Molecular Vision 2006; 12:389-398 <http://www.molvis.org/molvis/v12/a45/> Received 3 October 2005 | Accepted 13 April 2006 | Published 18 April 2006

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Technical Brief

Mouse opsin promoter-directed Cre recombinase expression in transgenic mice

Yun-Zheng Le,^1,2 Lixin Zheng,^2,3,4 Wei Zheng,^2,3 John D. Ash,^2,3 Martin-Paul Agbaga,^1,2 Meili Zhu,^2,3 Robert E. Anderson^1,2,3
 
 

Departments of ¹Cell Biology and ³Ophthalmology, University of Oklahoma Health Sciences Center, Oklahoma City, OK; ²Dean A. McGee Eye Institute, Oklahoma City, OK; ⁴West China Hospital, Sichuan University, Chengdu, Sichuan, China

Correspondence to: Yun-Zheng Le, Dean A. McGee Eye Institute, Room 409, 608 S. L. Young Boulevard, Oklahoma City, OK, 73104; Phone: (405) 271-1087; FAX: (405) 271-8128; email: Yun-Le@ouhsc.edu

Abstract

Purpose: Gene inactivation with homologous recombination in mice is a widely used tool to study gene function. However, many proteins play essential roles in a number of tissues and germline gene inactivation often results in embryonic lethality. To overcome this limitation and to dissect the functions of essential genes beyond embryonic development, we generated mouse rod opsin promoter-controlled cre transgenic mice with a goal of obtaining transgenic lines with a range of Cre activity in rod photoreceptors.

Methods: Transgenic mice expressing Cre recombinase directed by a long or short mouse opsin promoter were generated. Candidate Cre-expressing lines were identified with RT-PCR and Western blot analysis. Potentially useful Cre-expressing lines were characterized further with immunohistochemistry, PCR, and functional analysis using a Cre-activatable lacZ reporter mouse strain (R26R) to determine temporal and spatial patterns of Cre expression. Retinal function and morphology in these mouse lines were analyzed with electroretinography (ERG) and light microscopy of hematoxylin and eosin stained retinal sections.

Results: Transgenic mice expressing Cre in rod photoreceptors were generated. Characterization of candidate photoreceptor-specific Cre mice using immunohistochemistry and functional assays demonstrated that an efficient Cre-mediated recombination occurred in rod photoreceptor cells in one mouse line and a mosaic Cre-mediated recombination occurred in rod photoreceptors and rod bipolar cells in another mouse line. Further analysis of these mice with ERG and morphological examination suggested that the retinas of eight-month-old adults were normal.

Conclusions: We have generated transgenic mice expressing Cre recombinase in rod photoreceptors. One transgenic mouse line was capable of carrying out efficient Cre-mediated recombination in rod photoreceptors. Another transgenic mouse line was capable of carrying out mosaic Cre-mediated recombination in rod photoreceptors and bipolar cells across the whole retina. These mice will be useful tools for Cre/lox-based gene activation and inactivation, as well as genetic mosaics, in rod photoreceptors and rod bipolar cells.

Introduction

Gene targeting with homologous recombination in murine embryonic stem (ES) cells has generated much information in elucidating gene function and pathophysiology of human diseases. However, disruption of essential genes that are involved in multiple tissues often causes embryonic and neonatal lethality. This problem obscures the particular role of genes in a target tissue or in the adult, and can be overcome by using a conditional gene disruption strategy that only disrupts a gene of interest in a target tissue or at a particular time. Owing to its efficiency, the Cre/lox recombination system has become a widely used approach in conditional gene expression. The Cre recombinase of bacteriophage P1 is a 38 kDa protein that catalyzes site-specific DNA recombination between the target 34 bp loxP sites and is proficient in performing recombination in mice [1,2]. The Cre/lox-based gene disruption is based on the site-specific recombination between adjacent loxP sites that results in the deletion of the intervening sequence. This strategy often requires two genetic components: a target mouse carrying a loxP-flanked gene to be disrupted and a transgenic mouse that expresses Cre under the control of a temporal and/or spatial promoter. When these two components are combined in one mouse, the loxP-flanked gene will be deleted in Cre-expressing cells in a temporal or spatial fashion [3,4].

