T, Goldenring JR, Nam KT, Kurotani R, et al. (2007) Multiple dose-dependent roles for Sox2 in the patterning and differentiation of anterior foregut endoderm. Development 134: 2521–2531.
[25]
Ferri AL, Cavallaro M, Braida D, Di Cristofano A, Canta A, et al. (2004) Sox2 deficiency causes neurodegeneration and impaired neurogenesis in the adult mouse brain. Development 131: 3805–3819.
[26]
Dabdoub A, Puligilla C, Jones JM, Fritzsch B, Cheah KS, et al. (2008) Sox2 signaling in prosensory domain specification and subsequent hair cell differentiation in the developing cochlea. Proceedings of the National Academy of Sciences of the United States of America 105: 18396–18401.
[27]
Amaral PP, Neyt C, Wilkins SJ, Askarian-Amiri ME, Sunkin SM, et al. (2009) Complex architecture and regulated expression of the Sox2ot locus during vertebrate development. RNA 15: 2013–2027.
[28]
Griffiths-Jones S, Grocock RJ, van Dongen S, Bateman A, Enright AJ (2006) miRBase: microRNA sequences, targets and gene nomenclature. Nucleic acids research 34: D140–144.
[29]
Barrand S, Andersen IS, Collas P (2010) Promoter-exon relationship of H3 lysine 9, 27, 36 and 79 methylation on pluripotency-associated genes. Biochem Biophys Res Commun 401: 611–617.
[30]
Barrand S, Collas P Chromatin states of core pluripotency-associated genes in pluripotent, multipotent and differentiated cells. Biochem Biophys Res Commun 391: 762–767.
[31]
Alonso MM, Diez-Valle R, Manterola L, Rubio A, Liu D, et al. Genetic and epigenetic modifications of Sox2 contribute to the invasive phenotype of malignant gliomas. PLoS One 6: e26740.
[32]
Farthing CR, Ficz G, Ng RK, Chan CF, Andrews S, et al. (2008) Global mapping of DNA methylation in mouse promoters reveals epigenetic reprogramming of pluripotency genes. PLoS Genet 4: e1000116.
[33]
Wiebe MS, Wilder PJ, Kelly D, Rizzino A (2000) Isolation, characterization, and differential expression of the murine Sox-2 promoter. Gene 246: 383–393.
[34]
Funabashi H, Takatsu M, Saito M, Matsuoka H (2010) Sox2 regulatory region 2 sequence works as a DNA nuclear targeting sequence enhancing the efficiency of an exogenous gene expression in ES cells. Biochem Biophys Res Commun 400: 554–558.
[35]
Iwafuchi-Doi M, Yoshida Y, Onichtchouk D, Leichsenring M, Driever W, et al. (2011) The Pou5f1/Pou3f-dependent but SoxB-independent regulation of conserved enhancer N2 initiates Sox2 expression during epiblast to neural plate stages in vertebrates. Dev Biol 352: 354–366.
[36]
Miyagi S, Nishimoto M, Saito T, Ninomiya M, Sawamoto K, et al. (2006) The Sox2 regulatory region 2 functions as a neural stem cell-specific enhancer in the telencephalon. J Biol Chem 281: 13374–13381.
[37]
Miyagi S, Saito T, Mizutani K, Masuyama N, Gotoh Y, et al. (2004) The Sox-2 regulatory regions display their activities in two distinct types of multipotent stem cells. Mol Cell Biol 24: 4207–4220.
[38]
Sikorska M, Sandhu JK, Deb-Rinker P, Jezierski A, Leblanc J, et al. (2008) Epigenetic modifications of SOX2 enhancers, SRR1 and SRR2, correlate with in vitro neural differentiation. J Neurosci Res 86: 1680–1693.
[39]
Tomioka M, Nishimoto M, Miyagi S, Katayanagi T, Fukui N, et al. (2002) Identification of Sox-2 regulatory region which is under the control of Oct-3/4-Sox-2 complex. Nucleic Acids Res 30: 3202–3213.
[40]
Griffiths-Jones S (2006) miRBase: the microRNA sequence database. Methods in molecular biology 342: 129–138.
[41]
Fantes J, Ragge NK, Lynch SA, McGill NI, Collin JR, et al. (2003) Mutations in SOX2 cause anophthalmia. Nature genetics 33: 461–463.
[42]
Economides AN, Frendewey D, Yang P, Dominguez MG, Dore AT, et al.. (2012) Conditionals-by-Inversion: a novel universal method for the generation of conditional alleles. in preparation.
[43]
Albert H, Dale EC, Lee E, Ow DW (1995) Site-specific integration of DNA into wild-type and mutant lox sites placed in the plant genome. Plant J 7: 649–659.
[44]
Ekonomou A, Kazanis I, Malas S, Wood H, Alifragis P, et al. (2005) Neuronal migration and ventral subtype identity in the telencephalon depend on SOX1. PLoS biology 3: e186.
[45]
Nagy A (2000) Cre recombinase: the universal reagent for genome tailoring. Genesis 26: 99–109.
[46]
Ringrose L, Chabanis S, Angrand PO, Woodroofe C, Stewart AF (1999) Quantitative comparison of DNA looping in vitro and in vivo: chromatin increases effective DNA flexibility at short distances. Embo J 18: 6630–6641.
[47]
Testa G, Schaft J, van der Hoeven F, Glaser S, Anastassiadis K, et al. (2004) A reliable lacZ expression reporter cassette for multipurpose, knockout-first alleles. Genesis 38: 151–158.
[48]
Skarnes WC, Rosen B, West AP, Koutsourakis M, Bushell W, et al. (2011) A conditional knockout resource for the genome-wide study of mouse gene function. Nature 474: 337–342.
[49]
Seoighe C, Korir PK (2011) Evidence for intron length conservation in a set of mammalian genes associated with embryonic development. BMC Bioinformatics 12 Suppl 9S16.
[50]
Swinburne IA, Miguez DG, Landgraf D, Silver PA (2008) Intron length increases oscillatory periods of gene expression in animal cells. Genes Dev 22: 2342–2346.
[51]
Swinburne IA, Silver PA (2008) Intron delays and transcriptional timing during development. Dev Cell 14: 324–330.
[52]
Miyagi S, Masui S, Niwa H, Saito T, Shimazaki T, et al. (2008) Consequence of the loss of Sox2 in the developing brain of the mouse. FEBS letters 582: 2811–2815.
[53]
Smith AN, Miller LA, Radice G, Ashery-Padan R, Lang RA (2009) Stage-dependent modes of Pax6-Sox2 epistasis regulate lens development and eye morphogenesis. Development 136: 2977–2985.
[54]
Favaro R, Valotta M, Ferri AL, Latorre E, Mariani J, et al. (2009) Hippocampal development and neural stem cell maintenance require Sox2-dependent regulation of Shh. Nature neuroscience 12: 1248–1256.
[55]
Valenzuela DM, Murphy AJ, Frendewey D, Gale NW, Economides AN, et al. (2003) High-throughput engineering of the mouse genome coupled with high-resolution expression analysis. Nature biotechnology 21: 652–659.
[56]
Zhang Y, Buchholz F, Muyrers JP, Stewart AF (1998) A new logic for DNA engineering using recombination in Escherichia coli. Nat Genet 20: 123–128.
