Human Molecular Genetics, 2000, Vol. 9, No. 4 531-537
© 2000 Oxford University Press
Mapping of X-linked progressive retinal atrophy (XLPRA), the canine homolog of retinitis pigmentosa 3 (RP3)
1James A. Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA and 2Indian Institute of Chemical Biology, Department of Human Genetics, 4 Raja S.C. Mullick Road, Jadavpur, Calcutta 700 032, India
Received 15 September 1999; Revised and Accepted 13 December 1999.
DDBJ/EMBL/GenBank accession nos AF148798–AF148801.
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ABSTRACT |
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X-linked progressive retinal atrophy (XLPRA) in the Siberian husky dog is a naturally occurring X-linked retinopathy closely resembling X-linked retinitis pigmentosa (XLRP) in humans. In affected males, initial degeneration of rods is followed by cone degeneration and complete retinal atrophy; carrier females have random patches of rod degeneration consistent with random X chromosome inactivation. By typing the XLPRA pedigree with five intragenic markers [dystrophin, retinitis pigmentosa GTPase regulator (RPGR), tissue inhibitor of metalloproteinases 1, androgen receptor and factor IX], we established a linkage map of the canine X chromosome, and confirmed that the order of these five genes is identical to that on the human X. XLPRA was tightly linked to an intragenic RPGR polymorphism (LOD 11.7, zero recombination), thus confirming locus homology with RP3. We cloned the full-length canine RPGR cDNA and three additional splice variants. No disease-causing mutation was found in the RPGR-coding sequence of the four splice variants characterized, a finding similar to ~80% of human XLRP patients whose disease maps to the RP3 locus. In addition, there were no significant differences in the proportional expression of each splice variant in normal and pre-degenerate XLPRA-affected retina. Expression of all RPGR splice variants increased later in the disease, when retinas were undergoing active degeneration. The results provide further evidence of cross-species retention of a complex splicing pattern in the 3' portion of RPGR, the functional significance of which is unknown. In addition, the possibility of another disease locus in the RP3 region is supported.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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In 1996, mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene were identified as the cause of the RP3 form of X-linked retinitis pigmentosa (XLRP) (1). However, RPGR mutations account for only 20–30% of cases whose disease maps to the RP3 interval (1–3). RPGR is expressed at low levels, in particular in the retinal pigment epithelium (RPE) and retina (1,4), thus making it difficult to assess levels of the transcript in neuroretinal target tissues. Despite ubiquitous expression of RPGR, the disease caused by mutations in this gene is limited to the retina. RPGR has a complex splicing pattern in both human and mouse (4–6). Of the splice variants described, it is not known which, if any, are specific or most biologically relevant to the retina, although one retina-specific isoform has been described which, when deleted, results in the RP3 phenotype (6). Neither is it known what is the proportional expression of the different splice variants in the retina, or whether these proportions are altered in RP3.
The mechanism by which mutations in RPGR cause retinal degeneration is unclear. RPGR initially was thought to play a role as a guanine nucleotide exchange factor for a small G-protein in the RPE or retina (1,2). Because mutations in the RCC-1 (regulator of chromosome condensation) domain prevent binding to phosphodiesterase-
, which may play a role in membrane insertion or solubilization of prenylated proteins (7), a role for RPGR as a facilitator of intracellular protein trafficking has been proposed.
There is a lack of spontaneous animal models for X-linked retinal disease in general, and of XLRP in particular; currently, the Siberian husky dog is the only naturally occurring model of XLRP (8). In this study, we describe the development of XLPRA as a model for RP3. First, we established that XLPRA is a phenotypic correlate of XLRP. Next, we established the site of the disease locus on the canine X chromosome. A prerequisite for this was the development of a linkage map for the canine X chromosome, so that chromosomal locations of RP3 and XLPRA could be compared. Following the mapping to a region homologous to Xp21 in man, we began an extensive search for causative mutations within the canine RPGR cDNA and its splice variants. Analysis of these was critical since it has been proposed that the apparent deficiency in identifiable RPGR mutations in human RP3 patients might be due to the complex splicing pattern of this gene (1,3).
