Restriction enzyme-generated siRNA (REGS) vectors and libraries

Small interfering RNA (siRNA) technology facilitates the study of loss of gene function in mammalian cells and animal models, but generating multiple siRNA vectors using oligonucleotides is slow, inefficient and costly. Here we describe a new, enzyme-mediated method for generating numerous functional siRNA constructs from any gene of interest or pool of genes. To test our restriction enzyme-generated siRNA (REGS) system, we silenced a transgene and two endogenous genes and obtained the predicted phenotypes. REGS generated on average 34 unique siRNAs per kilobase of sequence. REGS enabled us to create enzymatically a complex siRNA library (>4 x 10(5) clones) from double-stranded cDNA encompassing known and unknown genes with 96% of the clones containing inserts of the appropriate size.     

A direct functional link between the multi-PDZ domain protein GRIP1 and the Fraser syndrome protein Fras1

Cell adhesion to extracellular matrix (ECM) proteins is crucial for the structural integrity of tissues and epithelial-mesenchymal interactions mediating organ morphogenesis. Here we describe how the loss of a cytoplasmic multi-PDZ scaffolding protein, glutamate receptor interacting protein 1 (GRIP1), leads to the formation of subepidermal hemorrhagic blisters, renal agenesis, syndactyly or polydactyly and permanent fusion of eyelids (cryptophthalmos). Similar malformations are characteristic of individuals with Fraser syndrome and animal models of this human genetic disorder, such as mice carrying the blebbed mutation (bl) in the gene encoding the Fras1 ECM protein. GRIP1 can physically interact with Fras1 and is required for the localization of Fras1 to the basal side of cells. In one animal model of Fraser syndrome, the eye-blebs (eb) mouse, Grip1 is disrupted by a deletion of two coding exons. Our data indicate that GRIP1 is required for normal cell-matrix interactions during early embryonic development and that inactivation of Grip1 causes Fraser syndrome-like defects in mice.     

Rb regulates proliferation and rod photoreceptor development in the mouse retina

The retinoblastoma protein (Rb) regulates proliferation, cell fate specification and differentiation in the developing central nervous system (CNS), but the role of Rb in the developing mouse retina has not been studied, because Rb-deficient embryos die before the retinas are fully formed. We combined several genetic approaches to explore the role of Rb in the mouse retina. During postnatal development, Rb is expressed in proliferating retinal progenitor cells and differentiating rod photoreceptors. In the absence of Rb, progenitor cells continue to divide, and rods do not mature. To determine whether Rb functions in these processes in a cell-autonomous manner, we used a replication-incompetent retrovirus encoding Cre recombinase to inactivate the Rb1(lox) allele in individual retinal progenitor cells in vivo. Combined with data from studies of conditional inactivation of Rb1 using a combination of Cre transgenic mouse lines, these results show that Rb is required in a cell-autonomous manner for appropriate exit from the cell cycle of retinal progenitor cells and for rod development.