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Identifier

000379003

Title

Generation of a conditional by inversion SOX2 allele

Alternative Title

Δημιουργία ενός επαγόμενου με αναστροφή SOX2 αλληλίου

Author

Mandalos, Nikolaos

Author

Μάνδαλος Νικόλαος

Thesis advisor

Ρεμπούτσικα Ευμορφία

Reviewer

Θερμού, Κυριακή
Ταβερναράκης, Ν.
Καρδάσης, Δ.
Πάχνης, Β.
Γραβάνης, Α.
Καραγωγέως, Δ.
Χαραλαμπόπουλος, Ι.

Abstract

Sox2 encodes a transcription factor that harbours a DNA binding HMG domain. It is considered one of the key gene players for the regulation of pluripotency and early embryonic development, and a determinant of cell fate during development and homeostasis. During early development, Sox2 is required for the formation of the epiblast. However, its role in stem cells is still elusive. Here, we performed a surgical ablation of Sox2 in the epiblast using a novel Conditional by Inversion Sox2 allele (Sox2COIN). 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. 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 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 Sox2ßgeo/ßgeo 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. Epiblast-inversion of the Sox2COIN allele generates normal heterozygote Sox2INV/+ adult animals and Sox2INV/+ (H) haploinsufficient mutant embryos. Sox2INV/+ (H) haploinsufficient and Sox2INV/mosaic embryos exhibit heart-looping defects, suffer from bradycardia, myocardium malformation and bleeding and die around E11. Sox2INV/+(H) haploinsufficient and Sox2INV/mosaic embryos exhibit hydrocephaly, exacerbated and aberrant migration of neural crest cells in the branchial arches and the frontonasal region, with no effect on cranial nerve formation but abnormal frontonasal development. We propose that Sox2 acts as a rheostat of the epithelial to mesenchymal transition during neural crest development.

Language

Greek

Subject

Biochemistry

Conditional alleles

Craniofacial development

Developmental biology

Heart development

Neural crest

SOX2

Soxes genes

Stem cells