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RESULTS |
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Retinal morphology
XLPRA is a disorder of rod photoreceptors. In hemizygous males, the rod outer segments initially are mildly disorganized, and have slightly irregular profiles (Fig. 1A and B). With progression, rod outer segments almost completely disappear, cone outer segment degeneration becomes apparent, and there is collapse of the interphotoreceptor space (Fig. 1C). Cone degeneration begins after significant rod loss has already occurred. Carriers exhibit two distinct cellular changes: uniform but moderate rod photoreceptor loss resulting in reduction of the outer nuclear layer to ~50% of its original width, and scattered foci of complete rod loss, presumably resulting from random X inactivation (Fig. 1D).
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Positioning of the XLPRA locus on the canine X chromosome
Five widely spaced canine X chromosome markers were used to type the XLPRA pedigree. Initial analysis using androgen receptor (AR), dystrophin (DMD) and factor IX indicated that the disease locus was located on the short arm of the canine X chromosome. To determine whether XLPRA was a locus homolog of RP2 or RP3, two additional intragenic markers in the canine tissue inhibitor of metalloproteinases 1 (TIMP-1) (9) and RPGR (10) genes were identified. The results of two-point linkage analysis are presented in Table 1. There were five of 36 recombinants between TIMP-1 and XLPRA (LOD 5.01,
= 0.13), thus placing XLPRA ~13 cM away from TIMP-1. Of 48 dogs examined, there was no recombination between an RPGR polymorphism and XLPRA (LOD 11.74, = 0); this establishes locus homology between XLPRA and RP3. Two-point linkage analysis between AR and the other markers indicates that the order of these five genes on the X chromosome is preserved between human and dog. Figure 2 illustrates comparative images of canine and human X chromosomes, with the canine markers used to map XLPRA, and identified human XLRP loci.
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Identification of canine RPGR splice variants
Four canine RPGR splice variants of 1.8, 2.4, 2.8 and 3.3 kb size have been cloned and characterized. All share the same 5' half of the coding region, namely exons 1–10 (Fig. 3). These contain two GTP phosphate-binding domains in exon 2 and the RCC-1 homology region in exons 3–10. Homology between the variants decreases from exon 10 to 19 as there is variable in-frame splicing of exons and introns resulting in variably sized cDNAs. A graphic comparison of putative splicing patterns in all of the canine RPGR variants is given in Figure 3. Alternative splice sites in the 2.4, 3.3 and 2.8 kb canine RPGR cDNA variants are clustered in the region encoding the hydrophilic (exons 14–16) portion of the protein.
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The characterized region of the 2.4 kb variant (GenBank accession no. AF148798) includes 2132 nucleotides of coding sequence, and 80 and 280 nucleotides, respectively, of 5'- and 3'-untranslated region (UTR). When compared with the human sequence (GenBank accession no. X97668), a 334 bp portion of the canine retinal RPGR cDNA is absent. At its 5' end, this segment corresponds precisely to the splice junction between exons 13 and 14 (nucleotides 1572–1573) of the human cDNA; the terminal 208 bp end of this portion has no significant homology to human exon 16, and appears to be unique to dog. Homology resumes at nucleotide 2114 (in exon 17) of the human cDNA sequence. Thus, it appears that in the 2.4 kb variant in the dog, regions corresponding to human exons 14 and 15 are missing (Fig. 3A).
In addition to the 2.4 kb splice variant, a 3.3 kb variant could be amplified from testes only. By nested PCR (Table 2, primers RPGR 20 and RPGR 21; Fig. 3B) and sequencing, an insert was identified between putative exons 13 and 16 of the canine 2.4 kb sequence. The 3.3 kb RPGR cDNA (GenBank accession no. 148801) is identical to the 2.4 kb sequence, except that there is an insertion of a large 960 nucleotide exon at nucleotide 1658 which encodes 320 amino acids which are in-frame with the predicted amino acid sequence for the 2.4 kb RPGR variant. The 960 nucleotide sequence consists of exons 14 and 15, with an intervening 619 nucleotide region flanked by splice donor and acceptor sites (GT..AG), and thus has the anatomical features of an intron. For ease of comparison with the human RPGR cDNA, we have termed this composite exon 14-14A-15.