Rods make up 97% of the photoreceptors in mice [5] and the rod-dominant mouse retina is an ideal model for biochemical, morphological, and phenotypic characterization of essential gene functions in the retina. We are interested in the roles of phosphoinositide signaling, glucose transport and metabolism, and neuro-protection in the retina. Establishment of transgenic lines that express Cre with a range of expression levels and recombination efficiencies in rod photoreceptors is a key to the successful dissection of the roles of essential genes in the retina. This report describes the generation and characterization of a short (0.2 kb) mouse opsin promoter-controlled cre (SMOP-cre) mouse line and a long (4.1 kb) mouse opsin-promoter controlled cre (LMOP-cre) mouse line. These mice are available for noncommercial research and can be obtained by contacting the corresponding author.

Methods

The use of animals in this study conformed to the guidelines established by the ARVO statement for the "Use of Animals in Ophthalmic and Vision Research" and was approved by the Institutional Animal Care and Use Committees of the University of Oklahoma Health Sciences Center, the Dean A. McGee Eye Institute, and the Oklahoma Medical Research Foundation.

Construction of a photoreceptor-specific cre transgene

The recombinant plasmid pLE103 carrying a short (0.2 kb) opsin promoter-controlled cre transgene (SMOP-cre) was constructed from plasmids pBS185 [2] and pMOPS [6]. The recombinant plasmid pLE111 carrying a long (4.1 kb) opsin promoter-controlled cre transgene (LMOP-cre) was constructed from pBS185 [2] and pRG3 [7]. Briefly, the 0.2-kb mouse opsin promoter on pMOPS was inserted in front of cre in the pBS185 to generate SMOP-cre plasmid pLE103 (Figure 1A). The 4.1-kb mouse opsin promoter on pRG3 was inserted in front of cre in the pBS185 to generate LMOP-cre plasmid pLE111 (Figure 1A). Both plasmids contained HindIII restriction sites flanking the transgene that carries the short or the long mouse ospin promoter, a translationally optimized cre [8], and an intron containing mouse metallothionein (MT-I) polyadenylation signal (Figure 1A). Junctions between different parts of the transgene were confirmed by DNA sequencing.

Generation and genotyping of transgenic mice

Cesium chloride double centrifugation was used to prepare the plasmid DNA carrying the transgene. The purified plasmid DNA was digested with the restriction enzyme HindIII to isolate the transgene (Figure 1A). The digested DNA was fractionated on an agarose gel and purified with Qiagen gel extraction kit (Valencia, CA). The purified DNA was used in zygote injection with FVB/N background mice at the Oklahoma Medical Research Foundation Microinjection Core Facility. PCR diagnostic for cre transgene was performed according to the procedures described by Le and Sauer [4] using primers (a) 5'-AGG TGT AGA GAA GGC ACT TAG C-3' and (b) 5'-CTA ATC GCC ATC TTC CAG CAG G-3' to detect a 411 bp product. Further confirmation of the presence of the transgene was performed with Southern hybridization using a 0.5-kb cre DNA fragment, according to an established procedure [9]. PCR detection of the Cre-activatable lacZ reporter gene in R26R mice was performed according to an established procedure [10] with primers (c) 5'-GAG TTG CGT GAC TAC CTA CGG-3' and (d) 5'-GGC TTC ATC CAC CAC ATA CAG G-3' to detect a 495 bp product. PCR detection of Cre-mediated lacZ reporter gene activation in the R26R mouse genomic DNA was performed using primers (e) 5'-AAA GTC GCT CTG AGT TGT TAT-3' and (f) 5'-CAT TCG CCA TTC AGG CTG CG-3' to detect a 710 bp product (25 to 30 cycles at 94 °C for 30 s, 64 °C for 30 s, and 72 °C for 60 s). PCR diagnostic for an internal DNA control, β-actin, was performed according to an established procedure [10] using primers (g) 5'-GAC GAG GCG CAG AGC AAG AGA GG-3' and (h) 5'-CTC TTT GAT GTC ACG CAC GAT TTC-3' to detect a 450 bp product.

