Generation of a Transgenic Rabbit Model of Retinal Degeneration

Abstract

purpose. To generate a transgenic (Tg) rabbit model of retinal degeneration and to characterize the pattern of degeneration by using histology and electrophysiology.

methods. Rhodopsin Pro347Leu Tg rabbits were generated by BAC transgenesis. Tg rabbits were identified by Southern blot analysis, and the expression levels were measured by quantitative RT-PCR. Retinal histology was examined by light and electron microscopy and immunohistochemistry. Retinal function was assessed by full-field electroretinograms (ERGs).

results. Six lines of Tg rabbits were generated, and two lines with higher levels of expression showed rod-dominant progressive retinal degeneration. Retinal histology indicated a marked regional variation in the loss of photoreceptors with the central retina more severely affected than the peripheral retina. The characteristics of the ERGs of transgenic rabbits indicated that the rod components of the ERGs were reduced to only 5% by 48 weeks, whereas the cone components remained at 35% in the wild-type at the same time point. The retinal ultrastructure of Tg rabbits showed a large number of small vesicles that accumulated in the extracellular space of the photoreceptors.

conclusions. To the best of the authors’ knowledge, this is the first rabbit model of progressive retinal degeneration. Because rabbits have large eyes and are easy to handle and breed, they will provide a useful animal model for the study of the pathophysiology of and new treatments for retinal degeneration.

Retinitis pigmentosa (RP) is the name given to a group of inherited retinal disorders characterized by a progressive loss of rod and cone photoreceptors and eventual atrophy of the entire retina. ^{1 2 3} The worldwide prevalence of RP is approximately 1 in 4000, meaning that more than 1 million individuals are affected worldwide. ³ RP is genetically heterogeneous; mutations in several photoreceptor-specific and some nonspecific genes are known to cause the condition. ⁴ Of these, mutations in the rhodopsin gene are the most prevalent class identified to date, causing approximately 25% to 40% of the autosomal dominant RP cases. ^{5 6}  

Animal models of RP are important for understanding the pathophysiology and for developing new treatments for these diseases. Various naturally occurring and genetically manipulated animal models of RP have been studied—for example, fruit flies, zebrafish, chickens, mice, rats, cats, dogs, and pigs (for reviews, see Refs. ⁷ , ⁸ ). Of these models, midsized and large animal models have become particularly important because their large eyes make it easier to test new treatments. These treatments may include surgical procedures such as intraocular devices, ^{9 10} subretinal injection of genes for gene therapy, ¹¹ and implantation of retinal prostheses. ¹² Several models of retinal degeneration have been identified or generated in cats, ^{13 14} dogs, ^{15 16} and pigs, ¹⁷ and colonies of affected animals have been established. However, a rabbit model of progressive retinal degeneration has not yet been produced, despite the fact that this animal has large eyes and is easy to breed and handle. In addition, there is a considerable accumulation of past works on the anatomy and physiology of the rabbit eye. ^{18 19 20 21}  

Recent advances in the use of bacterial artificial chromosomes (BACs) modified by homologous recombination have promoted the use of this powerful tool in the generation of transgenic (Tg) animals because this technique makes possible the precise and efficient engineering of large DNA fragments. ^{22 23 24 25} In the present study, we used BAC transgenesis to generate a rhodopsin Tg rabbit model of retinal degeneration. These Tg rabbits exhibited rod-dominant, progressive photoreceptor degeneration and striking regional variation in the pattern of photoreceptor loss. 

Methods

This study was conducted in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. All protocols were approved by the Institutional Review Board of the Nagoya University Graduate School of Medicine. 

Rabbit Rhodopsin BAC Clone

The rabbit rhodopsin BAC clone, LB1-7M22, was selected from the LBNL-1 New Zealand White Rabbit BAC library by hybridization of high-density arrayed nylon filters using a probe that consisted of a [P³²]-labeled fragment of exon 5 of the rabbit rhodopsin gene. The clone was obtained from the BACPAC Resources Center at the Children’s Hospital of Oakland Research Institute. The presence of a full-length rabbit rhodopsin genomic sequence was verified by Southern blot analysis with a probe that consisted not only of exon 4 of the rhodopsin gene but also exon 14 of the rabbit WDR10 gene and exon 25 of the rabbit PLXND1 gene. These latter two genes are the 5′- and 3′-flanking genes, respectively, of the rabbit rhodopsin gene (Fig. 1A) . The exon 4 genomic fragment of the rabbit rhodopsin gene, exon 14 of the rabbit WDR10 gene, and exon 25 of the rabbit PLXND1 gene were amplified by PCR and subcloned into the pGEM-T easy vector (Promega, Madison, WI) for labeling with [P³²]. 

