Construction of Targeting and Segmental Replacement Vectors. DNA segments flanking the mouse rhodopsin gene were isolated from a mouse genomic library of 129SvEv origin (1) (gift from Arthur Beaudet) by using plasmid DNAs SE6 and MOPS1 (gifts from Wolfgang Baehr) as probes (2). The 5'-flanking segment of the targeting vectors is a 4.05-kb SacI–SacI fragment, whose 3' boundary lies in the middle of the 5' untranslated region of the rhodopsin transcript at a SacI site that is conserved between the mouse and human rhodopsin genes (see Fig. 1A). In the targeting vector used initially to replace the mouse rhodopsin gene with an HPRT minigene not flanked by lox sites, the 3'-flanking segment was a 6.5-kb EcoRI–EcoRI fragment. All other targeting vectors for homologous replacement carried a 5.5-kb EcoRI–BglII fragment as their 3'-flanking segment. The 5' EcoRI site lies in the 3' untranslated region of the rhodopsin transcript upstream of all of the identified polyadenylation sites (2) (Fig. 1).

To replace the mouse rhodopsin gene with the HPRT minigene (3), three first-step targeting vectors were constructed by attaching the mouse 5'- and 3'-flanking segments to three forms of the HPRT minigene: the minigene alone, the minigene flanked by loxP and lox511 sites, and the minigene flanked by loxH and lox511 sites (Fig. 1). The herpes virus TK gene was attached to one end of the construct to allow selection against random integrants (4). The second-step homologous recombination targeting vector was constructed by attaching the 5'- and 3'-flanking segments to a 7.4-kb SacI–HindIII fragment of the human rhodopsin–GFP gene from plasmid pSRG, whose construction was described (5). Segmental replacement vectors were constructed by cloning the 7.4-kb SacI–HindIII fragment of the human rhodopsin–GFP gene into SacI and HindIII sites that were flanked by either a loxP or a loxH site and by a lox511 site. All cloning was done by using a pBluescriptSK plasmid backbone.

ES Cell Culture and Targeted Modification. The HPRT^– ES cell line AB2.2 123 was grown on g -irradiated SNL 76/7-4 feeder cells in knockout DMEM (Invitrogen) supplemented with 15% FBS (Invitrogen)/100 units/ml PenStrep/2 mM L-glutamine/0.1 mM 2-mercaptoethanol. Feeder cells were grown in knockout DMEM supplemented with 10% FBS, PenStrep, and L-glutamine. Feeder cells were treated in suspension with 60 Gy of g irradiation (Gammacell 1,000 model C) and added to gelatinized plates to make feeder plates to support embryonic stem cell (ES cell) growth.

For homologous gene targeting, ES cells at 10⁷ cells per 0.9 ml were electroporated in a GenePulser II (Bio-Rad) at 230 V and 500 m F in a 0.4-cm gap cuvette by using 15 m g of linearized targeting vector. For segmental replacement, ES cells at 5 × 10⁶ cells per 0.9 ml were electroporated under the same conditions in the presence of 25 m g of supercoiled segmental replacement vector plus 25 m g of the Cre expression vector pOG231. In various experiments, treated ES cells were plated out at 0.2 × 10⁶ to 3 × 10⁶ cells per 10-cm feeder plates in standard culture medium. Selection was applied after 24–48 h. For the first step in the targeting scheme (Fig. 1), HPRT⁺TK^– cells were selected by growth in HAT (0.1 mM hypoxanthine/0.4 m M aminopterin/16 m M thymidine) plus 0.2 m M 1-(2-deoxy-2-fluro-b -D)-arabinofuranosyl-5-iodouracil (FIAU). For the second step (Fig. 1), HPRT^– cells were selected by growth in 10 m M 6-thioguanine.

