Abstract:
The present study describes the genetic ablation of Pde6h and Kcnv2 in mice by methods of molecular biology with a view to generate a Pde6h-knockout and a Kcnv2-knockout-lacZ-knockin mouse line representing new animal models for hereditary retinal diseases. The Pde6h gene encodes the inhibitory gamma’-subunit of the cone phosphodiesterase, which is involved in the transformation of light into neuronal signals in phototransduction. When light strikes the photoreceptors, PDE is activated by the active form of transducin, which binds to the inhibitory subunit of cone PDE and sterically displaces the latter, thereby relieving its inhibitory effect on the catalytic subunits and permitting the hydrolysis of cGMP. This signaling results in closure of CNG-channels, followed by hyperpolarization of the cell membrane and a decrease in glutamate release at the cell synapse. There is still little known about Pde6h, but in the last years it was discussed as a potential disease-causing gene for hereditary retinal diseases. To investigate the function of Pde6h and the biochemical parameters of cone phototransduction in further detail as well as the expression and function of Pde6h in nonretinal tissues, the aim of the present study was to delete Pde6h in a constitutive and a conditional manner in mice. Subsequent comparison of the resulting phenotype of knockout mice with their wildtype littermates will reveal the biological function of Pde6h. The developed knockout strategy was based on the Cre/loxP system, enabling to generate a constitutive as well as a conditional knockout of Pde6h. The gene architecture was analyzed, choosing the exons 2-4 to be flanked by two loxP sites by homologous recombination in embryonic stem cells of mice. In numerous cloning steps, a suitable DNA construct was generated and finally electroporated into murine stem cells. The targeting construct was integrated into the stem cell genome, and the desired homologous recombinant stem cell clones were identified using selective medium and Southern blot analysis. After blastocyst injection of these clones and subsequent embryo transfer into pseudo-pregnant foster mice, several highly chimeric mice were born. Mating of chimeras yielded two progenies with proven germ-line transmission of the mutation. Crossing to transgenic CMV-“Cre-deleter”-mice and subsequent outcrossing of the transgene finally resulted in Pde6h knockout mice. Immunofluorescence analysis of retinal Pde6h-/- slices with an antibody specifically directed against the gamma-subunit of cone PDE confirmed the deletion of Pde6h. Pde6h-/- mice are viable and are now available for investigations of behavioural and physiological disorders. First functional analysis of the retina by ERG, SLO and OCT revealed no difference between Pde6h-/- mice and wildtype littermates, possibly indicating compensatory mechanisms. One possible explanation could be the expression of Pde6g not only in rods but also in Pde6h-/- cones. Further investigations for example with Pde6g/Pde6h double knockout mice or the generation of Pde6h-/-/Nrl-/- mice with a “cone-dominated” retina could help to reveal compensatory effects. By mating mice carrying exons flanked by two loxP sites with appropriate mice that express Cre recombinase, the Pde6h gene can also be deleted in a tissue specific or inducible manner. These methods provide further possibilities to investigate the physiological and pathophysiological role of Pde6h. Kcnv2 presumably encodes a modulatory subunit of a voltage gated K+-channel in the inner segments of photoreceptors, which is involved in the modulation of light induced signals. Mutations in KCNV2 in humans cause a particular form of retinal dystrophy. In the present study, a Kcnv2-knockout-lacZ-knockin targeting construct, based on the Cre/loxP-system and expressing lacZ under the control of the native Kcnv2 promotor, was introduced into embryonic stem cells of mice by homologous recombination. Using Cre recombinase, the selection cassette was deleted, and stem cell clones with homologous recombination as well as deletion of the selection cassette were identified by Southern blot analysis and PCR. Several selected clones were prepared for blastocyst injection, which in future will lead to the generation of chimeric mice. By mating with wildtype mice, the occurrence of germ-line transmission will be analysed. After the establishment of the desired mouse line, physiological and pathophysiological parameters will be investigated and the expression of Kcnv2 will be observed indirectly by analysing the expression of lacZ in nonretinal tissues.