BAC Tg Construct

A rhodopsin P347L BAC Tg construct harboring a C-to-T transition in exon 5 of the rabbit rhodopsin gene was generated by BAC recombineering (Fig. 1B) . A point mutation was introduced into the rabbit rhodopsin BAC clone. ²⁴ In brief, an rpsL-neo counter selection cassette, flanked by 40-nucleotide homologous arm sequences on either side of the C-to-T transition site of the rhodopsin gene, was amplified by PCR. The amplified rpsL-neo counter selection cassette was inserted into the rhodopsin gene of the rabbit BAC clone by Red/ET recombination. The subcloned exon 5 fragment of the rabbit rhodopsin gene was modified with a C-to-T transition at proline 347, and the serial restriction sites KpnI, PstI, and BglII in the 3′-untranslated region. The modified sequence was subcloned into the pGEM-T easy vector for sequencing. The rpsL-neo cassette inserted into the rhodopsin gene of the rabbit BAC clone was replaced by the modified exon 5 fragment by using rpsL counter selection. The BAC modification was verified by Southern blot analysis and sequencing (Fig. 1C) . The rhodopsin P347L BAC transgene was purified in a modified procedure. ²⁶ The BAC Tg construct was extracted from 250 mL of Escherichia coli culture. For purification, 10 μg of the BAC Tg construct was linearized overnight with PI-SceI endonuclease (New England Biolabs, Beverly, MA), which cleaves a unique site in the BACe3.6 vector sequence. The linearized BAC DNA was separated by pulsed-field gel electrophoresis (PFGE) and extracted from the preparative pulsed-field gel by electroelution. After dialysis against a TE buffer containing 0.1 mM EDTA, aliquots of DNA were subjected to PFGE for size and quality control. The BAC DNA concentration was adjusted to 1 ng/μL for microinjections. The aliquots of BAC DNA solution were stored at 4°C until the microinjections were performed. 

Rhodopsin P347L Tg Rabbits

Rhodopsin P347L Tg rabbits were generated by pronuclear injection of the BAC Tg construct into New Zealand White rabbit embryos. Transgenic founders and germline transmission of the BAC Tg construct were assessed by Southern blot analysis of BglII-digested ear DNA, which was probed with a [P³²]-labeled exon 4 fragment of the rabbit rhodopsin gene. 

DNA Fluorescence In Situ Hybridization Analysis

DNA FISH analysis was used to examine the actual site of the integrated transgene for each Tg line. Chromosome preparations were obtained with standard techniques and hybridized with a full-length rhodopsin P347L BAC Tg construct as a probe. The probe was labeled with biotin (Roche Diagnostics GmbH, Mannheim, Germany) and detected with avidin-FITC (Fluorescein Avidin D; Vector Labs, Burlingame, CA). The site of the transgene integration was determined by using a standard rabbit chromosome map. ²⁷  

Quantitative RT-PCR

One milligram of total retinal RNA (12 weeks of age) was incubated with 200 units of reverse transcriptase (SuperScript II; Invitrogen, Carlsbad, CA), and the cDNA was used for quantitative RT-PCR (qRT-PCR; QuantiTect SYBR Green PCR Kit; Qiagen, Valencia, CA) and a thermocycler (LightCycler 1.5; Roche Applied Science, Indianapolis, IN). Twenty-microliter reactions were loaded into the thermocycler containing 2 μL of the cDNA sample and 0.5 μM of primers specific for the mutated rhodopsin (forward: 5′-CTA CAT CAT GAT GAA CAA GCA G-3′ and reverse: 5′-TGG CTG GTC TCC GTC TTG GAA-3′) or common primers for wild-type (WT) and mutated rhodopsin (forward: 5′-CTA CAT CAT GAT GAA CAA GCA G-3′ and reverse: 5′-GCA GTG CAG ATC TGC AGG T-3′). For quantification, a standard curve was generated from a cDNA template for each gene. The relative levels of transgene expression were quantified as a ratio of the Tg to the endogenous rhodopsin mRNA. 

