This research followed the tenets of the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, and the guidelines of the University of Pennsylvania for Animal Care and Use. The
Rp1-myc targeting vector was constructed using segments of genomic DNA amplified by high-fidelity PCR from a mouse bacterial artificial chromosome (BAC) clone that contains the entire mouse
Rp1 gene. This BAC clone, 314A, was identified by screening high-density filters from the RPCI-22 (129S6/SvEvTac) Mouse BAC Library (BACPAC web site: http://www.chori.org/bacpac/libraryres.htm/ provided in the public domain by the National Center of Excellence in Genomics at Children’s Hospital Oakland Research Center, Oakland, CA). The 3′ arm of the targeting vector, including bases 16,110 to 16,879 of the
Rp1 gene (based on GenBank AF291754; http://www.ncbi.nlm.nih.gov/Genbank; provided in the public domain by the National Center for Biotechnology Information, Bethesda, MD), plus an additional 1,174 bases 3′ to the gene, was amplified with PCR primers containing
XhoI and
KpnI restriction sites. The purified, digested PCR product was then cloned into loxpneoBS, which contains a floxed pgk-neo cassette,
26 creating plasmid pSP2. The 5′ arm of the targeting vector including bases 6,822 to 12,003 of the
Rp1 gene was amplified and cloned into the
NotI and
SalI sites of the pCMV-tag5 vector (Stratagene) adjacent to the
myc tag to create plasmid pSP6. The 5′-
myc arm, including the
myc tag, stop codon and polyadenylation signal from pCMV-tag5, was then excised from pSP6, using the enzymes
NotI and
MluI, and cloned into the
NotI-
MluI sites of pSP2, upstream of the floxed pgk-neo cassette, creating the final
Rp1-myc targeting vector (see
Fig. 2 ).