The RICO mouse - a novel model of dominant uveal coloboma

Abstract

Purpose: Uveal coloboma is a potentially blinding congenital ocular malformation caused by failure of the optic fissure to close during the 5^th week of human gestation and at around 11.5 days in the mouse. Mutations in the developmentally-regulated genes have been described in some patients; however, the genetic cause of coloboma (if any) in many individuals has remained elusive. Here we describe the initial genetic and phenotypic characterization of the RICO (Retinal & Iris COloboma) mouse, a novel model of dominant uveal coloboma.

Methods: RICO was inadvertently created by the transgenic insertion of a human VEGF (hVEGF) expression vector. The insertion site was localized with FISH and commercially available BAC clones for mouse chromosome 13. Embryos and mice were genotyped using a combination of FISH and PCR and were phenotyped grossly and histopathologically. The insertion site was evaluated using a combination of comparative genomic hybridization (CGH), BAC library construction, shot gun sequencing of two BAC clones and ‘Whole-genome shotgun sequencing’ of RICO genomic DNA.

Results: The uveal coloboma in C57BL/6J-RICO mice was generally bilateral, affecting the iris, retina/choroid, and optic nerve. Transmission was autosomal dominant with nearly 100% penetrance. RICO mice breed well, do not express hVEGF, and display no apparent systemic abnormalities. CGH array of heterozygote RICO DNA shows no large duplications or deletions on Chr.13. BAC library construction resulted in two BAC clones containing the VEGF transgene (15Kb and 65Kb). Shotgun sequencing of BAC clones showed several contigs with Chr 13 sequence from two different regions (4Mb apart) suggesting a genomic rearrangement. ‘Whole-genome shotgun sequencing’ of RICO DNA suggested multiple copies of transgene insertion but identified the insertion in a well-conserved, noncoding site on chromosome 13.

Conclusions: Insertion of NSE-hVEGF transgene on mouse chromosome 13C results in a dominant uveal coloboma in C57BL/6J mice with homozygous lethality, reminiscent of human disease. The phenotype is not related to the expression of transgene. From the BAC library, shotgun sequencing of BAC clones and ‘Whole-genome shotgun sequencing’ of RICO genomic DNA, we propose a model for the genetic insertion/re-arrangement responsible for the phenotype. Further, analysis of this genomic region will be carried out to elucidate the key elements causing this disease in RICO mouse model.

Keywords: 539 genetics • 740 transgenics/knock-outs • 629 optic nerve