Clinical Ophthalmic Observations

Ophthalmic examinations were conducted every month after birth. Examinations of the cornea, anterior chamber, iris, and lens were performed by slit-lamp biomicroscopy. The vitreous and retina were examined by indirect ophthalmoscopy. A fundus camera (Kowa, Nagoya, Japan) was used for fundus photography and fluorescein angiography. 

Electroretinograms

Animals were dark-adapted for 60 minutes, then anesthetized with ketamine (25 mg/kg, IM) and xylazine (2 mg/kg, IM). ERGs were recorded with Burian-Allen bipolar contact lens electrodes (Hansen Laboratory, Iowa City, IA). The animals were placed in a Ganzfeld bowl and stimulated with stroboscopic stimuli of 2.2 log cd · s · m⁻² (photopic units) maximum intensity. Eight steps of stimulus intensities, ranging from −4.8 to 2.2 log cd · s · m⁻², were used for the scotopic ERG recordings, and four steps of stimuli, ranging from −0.8 to 2.2 log cd · s · m⁻², were used for the photopic ERGs. The photopic ERGs were recorded on a rod-suppressing white background of 1.3 log cd · m⁻². The signals were amplified, bandpass filtered between 0.3 and 1000 Hz, and averaged by a computer-assisted signal analysis system (MEB-9100 Neuropack; Nihon Kohden, Tokyo, Japan). The electrical activities of the rod and cone photoreceptors were assessed by the maximum response of the rod and cone a-waves. The maximum rod a-wave was extracted by waveform subtraction of the photopic ERG from the scotopic ERG at the maximum stimulus intensity of 2.2 log cd · s · m⁻². 

Rod and cone photoreceptor function was also assessed by the a-wave (P3)-fitting model of Hood and Birch. ²⁸ The a-wave was fitted with the following equation:  

\[\mathrm{P}3(i,\ t){=}{\{}1{-}\mathrm{exp}{[}{-}i\ {\cdot}\ S(t{-}t_{\mathrm{d}})^{2}{]}{\}}\ {\cdot}\ Rm\ (\mathrm{for}\ t{>}t_{\mathrm{d}})\]

where i is the flash energy (log cd · s · m⁻²); t _d is the time delay, t is the time after the flash onset, S is the sensitivity, and Rm is maximum response amplitude. 

Retinal Histology

Rabbit eyes were fixed overnight in a mixture of 10% neutral buffered formalin and 2.5% glutaraldehyde; F-G fixative), then transferred to 10% neutral buffered formalin. The tissues were trimmed, embedded in paraffin, sectioned vertically through the optic nerve (superior-inferior), and stained with hematoxylin and eosin. The thickness of the outer nuclear layer (ONL) was measured at 10 locations at 2-mm intervals. 

Immunohistochemistry

Freshly prepared rabbit eyes were fixed with 4% formaldehyde in phosphate buffer for 2 hours at 4°C. After fixation, the eyes were immersed in 20% sucrose, frozen in OCT compound (Sakura Finetechnical Co., Ltd., Tokyo, Japan), and sectioned at 15 μm. The tissue sections were processed for immunofluorescence staining with anti-rhodopsin antibody (RET-P1; Santa Cruz Biotechnology), followed by Alexa Fluor 488-conjugated anti-mouse IgG and Alexa Fluor 568-conjugated peanut agglutinin (PNA; Invitrogen), a lectin that binds specifically to rabbit cone photoreceptors. ²⁹ Specimens were observed with a fluorescence microscope (BX61 microscope with digital photograph system DP70-BSW; Olympus, Tokyo, Japan). 

Electron Microscopy

Eyes were enucleated from anesthetized rabbits (6-week-old WT and line 7 Tg rabbits). The anterior segment was removed, and the retina was fixed in 2.5% glutaraldehyde for 2 hours. After subsequent fixation in 1% osmium tetroxide for 90 minutes, the retina was dehydrated through a graded series of ethanols (50%–100%), and cleared in propylene oxide. Finally, the tissue was embedded in epoxy resin. Ultrathin sections were cut on an ultramicrotome (Ultracut E; Reichert-Jung, Vienna, Austria) and stained with uranyl acetate and lead citrate. The stained sections were observed by transmission electron microscopy (H-7650; Hitachi Co., Tokyo, Japan). 