Reverse transcriptase polymerase chain reaction analysis

RT-PCR analysis was performed similarly according to a documented procedure [10] using primer pairs (a) and (b), and (g) and (h) (described above) to detect a 411 bp cre and 450 bp β-actin transcripts, respectively.

β-Galactosidase assay

The function of Cre in transgenic mice was analyzed with β-galactosidase (β-gal) staining assays using the retinas of F1 double transgenic mice derived from Cre and Cre-activatable lacZ reporter mice [11]. The β-gal assay was carried out on a retinal whole mount using an established method [12]. For β-gal assays on frozen retinal sections, genotyped mice were euthanized and a permanent dye was injected at the superior pole of the cornea to maintain orientation. The dissected eyes were fixed in 2% paraformaldehyde and 0.25% glutaraldehyde in phosphate-buffered saline (PBS) at 4 °C for 1 h. After the removal of lens and vitreous, the remaining part of the eyes was cryoprotected, mounted in OCT medium, and cut at 10 μm on a cryostat. The sections were briefly fixed in 0.5% glutaraldehyde for 5 min, washed with PBS 3 times, and incubated overnight at room temperature in X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactoside) staining solution (1 mg/ml X-gal, 6 mM potassium ferricyanide, 6 mM potassium ferrocyanide, 2 mM MgCl₂, 0.02% NP-40, and 0.01% sodium deoxycholate in 1X PBS). Sections were then cover-slipped and observed under a microscope.

Immunohistochemistry and Western blot analysis

For immunohistochemistry (IHC), frozen retinal sections were prepared as described previously [12]. The anti-Cre polyclonal antibody (Novagen, San Diego CA) was diluted 1:200 for IHC and 1:2500 for Western blotting. The anti-protein kinase C-α monoclonal antibody (Sigma, St. Louis, MO) was diluted 1:500 for IHC. The anti-β-galactosidase polyclonal antibody (5 Prime-3 Prime, Boulder CO) was diluted 1:100 for IHC. The biotinylated peanut agglutinin (Vector Laboratories, Burlingame, CA) was diluted 1:500 for IHC. The anti-β-actin monoclonal antibody (Affinity Bioreagents, Golden, CO) was diluted 1:1000 for Western blotting. The procedures for primary and secondary antibody incubation followed the conditions described previously [12].

Quantification of Cre-expressing cells

Quantification of Cre-expressing cells was performed by counting the number of Cre-positive or Cre-negative nuclei in confocal images of anti-Cre antibody stained retinal sections. To ascertain the accuracy of the assay, three confocal images in peripheral and central areas of a retina were used and each image contained approximately 500 or more rod nuclei. At least three mice in each group were used in the calculation. The ratio of Cre-positive cells was expressed as the percentage±SD.

Electroretinography and retinal morphology

Overnight dark-adapted mice were used for scotopic and photopic electroretinography (ERG) with a UTAS-E 3000 ERG system (LKC technologies, Inc., Gaithersburg, MD), according to the conditions described previously [6,12,13]. For scotopic ERG, a strobe flash stimulus was presented to the overnight, dark-adapted, dilated eyes in a Ganzfeld with a 138 cd x s/m² flash intensity. For photopic ERG, a strobe flash stimulus was presented to 5-min, light-adapted, dilated eyes in a Ganzfeld with a 79 cd x s/m² flash intensity. For retinal morphology, the eyes were dissected after euthanization and fixed at room temperature overnight in Perfix (4% paraformaldehyde, 20% isopropanol, 2% trichloroacetic acid, 2% zinc chloride), paraffin-embedded, sectioned (5 μm thickness), and stained with hematoxylin and eosin. The thickness of the outer nuclear layer (ONL) was measured at a distance of 240 μm from the optic nerve to the inferior and superior ora serrata. Differences were assessed with Student's t-test. p<0.05 was considered significant. All results requiring statistic analysis were expressed as the mean±SD.