Colonies that survived selection were screened by Southern blotting (Fig. 1B). DNA from individual colonies was digested with BamHI, electrophoresed on 0.6% agarose gels, blotted onto Nylon membranes (Hybond-N+, Amersham Biosciences), and probed with the 1.1-kb BamHI–SacI mouse genomic fragment upstream of the 5'-flanking homology used in the targeting vectors. The 3' structures of targeted colonies were verified by cutting genomic DNA with HindIII and hybridizing with a 3' probe, either the entire 6.5-kb EcoRI–EcoRI fragment corresponding to the 3'-flanking homology in the targeting vectors, or the 3' 1.0-kb BglII–EcoRI subfragment. All probes were labeled by random priming in the presence of a -³²P-dCTP (Megaprime DNA labeling system, Amersham Biosciences).

Mouse Genotyping. Mice were cared for and handled by following an approved protocol from the Animal Research Committee of Baylor College of Medicine and in compliance with National Institutes of Health guidelines for the care and use of experimental animals. Mice were genotyped by Southern blotting or PCR analysis by using tail DNA (Fig. 1 B and C). Southern blotting was essentially as described above for ES cells. For mice carrying the human rhodopsin–GFP fusion, PCR analysis was performed by using primers that anneal to both mouse and human sequences located on either side of the GFP insertion [5'-GTTCCGGAACTGCATGCTCACCAC (exon 5) and 5'-GGCGCTGCTCCTGGTGGG (3' untranslated)]. Each primer has a single-base mismatch to the mouse sequence. The product for mouse rhodopsin is 194 bp and the product for human rhodopsin–GFP is 933 bp (Fig. 1C). rd mice were genotyped by PCR, using the primers described (6). Mice were analyzed for the HPRT knock-in by using a primer specific for the 5'-flanking region (5'-CAGTGCCTGGAGTTGCGCTGTGG) paired with a second primer specific for exon 1 of the HPRT minigene (5'-CACTAATCACGACGCCAGGGCTGC), which gives a 0.7-kb band.

Eye Fixation and Staining. Eyes were removed from killed mice and fixed in 4% paraformaldehyde (EM Science) in PBS for 1 h at room temperature with gentle shaking. For sections, eyes were soaked in 30% sucrose in PBS at room temperature with gentle shaking until eyes sank. Eyes were frozen in 75% Tissue-Tek OCT (Sakura Finetek, Torrance, CA) and 25% Immu-Mount (Shandon, Pittsburgh) in liquid nitrogen. Sections (10-20 m m thick) were cut with a Microm HM 500 microtome (Microm Instruments, Heidelberg, Germany), air dried, washed three times in PBS at room temperature for 10 min each, mounted with Gel/Mount (Biomeda, Foster City, CA), and examined by fluorescent or confocal microscopy (LSM 510, Zeiss). For retinal wholemounts, the cornea, lens, and vitreous were removed and the retina was dissected from the eye cup. The retina was placed on a slide with the photoreceptors up, flattened by making radial incisions, and mounted with Gel/Mount.

For cone staining, thawed eye sections were post fixed with methanol/acetone (1:1) at room temperature for 10 min and then rehydrated by soaking in PBS three times, each for 10 min. Sections were blocked by incubation with 10% sheep serum in PBS for 1 h at room temperature in a humidifying chamber. After rinsing with water and air drying, sections were stained for cones with rhodamine peanut agglutinin (Vector Laboratories) (1:100 dilution in the blocking solution) at room temperature for 1 h in a humidifying chamber. Retinal wholemounts were fixed with 4% paraformaldehyde in PBS for 1 h, washed in 0.1 M phosphate buffer plus 0.5% Triton X-100 and 0.1% NaN₃ (PB++) for 2 h with four buffer changes, and blotted with 10% sheep serum in PB++ for 2 h at room temperature. Cones were stained with rhodamine peanut agglutinin (1:100 dilution in PB++ with 1% sheep serum) overnight at 4°C, washed with PB++ for 3 h with six buffer changes, and mounted with Gel/Mount.