Results

Generation of Tg Rabbits

We identified a rabbit rhodopsin BAC clone that included sequences approximately 150 kb upstream of the transcription initiation codon, the entire rhodopsin structural gene, and sequences downstream of the termination codon of the gene (Fig. 1A) . Assuming that these genomic sequences would lead to correct expression of the rhodopsin gene, we inserted a C-to-T transition into the BAC clone in the codon of proline 347 by using BAC recombineering (Fig. 1B) . The mutation introduced into the BAC Tg construct was then confirmed by sequence analysis (Fig. 1C) . The C-to-T transition in exon 5 of the rabbit rhodopsin gene locus resulted in a proline-to-leucine substitution at codon 347. 

After the BAC modification in E. coli, the linearized BAC Tg construct was purified and injected into rabbit embryos at the pronucleus stage. PFGE showed that the purified rhodopsin P347L Tg construct was approximately 150 kb (Fig. 2A) . Southern blot analysis showed that 12 of 80 newborn rabbits (15%) were transgene positive, and 10 of the 12 survived (Table 1) . These 10 founders were bred with WT rabbits, and six founders transmitted the transgene to their offspring. 

Characteristics of Each Line of Tg Rabbit

FISH analysis showed that five founders, rabbits 7, 8, 10, 11, and 14, had a single site of transgene integration, and one founder, rabbit 16, had integrations at two sites (Table 2) . Three lines, 10, 11, and 14, carried low copy numbers of the transgene, and two lines, rabbits 7 and 8, carried high copy numbers (Fig. 2B) . The founder of line 16 carried both high and low copy numbers with transgene insertions on different chromosomes that yielded two different lines, 16a and 16b (Fig. 2B) . 

The transgene copy number estimated by Southern blot analysis correlated roughly with the level of transgene expression and the degree of photoreceptor degeneration as determined by the ERG a-wave amplitude (Table 2) . Lines 7 and 8, which had higher transgene copy numbers, had higher levels of transgene expression and showed a rapid, progressive reduction in the a-wave amplitude. In contrast, lines 10, 11, and 14, which had lower copy numbers, had lower transgene expression levels, and the a-wave amplitude was not significantly different from that of age-matched (12 weeks of age) WT rabbits. The a-wave amplitudes for these three lines remained within the normal range, even at 48 weeks (data not shown). 

Because of a restriction in the number of rabbits that could be housed in our animal facilities, we mainly produced and investigated line 7, which had the highest level of transgene expression and the most severe photoreceptor degeneration. 

Clinical Findings

Tg rabbits from all lines had normal corneas, anterior chambers, and clear lenses. There was no difference in the fundus appearance or fluorescein angiograms between WT and Tg rabbits at any age up to 40 weeks (Fig. 3) . However, it should be noted that Tg animals were on an albino background, and the characteristic bone spicule pigmentation of the retina seen in RP eyes would therefore not be expected. 

Retinal Histology and Immunohistochemistry

Retinal histology in the area of the visual streak, the central area of the rabbit retina, of a WT and a line 7 Tg rabbit at different ages are shown in Figure 4A . At 2 weeks of age, the retinal histology of Tg rabbits was nearly indistinguishable from that of WT rabbits. Both types of rabbits had six or seven layers of nuclei in the ONL. Thereafter, the thickness of the ONL in Tg rabbits progressively decreased (Fig. 4B) . At 48 weeks, only a single row of nuclei remained in the ONL of the retina of Tg rabbits. In contrast, the architecture and thickness of the middle and inner retinal layers were relatively well preserved even at 48 weeks of age. 

We also examined the retina of Tg rabbits by immunohistochemistry using an anti-rhodopsin antibody and PNA lectin. There was no detectable rhodopsin labeling in the retina of 48-week-old Tg rabbit in the area of the visual streak (Fig. 5) . The cone inner and outer segments were stained by PNA, but their structures were severely disrupted in the 48-week-old Tg rabbit. 

There were distinct regional differences in the degree of photoreceptor loss in the older Tg rabbits. The retinal sections from 12-week-old WT and Tg rabbits at three locations along the vertical meridian are shown in Figure 4C . It is known that in normal rabbits, the density of rod and cone photoreceptors is highest at the visual streak located inferior to the optic nerve head. ²⁰ Consistent with previous reports, the thickness of the ONL in WT rabbits was at its maximum near the visual streak. In contrast, the ONL in Tg rabbits was thinnest near the visual streak and was relatively preserved in the peripheral retina (Fig. 4D) . 