Results & Discussion

Generation of opsin promoter-controlled cre transgenic mice

Previous studies have shown that a 4.1-kb or 0.2-kb mouse opsin promoter was capable of directing transgenic expression in rod photoreceptors [6,7]. However, indirect comparison of different lengths of mouse or bovine opsin promoters in transgenic studies suggested that the longer opsin promoter is likely to confer a more uniformed transgenic expression in rod photoreceptors, while the short opsin promoter is likely to achieve a mosaic expression pattern [7,14]. With a goal of obtaining a range of Cre activity in the rod photoreceptors of mice, we generated transgenic mice with the 4.1-kb and the 0.2-kb mouse opsin promoters to direct Cre expression (Figure 1A). The 4.1-kb long mouse opsin promoter-controlled cre (LMOP-cre) and 0.2-kb short mouse opsin promoter-controlled cre (SMOP-cre) constructs were used in generating transgenic mice. The transgenic mice were identified by PCR analysis and confirmed with Southern blot hybridization (data not shown). The cre-positive mice were mated to obtain germline transmitted mice. All germline transmitted mice appeared normal in size, morphology, and behavior, and were screened for cre expression with reverse transcription (RT)-PCR using 7 to 15-day-old retinas (Figure 1B). Mice expressing cre mRNA were subjected to functional analysis using an existing Cre-activatable lacZ reporter mouse line R26R in C57B6 background [11]. Since the F1 double transgenic Cre/R26R mice did not have inherited retinal degeneration, all further characterization was performed using this type of mice. Our preliminary characterization of all candidate lines with the F1 double transgenic Cre/R26R mice indicated that one LMOP-cre (LMOPC1) and one SMOP-cre (SMOPC1) transgenic line had productive Cre-mediated lacZ reporter expression across the retina, as well as normal retinal morphology and function (data not shown, see detailed discussion below). These mice were characterized further. Cre expression at the protein level in these mice was analyzed by Western blotting using retinal homogenates from F1 double transgenic Cre/R26R mice, as shown in Figure 1C.

Localization and functional analysis of Cre expression

As discussed earlier, an existing Cre-activatable lacZ reporter mouse line R26R [11] was utilized to identify potentially useful mouse lines. The R26R reporter mice carry a loxP-flanked transcriptional "STOP" sequence that prevents the transcription of the lacZ gene (Figure 2A). In the double transgenic cre/lacZ mice, this transcriptional "STOP" sequence can be removed only in cells expressing Cre. The β-galactosidase (β-gal) will then be expressed under the generalized promoter ROSA26. Thus, the Cre-expressing cells will produce a blue color using β-gal staining assay.

In the F1 double transgenic LMOPC1/R26R mice, a weak β-gal reporter expression was detected at postnatal day 7 (P7). Expression increased gradually until approximately six weeks of age when no further change was detected. Strong β-gal expression was localized to the outer nuclear layer where photoreceptors are located (Figure 2B,D,F). Immunohistochemistry (IHC) analysis of Cre expression in the retinal sections with an anti-Cre antibody suggested that Cre was mainly localized within the perinuclear cytoplasm and the membrane of the rod photoreceptor nuclei (Figure 3A), which supports our earlier observation that Cre carried a eukaryotic nuclear localization signal [15]. Confocal microscopic analysis of the retinal sections stained for Cre with an anti-Cre antibody and the cone photoreceptor marker stained with peanut agglutinin (PNA) showed that PNA-positive cones were not Cre positive (Figure 3D), suggesting that Cre was exclusively localized to the rod photoreceptors. The onset of Cre expression in the LMOPC1 mice was also determined by PCR analysis diagnostic for Cre-mediated activation of lacZ reporter gene with the genomic DNA isolated from the F1 double transgenic retinas of LMOPC1/R26R mice. The PCR analysis suggested that Cre-mediated recombination was detected at P7 (Figure 4A), when a weak β-gal stain was observed in the retinal sections.