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A 2.8 kb variant (GenBank accession no. AF148800) was also identified. It is identical to the 3.3 kb variant until the 3' terminus of canine exon 15 (nucleotide 2625); at this point, 225 bp of unique sequence, termed exon 15A, is spliced in-frame. No stop codon was identifiable and the sequence was terminated by a series of 12 adenine residues. Using primers RPGR 27 and RPGR 33, fragments of identical size and sequence could be amplified from both reverse-transcribed RNA and genomic DNA. This finding, together with the observation that exon 15A commences with a putative splice donor site (GT), indicates that exon 15A is derived from intron 15. The identity of the 2.4, 2.8 and 3.3 kb RPGR splice variants was determined initially by Southern blot, and subsequently was confirmed by nested PCR and sequencing. Hybridization to a common probe derived from the RCC-1 homology region (exons 3–10) suggested that each variant consisted of a common 5' portion, spliced to a unique 3' region. This was confirmed subsequently by sequencing.
An additional splice variant of 1.8 kb was amplified from poly(A) RNA using the Marathon RT–PCR kit (GenBank accession no. AF148799). When aligned with the human RPGR cDNA (GenBank accession no. X97668), the first 1326 nucleotides had >85% homology up to the 3' end of human exon 10 (nucleotide 1246). The subsequent sequence begins with a putative splice donor site (GT), and is presumably derived from intron 10. A stop codon is present nine nucleotides 3' to the termination of exon 10, and the remaining 465 bp sequence constitutes the 3'-UTR.
Tissue expression of RPGR variants
A northern blot using 20 µg of total retinal RNA from normal and affected male animals did not produce a signal; this was probably the result of the reported low level of RPGR expression and instability of the transcript (1,4). For that reason, the expression of all four variants was assessed by RT–PCR in nine tissues (liver, lung, heart, kidney, bone marrow, ovary, testes, brain and retina) followed by hybridization to a canine RPGR clone containing the RCC-1-homologous region of the cDNA (exons 3–10). The 2.4 kb variant could be amplified most readily from testes, retina, bone marrow, brain and kidney, and inconsistently from heart and lung (data not shown). The 1.8 and 2.8 kb variants showed a similar distribution of expression and were amplified readily from brain, bone marrow, retina and testes (data not shown). All variants could be amplified easily from testes, indicating that levels of RPGR expression in the dog are highest in this tissue.
Amplification and sequencing of RPGR splice variants in normal and XLPRA-affected tissues
The expression and sizes of the 1.8, 2.4 and 2.8 kb variants were assessed by RT–PCR in normal and affected retinas, and in affected RPE/choroid. Bands of similar size were present in both genotypes for these three splice variants, and we found no sequence abnormalities in the coding region (data not shown). Two nucleotide variations were identified by direct sequencing of overlapping PCR products; both were located in the composite exon 14-14A-15-15A. One was a previously described single nucleotide polymorphism in exon 14A (nucleotide 2133 of the 2.8 kb variant) (10), and the other was a single base change in exon 15A (nucleotide 2597 of the 2.8 kb variant). The first nucleotide change (A
G) resulted in a valine for methionine substitution in the predicted amino acid sequence. The polymorphism was present at an allele frequency of ~50% in the normal canine population (10). The second nucleotide change (CT) did not change the encoded amino acid (aspartic acid) and was in-phase with the first polymorphism. In addition, the UTRs for all splice variants were also examined for sequence differences between affected and normal dogs, and none were found.
Semi-quantitative RT–PCR
Having found no causative mutations within the splice variants identified, we tested the hypothesis that an alteration in the proportional expression in any one of the four splice variants could result in the XLPRA phenotype. We examined the expression levels of each variant in retinal tissues from three normal and three affected dogs of differing ages and disease severity. Of the affected dogs, one was 2 months old, an age when the retina is histologically normal; the other two were older (15 and 18 months) and had moderate to advanced degeneration, respectively. No significant decrease in any of the splice variants with respect to one another was present in retinal RNA of normal and affected dogs of any age; i.e. dogs with pre-degenerate or actively degenerating retinas. However, expression of all RPGR splice variants increases in the older affected dogs at the time that the retinas are actively degenerating (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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XLPRA in the Siberian husky dog is a close phenotypic correlate to XLRP in man. Affected males begin to show clinical signs of disease in the period between sexual maturity and young adulthood (8). The histology of XLPRA in affected males is characterized by diffuse progressive degeneration of rods with secondary and late loss of cones. Female carriers demonstrate the rod specificity of the disease most clearly, with patchy foci of selective rod degeneration. The findings in hemizygous males and heterozygous females with XLPRA resemble those seen in human XLRP (11,12).