ERGs of Tg Rabbits

To evaluate the retinal function of the rod and cone systems of Tg rabbits, we recorded full-field scotopic and photopic ERGs. The scotopic and photopic ERGs elicited by different stimulus intensities from a 12-week-old WT and 12- and 48-week-old Tg rabbits are shown in Figures 6A and 6B , respectively. Compared with the ERGs recorded from 12-week-old WT rabbits, the ERG amplitudes of Tg rabbits were clearly reduced at 12 weeks, and the degree of reduction became more severe at 48 weeks. The amplitude of the maximum rod a-wave, which reflects rod photoreceptor activity, was 28% of the WT at 12 weeks and reduced to 5% at 48 weeks (Fig. 6C) . In contrast, the maximum cone a-wave amplitude was 65% of the WT at 12 weeks and remained at 35% even at 48 weeks (Fig. 6D) . The a-wave fitting model of Hood and Birch ²⁸ also revealed that not only the maximum response amplitude (Rm), but also the transduction sensitivity (S) were abnormal in both rod and cone photoreceptors of Tg rabbits (Table 3) . These results indicated a rod-dominant, progressive photoreceptor dysfunction in the retina of this line of Tg rabbits. 

Presence of Extracellular Vesicles in the Tg Rabbit Retina

Finally, we compared the retinal ultrastructure of 6-week-old WT and line 7 Tg rabbits. The outer segments of the photoreceptors were slightly shorter and less organized in the retinas of 6-week-old Tg rabbits, although the outer segments still contained many well-packed discs at this age (Fig. 7A) . 

A striking finding in the Tg rabbit retina was the large number of small vesicles that accumulated in the extracellular space of the photoreceptors (Fig. 7A , asterisks). The vesicles were 50 to 300 nm in size and bound to a single membrane. We also found that these vesicles were cleaved from the membranes of the inner segments of the photoreceptors (Fig. 7B , arrows). 

Discussion

The purpose of this study was to generate a rabbit model of progressive retinal degeneration and to characterize the pattern of degeneration by using histology and electrophysiology. For this purpose, we used rabbit BAC transgenesis, which permitted us to produce a point mutation with no effect on the rest of the large rhodopsin gene, including the regulatory regions of the rhodopsin gene. ^{22 23 24} BAC transgenesis is known to provide high tissue- and stage-specific transgene expression that is independent of the site of integration and dependent on the number of integrated copies. ²⁵  

We succeeded in generating six lines of Tg rabbits with different expression levels. Two lines showed high transgene expression levels and progressive retinal degeneration. Retinal histologic and ERG studies showed early loss of rod function associated with relatively preserved cone function, which is very similar to the clinical findings of human RP patients with the rhodopsin P347L mutation. ^{30 31} To the best of our knowledge, this is the first rabbit model of progressive retinal degeneration. Because rabbits have large eyes and are easy to handle and breed, we believe that our Tg rabbits are useful animal models for testing various new treatments, including surgical procedures. 

The fundus appearance and fluorescein angiograms were nearly normal in our Tg rabbits. The blood vessel diameters and optic disc appearance were examined monthly, and they were indistinguishable between WT and Tg rabbits at ages up to 40 weeks. An early sign of RP in human patients is an attenuation of blood vessel diameters in the eye. The normal diameter of the retinal vessels in our Tg rabbits may be due to the characteristics of rabbit retina, because retinal vessels in rabbits are confined to the horizontal myelinated bands, comprising optic axons, oligodendrocytes, and astrocytes, and are not associated with the inner retinal layers, as in vascular retinas. 

In this study, we generated the transgenic rabbits on an albino background (NZW), because the rabbit BAC library was available only for NZW rabbits. However, this albino background may limit the model’s usefulness. First, normal fundus coloring without any pigmentation in our Tg rabbits may have occurred because we used the nonpigmented NZW rabbits. Second, it is known that there are other anomalies in the visual system of albino rabbits, including lower ganglion cell densities ³² and aberrant optic decussation and retinal projections. To overcome these limitations, we are currently producing a pigmented line of Tg rabbits by mating our NZW Tg rabbits with pigmented Dutch rabbits. 