In the F1 double transgenic SMOPC1/R26R mice, a weak Cre-activated β-gal reporter expression was detected at P11 when Cre-mediated recombination at the DNA level was observed (Figure 4A). Expression increased gradually until approximately eight weeks of age, when no additional change was detected (Figure 2C,E,G). The expression was localized to the presumptive photoreceptor cells and a subset of cells in the inner nuclear layer across the whole retina (Figure 2E,G). However, the intensity of the β-gal staining in the rod photoreceptors from SMOPC1/R26R mice was lower than in the rod photoreceptors obtained from LMOPC1/R26R mice in a side-by-side comparison of age-matched retinal sections (Figure 2D-G), suggesting that the efficiency of Cre-mediated recombination in the SMOPC1 mouse line was relatively lower than in the LMOPC1 mouse line. This was indeed reflected in the lower level of immunoreactivity measured in a side-by-side assay with the same anti-Cre antibody used to localize Cre expression in the LMOPC1 mice (Figure 3). Nevertheless, the Cre-expression was mainly localized within the perinuclear cytoplasm and the membrane of the rod photoreceptor nuclei, indicating that Cre was expressed in the rod photoreceptors of the SMOPC1 mice. However, our IHC assay did not detect any anti-Cre immunoreactivity in the inner nuclear layer of the SMOPC1 mice, suggesting that a low level of Cre was present in the β-gal-positive stained cells in this region, or Cre could be expressed early in the precursor cells that ultimately give rise to the bipolar cells.

To localize Cre-activatable lacZ expression in the inner nuclear layer, we performed a combination of β-gal staining and immunostaining on retinal sections with anti-synaptophysin and anti-calbindin antibodies, markers for photoreceptor terminals in the outer plexiform layer and horizontal cells [16,17]. The β-gal stained cells were clearly in the inner nuclear layer and were not the terminals of photoreceptors. In addition, β-gal staining did not co-localized with calbindin-positive horizontal cells (data not shown). Double labeling retinal sections with anti-β-galactosidase and anti-protein kinase C (PKC) antibodies demonstrated that β-gal and PKC, a rod bipolar marker [18], co-localized in the inner nuclear layer (Figure 5), indicating that Cre activated the lacZ reporter in rod bipolar cells (white arrows in Figure 5E-G). This result suggested that Cre was expressed in rod bipolar cells in the SMOPC1 mice at a level sufficient for productive Cre-mediated recombination. An alternative approach to localize Cre expression in the INL of SMOPC1 could be to use Cre-activatable fluorescent reporter mice, such as Z/EG reporter mice [19], in conjunction with immunostaining. The Z/EG reporter mice could be beneficial in precise localization of Cre expression in transgenic mice.

Although we only characterized one line from this transgenic construct and thus it is not possible to conclude whether the expression in the rod bipolar cells was caused by regulatory elements on the 0.2-kb opsin promoter or a positional effect in transgenics, the following scenario could account for the observed expression pattern in SMOPC1 mice. It was reported that the human blue cone promoter was capable of directing transgene expression in a subset of bipolar cells [20,21]. In these studies, transgenic expression in the bipolar cells is likely controlled by the interaction of the cis-acting element with transcription factors, such as OTXs or CRX, that are capable of directing photoreceptor- and bipolar cell-specific gene expression under normal conditions [22-26]. The loss of up-stream repressive element(s) could cause the 0.2-kb opsin promoter to direct the transgene expression in bipolar cells.

