Generation of Q344ter transgenic mice.

<p>(<b>A</b>) Construct used to generate the Q344ter transgenic mice. In an 11-kb BamHI-flanked genomic clone containing the murine rod opsin gene, codon Q344 was mutated to an early stop signal (bottom *). The resulting rhodopsin mutant is missing the QVAPA domain but retains the six known potential phosphorylation sites (underlined). An AvrII site within the Q344ter transgene was generated by two silent mutations. Capitalized nucleotides denote the introduced point mutations. (<b>B</b>) Scheme to establish transcript expression level of the Q344ter transgene. The AvrII site is used to differentiate between Q344ter transgenic and WT transcript species. (<b>C</b>) Phosphor-image used to establish transgene-to-total rhodopsin transcript ratio. Total rhodopsin transcripts from each murine retina were amplified by RT-PCR and divided into two equal fractions. After AvrII digestion of one fraction, the enzyme-resistant 250 bp band is compared to the corresponding band from the undigested fraction. (<b>D</b>) Detection of the Q344ter mutant by western blot. Equal fraction (1/800) of a retina was loaded onto each lane. R2-12N monoclonal antibody recognizes the amino-terminus and identifies endogenous and Q344ter rhodopsin, while the 1D4 antibody recognizes only full length endogenous rhodopsin. Q344ter expression in rho−/− background was confirmed by R2-12N (left panel). As expected, these species were not detected by 1D4 (right panel). Monomeric and dimeric rhodopsin migrate at 33 kD and 66 kD, respectively.