By measuring the ONL thickness at different locations along the vertical meridian, we found a marked regional variation in the loss of photoreceptors in the Tg rabbit retina. The loss of photoreceptors was at its maximum near the visual streak, where the photoreceptor density is highest in WT rabbits. In contrast, the ONL thickness in Tg rabbits was relatively preserved in the peripheral retina (Figs. 4C 4D) . Similar regional variations in photoreceptor loss have been reported in other large animal models, including pigs and dogs. ^{16 17} Such regional variation in photoreceptor loss may be due to topographic variations in opsin expression, as reported by Timmers et al., ³³ and van Ginkel et al., ³⁴ who showed a central-to-peripheral gradient of rhodopsin mRNA levels in bovine retinas. 

A distinct ultrastructural observation in the retina of Tg rabbits was the accumulation of numerous extracellular vesicles that were cleaved from the inner segments of the photoreceptors. At this stage, the outer segments still contained well-packed discs. These findings are consistent with findings in Tg mice with the P347S rhodopsin mutation. ³⁵ Using two monoclonal antibodies against rhodopsin, Li et al. ³⁵ demonstrated that these small vesicles contain rhodopsin, and they proposed that they were produced as a consequence of a defect in the transport of rhodopsin from the inner segment to the disc membranes of the outer segments. Although we have not yet examined whether the vesicles contain rhodopsin in our Tg rabbits, the similarity in the ultrastructural findings and site of rhodopsin mutation suggested that the defective delivery of opsin to the outer segment may be one of the causes of photoreceptor cell death in our Tg rabbits. However, other factors, including an overexpression of rhodopsin, ^{36 37} prolonged activation of phototransduction, ³⁸ or activation of mislocalized opsin, ³⁹ may be involved. 

In conclusion, we have succeeded in generating a Tg rabbit model of retinal degeneration. Although further studies are needed to determine the exact mechanism of photoreceptor death observed in our model, we believe that our Tg rabbits will serve as a useful midsized animal model with which to study the pathophysiology of RP and develop novel treatments. 

 Supported by Health Sciences Research Grant H16-sensory-001 from the Ministry of Health, Labor, and Welfare of Japan, and Grants 18591913 and 18390466 from the Ministry of Education, Culture, Science, and Technology of Japan.

 Submitted for publication September 12, 2008; revised November 9, 2008; accepted January 2, 2009.

 Disclosure: M. Kondo, None; T. Sakai, None; K. Komeima, None; Y. Kurimoto, None; S. Ueno, None; Y. Nishizawa, None; J. Usukura, None; T. Fujikado, None; Y. Tano, None; H. Terasaki, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Figure 1.

 

Rhodopsin P347L BAC construction by recombineering. (A) Presumed structure of the rabbit rhodopsin BAC clone. The BAC clone contains the full-length rabbit rhodopsin genomic sequence, as determined by Southern hybridization probed by the 5′-flanking gene (left), rhodopsin gene (middle), and 3′-flanking gene (right). Underbars: positions of the Southern hybridization probes used. (B) The transgene construct. The sequence of exon 5 of the WT rhodopsin gene (top) was replaced by the rhodopsin P347L exon 5 sequence (middle) by Red/ET recombination. The rhodopsin P347L gene (bottom) has a BglII restriction site for Tg. (C) Sequence analysis for the rhodopsin P347L mutation. The rhodopsin P347L exon 5 fragment was amplified from the rhodopsin P347L BAC construct by PCR and sequenced.

Figure 2.

 

Generation of rhodopsin P347L Tg rabbits. (A) Purified rhodopsin P347L transgene construct. PFGE showed that the purified rhodopsin P347L transgene construct was almost 150 kb in size. (B) Southern blot analysis of F1 rabbits of rhodopsin P347L Tg lines. The endogenous rhodopsin WT gene and the rhodopsin P347L transgene were detected as 6 and 3 kb BglII fragments that hybridized to an exon 4 probe. The copy numbers of the integrated transgene for each line were determined by comparing with a control copy number signal intensity.

Table 1.

 

Number of Animals and Zygotes Used to Generate Tg Rabbits

Donor rabbits (total)	36
Zygotes recovered (total)	800
Fertilized zygotes (%)	540 (68)
Zygotes microinjected (%)	456 (84)
Zygotes implanted (%)	456
Zygotes implanted per recipient rabbit (mean ± SD)	27 ± 3
Recipient rabbits	17
Pregnancy rate (%)	12 (71)
Gestation period (days, mean ± SD)	32 ± 1
Rabbits born (total)	80
Transgenic positive (F0) rabbit (surviving)	12 (10)
Founders that passed the transgene onto their offspring	6

Table 2.