Of the genes included in the linkage analysis, the approximate physical location on the canine X chromosome of two, AR (13) and factor IX (14), is known. Using two-point linkage, the chromosomal location of the other three genes was inferred by their linkage distances from AR. The results indicate that the order of these genes on the X chromosome is similar in dog and man, and that rearrangements of large portions of the X chromosome, as seen in rodents (15), are not present in the dog. XLPRA is tightly linked to an intragenic marker in the canine RPGR gene—this establishes XLPRA as the locus homolog of RP3, and places XLPRA at the canine equivalent of human Xp21.
Comparison of the deduced amino acid sequences of the four canine RPGR splice variants, and the published human (1,2) and mouse (4) RPGR sequences, reveals extensive preservation of amino acid homology over the RCC-1 repeat region. Homology in all three species drops over the hydrophilic portion of the protein, encoded by exons 14–16, but is resumed over the terminal 120 amino acids. The open reading frame of the canine 2.4 kb splice variant was characterized first among all the splice variants identified in dog. This variant shares 82 and 79% nucleotide sequence identity, respectively, with the human (GenBank accession no. X97668) and mouse (GenBank accession no. AF044677) RPGR nucleotide sequences. Highly conserved regions in all three species include the two GTP phosphate-binding domains, the RCC-1 homology region and the terminal isoprenylation signal (Fig. 3A).
In the dog, human and mouse, all of the characterized canine RPGR splice variants share the same 5' half of the coding region. In fact, the 1.8 kb variant consists almost exclusively of the first 10 exons, suggesting that the critical functional portion of the coding sequence resides within the RCC-1 repeat regions. Homology to the RCC-1 region prompted speculation that RPGR was a guanine nucleotide exchange factor (1). However, a BLAST search of the predicted amino acid sequence from the 2.4 kb canine RPGR cDNA against the Swiss protein database indicates that the canine RPGR has ~30% amino acid identity to a protein with a putative role in ubiquitin conjugation (KIAA0032; GenBank accession no. 2495699), and slightly lower (25–30%) identity with RCC-1 proteins of several species.
The portions of the human RPGR protein encoded by exons 14–16 are highly positively charged, but their function is unknown (16). The corresponding region in dog appears to be the major site of alternative splicing in the canine RPGR gene, suggesting that it may play a role in cellular localization or substrate specificity of the protein products of individual splice variants. Apart from the 1.8 kb variant, the other canine isoforms are characterized by variable in-frame inclusion of sequence encoded by exons 14, intron 14, exon 15 or intron 15. In the 2.8 kb variant, the coding sequence is terminated by a series of lysine residues. Despite much effort, we were unable to obtain the true 3'-UTR of this variant; it is likely that a string of adenine residues served as template for the oligo(dT) primer used to perform RT–PCR in making the retinal cDNA library, with resultant premature termination of the transcript obtained by subsequent PCR. Reported sites of alternative splicing in the human RPGR gene include coding regions presumably derived from intron 15, and sequences downstream of the published 3'-UTR (5,6).
The inclusion of the composite exon 14-14A-15 to create the 3.3 kb variant in testes appears to be developmentally regulated, and coincides with the onset of sexual maturity. The presence of similarly sized splice variants in the testes of both mouse (4,6) and dog, as well as relatively abundant expression of RPGR in the testes of both species, suggests a significant role for RPGR in this organ. The 3.3 kb band was faintly visible on Southern blots of retinal RT–PCR products (data not shown), and could be amplified by semi-quantitative RT–PCR, thus indicating that this variant is expressed at very low levels in the retina.