For mice expressing rhodopsin–GFP, nuclei were counted by using the trace amounts of fluorescence in the nuclear layer. For other mice, nuclei were stained by using TO-PRO-3 iodide (Molecular Probes) in 0.1 M phosphate buffer plus 0.5% Triton X-100, 0.1% NaN₃, and 1% sheep serum for 1 h at room temperature. Sections were then washed with PBS three times for 10 min each, rinsed with water, and mounted with Gel/Mount. Images were captured from different locations in the retina, excluding areas around the optic nerve and the periphery. We counted 30–100 columns of nuclei for each retina and averaged them for each time point.

Northern and Western Blotting. RNA was isolated from retinas homogenized in TRIzol Reagent (Invitrogen) by using RNase-free plastic tubes and pestles. Genomic DNA was sheared by two passes through a 23–26 gauge needle, chloroform (20% by volume) was added, and the sample was shaken vigorously and then centrifuged at 12,000 × g for 15 min at 4°C. RNA was extracted from the aqueous phase by using an RNeasy minikit (Qiagen, Valencia, CA), according to the manufacturer’s recommendations. Samples were electrophoresed on 1% agarose denaturing formaldehyde gel (0.22 M formaldehyde/20 mM Mops buffer, pH 7.0/5 mM sodium acetate/1 mM EDTA), transferred to a Nylon membrane (Hybond-N+, Amersham Biosciences), and probed with bovine rhodopsin cDNA that was labeled by random priming in the presence of a -³²P-dCTP (Megaprime DNA labeling system). To normalize for sample loading, blots were striped and rehybridized with labeled probe from b -actin cDNA or GAPDH cDNA. Samples were quantified by PhosphorImager analysis using IMAGEQUANT 5.2 software (Molecular Dynamics).

For Western blotting, retinas were homogenized in 100 m l of sample application buffer (5% SDS/15% sucrose/50 mM Na₂CO₃/50 mM DTT/1% 2-mercaptoethanol/bromophenol blue), sonicated briefly, and centrifuged to remove debris. Proteins were separated by electrophoresis on a 10% SDS/PAGE (pH 8.8) with a 4% stacking gel (pH 6.8). Proteins were transferred to nitrocellulose membranes by using Transfer Buffer (Bio-Rad) by electrophoresis at 350 mA for 1 h. Membranes were blotted with 5% dry milk in Tris-buffered saline Tween 20 (TBST) (0.05 M Tris·HCl, pH 8.0/0.15 M NaCl/0.05% Tween 20) at 4°C overnight, rinsed twice with TBST, and incubated with 1/300 B6-30N mAb (gift from P. A. Hargrave), which binds the N-termini of mouse and human rhodopsins, in 0.5% dry milk in TBST for 4 h at room temperature. Membranes were washed three times with TBST, incubated with 1/2,000 anti-mouse IgG horseradish peroxidase (HRP) conjugate (Promega) in 0.5% dry milk in TBST for 1 h at room temperature, and washed three times with TBST. Bands were detected with enhanced chemiluminescence Western blotting detection reagents (Amersham Biosciences). Bands were quantified by densitometry by using a Personal Densitometer SI and IMAGEQUANT 5.2 software (Molecular Dynamics).

1. Zheng, B., Mills, A. A. & Bradley, A. (1999) Nucleic Acids Res. 27, 2354–2360.

2. al-Ubaidi, M. R., Pittler, S. J., Champagne, M. S., Triantafyllos, J. T., McGinnis, J. F. & Baehr, W. (1990) J. Biol. Chem. 265, 20563–20569.

3. Harriman, G. R., Bradley, A., Das, S., Rogers-Fani, P. & Davis, A. C. (1996) J. Clin. Invest. 97, 477–485.

4. Mansour, S. L., Thomas, K. R. & Capecchi, M. R. (1988) Nature 336, 348–352.

5. Intody, Z., Perkins, B. D., Wilson, J. H. & Wensel, T. G. (2000) Nucleic Acids Res. 28, 4283–4290.

6. Pittler, S. J. & Baehr, W. (1991) Proc. Natl. Acad. Sci. USA 88, 8322–8326.