Both LMOPC1 and SMOPC1 mice were capable of performing productive Cre-mediated recombination. However, the expression of both Cre and Cre-activatable β-galactosidase in the SMOPC1/R26R mice was lower than that in the age-matched LMOPC1/R26R mice, suggesting that SMOPC1 mice produced relatively less Cre than LMOPC1 mice. These results were supported by the observation that Cre-mediated recombination in the SMOPC1/R26R mice was detected at P11 (Figure 4A), four days later than in the LMOPC1/R26R mice. These observations also correlated with a level of Cre protein demonstrated by Western blot analysis of retinal homogenates (Figure 1C). In our hands, SMOPC1 mice consistently showed a lower level of Cre protein (Figure 1C). Our results also suggested that from P30 to P60, there were no significant increases in the amounts of retinal Cre protein in both LMOPC1 and SMOPC1 mice (Figure 1C).

To determine the ratio of Cre-positive photoreceptors in LMOPC1 and SMOPC1 mice, a direct assay with quantitative confocal microscopy (as shown in Figure 3D) was performed by calculating the percentage of Cre-positive rod nuclei in the anti-Cre antibody-stained retinal sections from three-month-old mice. Our results showed that there were no significant changes in the ratio of Cre-positive photoreceptors between the peripheral and the central retina in each mouse and 77.5% of LMOPC1 rods and 43.3% of SMOPC1 rods had detectable Cre expression (Figure 3E). However, no apparent increases in the ratio of Cre-positive rods were observed in six-month-old mice (data not shown), suggesting that the number of Cre-expressing cells was relatively stable after early development. Since a successful Cre-mediated recombination requires only four Cre recombinase molecules [27] and immunohistochemistry is not likely to detect cells expressing a very low level of Cre, the ratios of Cre-expressing rods in these mice were likely a conservative estimation.

Ectopic expression of Cre

The level of ectopic Cre expression was examined in the following representative tissues: brain, heart, liver, muscle, kidney, and spleen in both two-month-old F1 double transgenic LMOPC1/R26R mice and two-month-old F1 double transgenic SMOPC1/R26R mice. PCR analysis diagnostic for Cre-mediated recombination in the R26R mouse genomic DNA suggested that there was no apparently productive Cre-mediated recombination in these representative tissues in either the LMOPC1 mice or the SMOPC1 mice (Figure 4B). This result was also supported by the observation that no detectable Cre-mediated lacZ reporter expression was observed beyond the negative control level in these representative tissues from either the F1 double transgenic LMOPC1/R26R mice or the F1 double transgenic SMOPC1/R26R mice (data not shown). Therefore, the LMOPC1 mice and the SMOPC1 mice are not likely to cause any phenotypic changes in non-ocular tissues in conditional gene knockout studies.

Integrity of rod photoreceptors

Several lines of evidences suggest that over-expression of Cre could cause unwanted chromosomal rearrangement in mammals [28,29]. Over-expression of Cre in photoreceptors may induce retinal degeneration and the transgenic mice may not be suitable for gene function studies. This concern was supported by previous observations that over-expression of Cre in rod photoreceptors correlated with retinal degeneration (data not shown) [30]. Thus, the rod photoreceptor integrity was a major criterion in our characterization of potentially useful Cre mice. ERG and morphologic analysis for LMOPC1 and SMOPC1 mice were performed using eight-month-old F1 double transgenic Cre/R26R mice. The scotopic and photopic ERG analysis suggested that both LMOPC1 mice and SMOPC1 mice had normal rod and cone function when they were eight-month-old (Figure 6). Hematoxylin and eosin staining of the retinal sections from eight-month-old F1 double transgenic Cre/R26R mice showed that Cre expression did not cause retinal degeneration and that the retinas of both the LMOPC1 transgenic mice and the SMOPC1 transgenic mice were normal (Figure 7). Since Western blot analysis of Cre expression with retinal homogenates showed that there was no apparent increase in Cre protein accumulation in either the LMOPC1 mice or the SMOPC1 mice from P30 to P60 (Figure 1C), the effect of Cre toxicity is not likely to increase over time in the adult mice. Therefore, these mice are potentially useful for gene function studies.