Approximately 70% of human X-linked retinitis pigmentosa patients map to Xp21.1 and thus fall into the RP3 category; of these, only 20% have exonic mutations or splice defects in the RPGR gene (1,2,16). Possible reasons for failure to identify mutations within those RP3 families without RPGR mutations include the following. (i) There is genetic heterogeneity within the RP3 locus; i.e. the presence of additional disease gene(s) in the RP3 interval. (ii) There are mutations in as yet uncharacterized splice variants of RPGR. It is possible that there may be a cluster of mutations affecting a particular splice variant(s) which is expressed exclusively in the retina and/or RPE. This has been reported recently for a retina-specific isoform which has intron 15a followed by a premature stop codon. However, disease caused by deletion of this exon has been recognized only in one family (6). (iii) There is a promoter defect resulting in abnormal expression of RPGR. (iv) There are mutations in another gene which alter RPGR expression.
In order to identify possible mutations in the coding sequence of the canine RPGR gene, we sequenced all four splice variants amplified by RT–PCR from retinal RNA of normal and affected dogs. No mutations were found. Next, we assessed the proportional expression of individual RPGR splice variants in normal and affected retinas by semi-quantitative RT–PCR. Expression of each splice variant was similar in normal and pre-degenerate affected tissues. Thus, abnormal RPGR expression, either from a promoter defect or a positional effect, is excluded from causal association with XLPRA. It is possible that mutations causing disease in the XLPRA colony, and in the balance of human patients whose disease maps to the RP3 region, reside in a yet-to-be-discovered novel exon(s) of the RPGR gene. Complete sequencing of the RPGR gene in normal and affected dogs would be the most complete way of identifying such a mutation. This is a more realistic undertaking in a defined genetic isolate, such as the XLPRA canine model in which a single mutation is segregating, than in multiple human families with distinct mutations. Alternatively, mutations may reside not in RPGR, but in a nearby novel gene(s) in the ~500 kb region that defines the RP3 interval.
Further localization of the XLPRA locus will require more markers in the region—these can be developed most efficiently within the canine homologs of the human expressed sequence tags, in particular those which are expressed in the retina and map to the RP3 interval. Refining the mapping of the XLPRA locus may strengthen evidence for involvement of RPGR in XLPRA, and thus support continued search for a mutation in this gene. Alternatively, RPGR may be excluded, with subsequent identification of a novel gene in the interval which may harbor mutations causing X-linked photoreceptor disease in man and animals.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Animals
We developed the XLPRA colony from one affected male dog, by outbreeding to laboratory beagles known to be free from retinal degeneration alleles (8). Animals were only included in the linkage study if clinically evident disease status could be confirmed unequivocally by clinical examination (ophthalmoscopy and electroretinogram) and/or histology (8). The genotype of dogs maintained through adulthood for breeding was established by clinical methods, combined with disease status of their progeny.
DNA samples and retinal histology
Genomic DNA was isolated using standard techniques (17) either from blood samples collected in citrate anticoagulant tubes or from splenic samples from deceased dogs. Eyes were obtained by enucleation prior to euthanasia of dogs deeply anesthetized with intravenous pentobarbital. For extraction of RNA, retina was isolated under sterile conditions, placed in sterile RNase-free tubes, frozen by immersion in liquid nitrogen and stored at –70°C until utilized. Eyes were prepared by fixation in a triple aldehyde/osmium tetroxide protocol and embedded in plastic as previously described (18). For each eye, complete sections extending from the optic nerve to the ora serrata of the superior, inferior and temporal meridians were examined.
Typing and linkage analysis
Animals in the pedigree were typed with five intragenic markers [factor IX (19), AR (20), TIMP-1 (9), RPGR (10) and DMD (21)] spanning the X chromosome. Two-point linkage analysis was performed between the XLPRA locus and each marker, and between each pair of markers using the LINKAGE package of programs (22–24). The disease trait was coded as sex-linked recessive with full penetrance.
RT–PCR
Total RNA was prepared from retinal tissues of four normal and four XLPRA-affected male dogs by standard guanidinium thiocyanate extraction (25). Total RNA was also extracted from eight normal non-retinal tissues (liver, lung, kidney, heart, brain, testes, ovary and bone marrow). A 3 µg aliquot of total RNA was reverse transcribed using random hexamers or a reverse primer specific for the poly(A) tail. Primer sequences, primer locations, annealing temperatures and expected product sizes for selected RPGR PCRs are given in Table 2 and Figure 3B. A MgCl2 concentration of 1.5 mM was used for all reactions.