 

Characteristics of Six Lines of Tg Rabbits

Line	Site of Insertion	Estimated Copy Number	Ratio of Transgene to Endogenous Opsin mRNA	Amplitude of Maximum Scotopic ERG a-Wave at 12 Wk (μV)
7	13q	30	4:1	49.6 ± 12.7 (n = 5)
8	12q	10	1:1	96.5 ± 20.1 (n = 5)
10	2q	1	0.15:1	180.2 (n = 1)
11	Xq	3	0.07:1	165.1 (n = 1)
14	9p	3	0.1:1	159.3 (n = 1)
16*	6q, 3p	Variable	Variable	Variable

 Maximum scotopic ERGs (mixed rod and cone response) were measured with high intensity flash stimulus of 2.2 log cd · s · m⁻². Results are expressed as the mean ± SD. The a-wave amplitude of WT NZW rabbits was 170 ± 28 μV (n = 5).

*  Double copy line.

Figure 3.

 

Fundus photographs (top) and fluorescein angiograms (bottom) obtained from a 40-week-old WT and a rhodopsin P347L Tg rabbit from line 7. The appearances of the fundus and the angiogram of the Tg rabbit were indistinguishable from those of the WT rabbit, even at 40 weeks.

Figure 4.

 

Retinal histology of Tg rabbits (line 7). (A) Retinal sections of WT and Tg rabbits at 2, 6, 12, and 48 weeks of age. (B) Changes in the thickness of the ONL at different ages (in weeks) for WT and Tg rabbits. The number of animals examined is shown in parentheses. (C) Vertical retinal sections 6 mm superior to the optic nerve head (ONH), at the visual streak, and 8 mm inferior to the ONH of 12-week-old WT and Tg rabbits. (D) Thickness of the ONL along the vertical meridian measured at 10 retinal locations at 2-mm intervals. Mean ± SEM of five WT and five Tg rabbits are plotted.

Figure 5.

 

Immunohistochemical analysis of rod and cone photoreceptors double labeled with rhodopsin (green) and PNA (red) at the visual streak of 48-week-old WT (left) and transgenic (right) rabbits. Bar, 50 μm.

Figure 6.

 

ERGs recorded from 12-week-old WT and 12- and 48-week-old rhodopsin P347L (line 7) Tg rabbits. (A) Scotopic ERGs elicited by eight different stimulus intensities. (B) Photopic ERGs elicited by four different stimulus intensities. (C) Rod maximum a-waves elicited by 2.2 log cd · s · m⁻². These responses were obtained by waveform subtraction of photopic ERGs from scotopic ERGs. (D) Cone maximum a-waves elicited by 2.2 log cd · s · m⁻² on a rod-suppressing white background of 1.3 log cd · s · m⁻².

Table 3.

 

Summary of Photoreceptor Function Parameters in Tg Rabbits

	12 Wk			48 Wk
		WT	Tg	WT	Tg
Rod log Rm (maximum response)	2.23 ± 0.08	1.63 ± 0.06*	2.06 ± 0.10	—, †
Rod log S (sensitivity)	3.50 ± 0.04	3.19 ± 0.11*	3.42 ± 0.06	—, †
Cone log Rm (maximum response)	1.73 ± 0.09	1.47 ± 0.07*	1.64 ± 0.08	1.21 ± 0.23*
Cone log S (sensitivity)	2.97 ± 0.05	2.84 ± 0.14	2.93 ± 0.10	2.68 ± 0.18*

 Data are expressed as the mean ± SD. n = 5 for all.

*   P < 0.05 (unpaired t-test).

†  Rod photoreceptor responses in 48-week-old Tg rabbits were too small for the fitting model to be applied.

Figure 7.

 

Ultrastructural analyses of 6-week-old WT (left) and Tg (right) rabbit retinas. (A) Tg rabbit retina showing many small vesicles accumulated in the extracellular space ( ). (B) These abnormal vesicles were cleaved from the inner segment of photoreceptors (arrows).

The authors thank Kensaku Kitada (Kitayama Labes Co., Ina, Japan) for breeding the Tg rabbits; Akira Shiota (PhoenixBio Co., Ltd. Utsunomiya, Japan) for technical help with BAC transgenesis; Robert E. Marc and Bryan W. Jones of Utah University for critical comments on the manuscript; and Duco I. Hamasaki of Miami University and Yozo Miyake of Shukutoku University for discussions of the manuscript.