Summary

To dissect gene function in rod photoreceptors, several laboratories have made tremendous efforts to generate and characterize rod-specific cre mice [30-32]. A common goal of these studies was to generate a resource of rod-specific Cre mouse lines with a range of expression levels to suit the requirement for each individual studies. At this time, there are two published potentially useful rod photoreceptor-specific Cre mouse lines [31,32]. Regarding any possible redundancy among these Cre-expressing animals, having multiple Cre lines is definitely beneficial, as no two Cre-expressing lines are the same, and subtle variations in the expression of Cre is useful in understanding the function of a floxed gene. Thus, there is indeed great importance and necessity for several Cre mouse lines. Here we contribute two new novel and useful mouse lines expressing Cre in rod photoreceptors. Mice have 20 chromosomal pairs, thus there is a chance of 5% that a cre transgene and a floxed gene of interests are located on the same chromosome. Our LMOPC1 mice expressed a detectable level of Cre in at least 77.5% of the rods and thus can be utilized for studies requiring efficient Cre-medicated gene activation or inactivation in rod photoreceptors (Figure 3E). The SMOPC1 mice expressed a detectable level of Cre in 43.3% of the rods across the whole retina. The mosaic Cre-mediated recombination in the SMOPC1 mice (Figure 2E,G, Figure 3E) could result in a less detrimental phenotype in mice and may provide an advantage for some gene knockout studies. Owing to the relatively little effort and a high reproducibility in generating desired phenotypes, genetic mosaics have become a method of choice to investigate cell lineages, patterns of growth and gene function, and to provide a means to circumvent the challenge in phenotypic analysis of mice generated with an all-or-none genetic approach [33]. Since the conditional gene knockout of Bcl-x with SMOPC1 mice resulted in detectable functional changes [34], the level of Cre expression in SMOPC1 mice is sufficient for photoreceptor gene inactivation. In addition, the amount of Cre protein present in the SMOPC1 mouse retina was much lower than in the LMOPC1 mouse retina (Figure 1C), the long-term Cre toxicity, if any, should be much lower in the SMOPC1 mouse line. Finally, the SMOPC1 mice expressed Cre in rod photoreceptors and rod bipolar cells; therefore, this novel mouse strain may also be useful for dissecting the function of genes that are involved in multiple neuronal processes in the rod photoreceptors and/or rod bipolar cells.

An additional advantage of having more than one characterized rod-specific cre mouse line is that it will circumvent a situation where disruption of a particular gene is located on the same chromosome of the cre transgene insertion. For conditional gene knockout studies, a low cost experiment to determine whether a floxed gene is located on the same chromosome of cre transgene insertion is to simply breed Cre mice with the mice carrying a floxed gene for two generations. The genotyping results from F2 mice will tell if the floxed gene is on the same chromosome of cre transgene insertion.

Acknowledgements

The authors declare that there are no commercial relationships in any part of this study. We thank Cassandra Kontas, Linden Cowley, and Yuwei Le for technical assistance; Dr. Mathew LaVail for preliminary characterization of the retinal morphology; Drs. Muayyad R. Al-Ubaidi, Janis Lem, and Brian Sauer for providing the plasmids used in this study; and the Transgenic Core Facility at the Oklahoma Medical Research Foundation for generating the transgenic mice. This study was supported by OCAST contract HR01-083; NIH grants RR17703, EY00871, EY12190, and EY04149; Research to Prevent Blindness, Inc., and the Foundation Fighting Blindness, Inc.

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