Figure 1.

Generation of CNGB1ΔCaM mice. A, Schematic of the domains within the rod CNGβ1 subunit: GARP, Glutamic acid-rich protein; CaM, calmodulin binding domain; CNBD, cyclic nucleotide binding domain. Fourteen amino acid residues (red) were deleted within the CaM binding domain in the targeting construct. B, The calmodulin-binding domain is coded within exon 20 (asterisk). The 1.8 kb neoloxP selection marker was inserted into intron 19. DNA extracted from neomycin-resistant ES cell colonies was digested with HindIII (H). The endogenous fragment is predicted to be 2.9 kb, whereas the correctly targeted homologous recombination event will generate a 4.7 kb fragment (2.9 kb + 1.8 kb from the neomycin cassette) when the indicated probe is used in a Southern blot. C, Southern blot of representative colonies that did not (lanes 1 and 3) and did (lane 2) contain the correct homologous recombination event. D, Representative Western blot (N = 3–5) of retinal homogenates from 1-month-old WT (lane 1), CNGB1ΔCaM (lane 2), CNGB1ΔCaM/GCAPs^−/− (lane 3), and GCAPs^−/− (lane 4) mice. No changes in levels of indicated retinal proteins were detected except for the absence of GCAP1 and GCAP2 when their genes were knocked out. E, CNGB1ΔCaM is correctly localized to the outer segment (left). The mutation did not affect the retinal morphology (right). RPE, Retinal pigmented epithelium; OS, outer segment; IS, inner segment; ONL, outer nuclear layer.