Cloning of the normal canine RPGR cDNA and characterization of additional RPGR splice variants
Due to the low expression of RPGR transcripts, cloning and characterization of all splice variants was done using PCR-based methodology rather than in situ cloning and northern blot analysis. The canine RPGR cDNA was obtained by a combination of RT–PCR, 5' RACE and 3' RACE (Gibco BRL, Gaithersburg, MD) from brain RNA. Primer combinations used to amplify all canine RPGR variants are given in Table 2.
The identity of full-length PCR products of the 2.4, 2.8 and 3.3 kb splice variants amplified from retina and other tissues was confirmed by Southern blotting using a 1075 bp [32P]dCTP-labeled canine RPGR DNA fragment derived from putative canine exons 3–10 as a probe, and by sequencing. The identity of the smaller 1.8 kb fragment was determined by sequencing alone. The expression of all four variants was assessed by RT–PCR in nine tissues (liver, lung, heart, kidney, bone marrow, ovary, testes, brain and retina), followed by hybridization to a canine RPGR clone containing exons 3–10. Alignment of canine RPGR sequences to human and mouse (GenBank accession nos X97668 and AF044677) RPGR cDNA sequences was performed using the BLAST two sequence analysis function (http://www.ncbi.nlm.nih.gov/gorf/bl2.html ).
Comparison of all RPGR splice variants in normal and XLPRA-affected retina
The size and expression of all splice variants in normal and XLPRA-affected retinas were determined by RT–PCR. In addition, all transcripts were examined in RPE/choroid from an XLPRA-affected dog. Primers and conditions used are described in Table 2, and locations of primers indicated in Figure 3B. RPGR sequence from each variant was assessed by direct sequencing of PCR products. In order to minimize sequencing errors, all fragments were sequenced in both directions from at least two normal and two affected dogs. Primers were selected so that overlapping sequence from both coding and non-coding strands was obtained. Full-length sequence was assembled and compared for base differences.
Evaluation of relative expression levels of each splice variant in normal and XLPRA-affected retinal tissues by semi-quantitative RT–PCR
To assess differences in the proportional expression of each splice variant in normal and XLPRA-affected retina, semi-quantitative RT–PCR was performed using a Perkin Elmer (Foster City, CA) 7700 PCR instrument. RPGR probes and primers were selected to span splice junctions which were specific for each splice variant. RPGR amplimers were normalized to 18S rRNA. Quantitative PCR of RPGR and 18S rRNA was performed in separate tubes using the comparative Ct method; all procedures were performed according to the manufacturer’s instructions as detailed in user bulletin #2 (http://www2.perkin-elmer.com/ab/techsupp/7700.html ), the instructions accompanying the PCR Core reagent, the TaqMan reverse transcription reagent kits (Perkin Elmer) and published material using this technique (26). Linearity of the amplification reaction was verified by a control experiment examining the amplification efficiencies of experimental (RPGR) and control (18s rRNA) genes.
Each RPGR variant was amplified from retinal cDNA of three normal (7, 12 and 15 months of age) and three affected (2, 15 and 18 months of age) dogs, and normalized using the 18S rRNA amplification. As RNA was limiting, not all dogs were tested for each variant, but at least two normal and two affected dogs were assessed for each. For the majority of samples, reactions were performed by amplification of each splice variant from cDNA as well as using reverse transcriptase negative controls. PCRs were run as duplicates or triplicates, and, where appropriate, the mean and standard deviation was determined. As the 1.8 kb variant and the 2.8 kb variants would be amplified from genomic DNA (Fig. 3B), RNA for all PCRs was treated with DNase prior to reverse transcription.
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ACKNOWLEDGEMENTS |
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The authors are grateful to Vicki Baldwin for her assistance in the molecular biology laboratory, Julie Alling and Sue Pearce-Kelling for their assistance in preparation of tissues for microscopy, and Jill Czarnecki and Jennifer Johnson for graphics. The authors also thank the staff of the RDS Facility for assistance with breeding and rearing of the research dogs. This work was supported in part by the Foundation Fighting Blindness, NEI/NIH grant EY 06855, American Kennel Club–Canine Health Foundation, the Morris Animal Foundation/The Seeing Eye Inc. and the Siberian Husky Club of America.
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: +1 607 256 5620; Fax: +1 607 256 5689; Email: gda1@cornell.edu
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REFERENCES |
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