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Purpose: Germline substitutions in the endothelial cell tyrosine kinase receptor TIE2/TEK cause a rare inherited form of venous anomalies, Mucocutanous Venous Malformation (VMCM). We showed that at least 50% of common sporadic venous malformations (VM) are also caused by somatic activating mutations in TIE2. The identification of the etiopathogenic cause provides us with a means by which to create an in vivo mouse model of the disease. Methods: “Knock-in” mouse lines carrying the most frequent VM-causing Tie2 mutations, inherited R849W and somatic L914F, are being created. The targeting constructs replace wild-type exon 15 (R849) or 17 (L914) with cDNA starting at exon 15 or 17 respectively, followed by the remaining 3’ coding region flanked by loxP sites, and then the mutant exon. Upon Cre-expression, the cDNA containing the wild-type exon will be floxed out, leaving only the mutant exon and the endogenous 3’ exons, to be expressed. After electroporation, homologously recombined murine ES cells are identified using PCR and Southern blot, and selected clones injected into blastocysts to generate targeted mice. Cre-expression and wild-type Tie2 excision will be induced ubiquitously by crossing these lines with PGK-Cre transgenic mice. Replacement specifically in endothelial cells will be obtained by crossing them with VECad-Cre-ERT2 transgenics expressing Cre recombinase in a tamoxifen-dependent, EC specific manner. Results: The targeting constructs for L914F and R849W have been assembled, and the L914F construct has been electroporated into ES cells. Five positive clones have been identified, and will be injected into blastocysts. Conclusions: The knock-in strategy replaces wild-type Tie2 with VM-causative mutations, creating an in vivo model of the disease. This will allow us to further study these mutations and understand their etiopathogenic mechanisms, as well as test potential therapeutic measures for their efficacity.
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Uebelhoer, M., Limaye, N., Brouillard, P., & Vikkula, M. (2010). Towards a Venous Malformation Mouse Model. 18th Workshop on Vascular Anomalies, Brussels, Belgium. https://hdl.handle.net/2078.5/53067