April 2006
Volume 47, Issue 4
Free
Biochemistry and Molecular Biology  |   April 2006
Disruption of the Gene Encoding the β1-Subunit of Transducin in the Rd4/+ Mouse
Author Affiliations
  • Eiko Kitamura
    From the Jules Stein Eye Institute, the
  • Michael Danciger
    Biology Department, Loyola Marymount University, Los Angeles, California; and
  • Clyde Yamashita
    From the Jules Stein Eye Institute, the
  • Nagesh P. Rao
    Department of Pathology, David Geffen School of Medicine at UCLA, Los Angeles, California; the
  • Steven Nusinowitz
    From the Jules Stein Eye Institute, the
  • Bo Chang
    The Jackson Laboratory, Bar Harbor, Maine.
  • Debora B. Farber
    From the Jules Stein Eye Institute, the
    Molecular Biology Institute, and the
Investigative Ophthalmology & Visual Science April 2006, Vol.47, 1293-1301. doi:10.1167/iovs.05-1164
  • Views
  • PDF
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      Eiko Kitamura, Michael Danciger, Clyde Yamashita, Nagesh P. Rao, Steven Nusinowitz, Bo Chang, Debora B. Farber; Disruption of the Gene Encoding the β1-Subunit of Transducin in the Rd4/+ Mouse. Invest. Ophthalmol. Vis. Sci. 2006;47(4):1293-1301. doi: 10.1167/iovs.05-1164.

      Download citation file:


      © ARVO (1962-2015); The Authors (2016-present)

      ×
  • Supplements
Abstract

purpose. The Rd4/+ mouse inherits an autosomal dominant retinal degeneration that cosegregates with a large inversion spanning nearly all of mouse chromosome 4 (Chr 4). This inversion is homozygous lethal. The hypothesis for the study was that disruption of a gene at one of the two breakpoints in the Rd4 chromosome is responsible for the retinal degeneration. The purpose was to identify the disrupted gene.

methods. Genotyping was performed by PCR and gel electrophoresis. The Rd4/+ phenotype was confirmed by ERG. Fluorescence in situ hybridization (FISH) analysis was performed with bacterial artificial chromosome (BAC) probes. Northern and quantitative PCR procedures were used to evaluate Gnb1 mRNA expression. Protein expression was measured by Western blot.

results. To identify the Rd4 gene defect, the breakpoints were first localized with a testcross and the locus refined by using FISH. Genetic testcross data revealed that the inversion breakpoints are located within a few centimorgans of both the telomeric and centromeric ends of Chr 4. Initial FISH analysis showed the proximal breakpoint of the inversion to be in the centromere itself. Therefore, we focused on the distal breakpoint and found that it lies in the second intron of the gene Gnb1, coding for the transducin β1-subunit (Tβ1) protein that is directly involved in the response to light of rod photoreceptors. Before the beginning of retinal degeneration in Rd4/+ retina, the levels of Gnb1 mRNA and Tβ1 protein are 50% of those in wild-type retina.

conclusions. The results suggest that disruption of the Gnb1 gene is responsible for Rd4 retinal disease.

Retinal degeneration is a major cause of visual impairment and blindness in humans. In recent years, remarkable progress has been made in the identification of genes responsible for various forms of retinal degeneration, and, to date, more than 160 loci have been discovered. The defective gene has been identified in more than 110 of these loci (Retinal Information Network; www.sph.uth.tmc.edu/RetNet/home.htm, provided in the public domain by the University of Texas Houston Health Science Center, Houston, TX). Some of these genes were first identified in mouse models and then were found to be associated with human disorders. For example, the discovery that a mutant cGMP-phosphodiesterase β-subunit gene (Pde6b) was responsible for the rd1 mouse retinal degeneration 1 2 3 led to the identification of mutations in the β-subunit of cGMP-phosphodiesterase in individuals affected with autosomal recessive retinitis pigmentosa. 4 5 6 Furthermore, finding the defective rd7 gene 7 created the opportunity to study the molecular pathways that regulate normal retinal development and to understand some of the consequences of mutations in NR2E3 that cause enhanced S-cone syndrome in humans. 8 9 10 The Rd4/+ degeneration was found in a stock carrying the chromosomal inversion In(4)56Rk, which was induced in a DBA/2J male 11 and bred onto the C57BL/6J background. As reported in this study, in the affected mouse, the photoreceptor layer of the retina had degenerated completely and the electrophysiological response to light (as measured by the electroretinogram [ERG]) had completely disappeared by 6 weeks of age. The phenotype is inherited in an autosomal dominant fashion and is always associated with an inversion encompassing nearly all of chromosome (Chr)4. Homozygous inheritance of the Rd4 chromosome is lethal. In this article, we describe the identification of the gene disrupted at the distal breakpoint of Chr 4 in the Rd4 mouse. Because our initial experiments demonstrated that the proximal breakpoint was in the centromere itself and that the distal breakpoint was in the subtelomeric region of Chr 4, we used FISH to first flank and then identify this distal breakpoint. Bacterial artificial chromosomes (BACs) that hybridized to the remaining, uninverted telomeric region and those that hybridized to the proximal inverted region of the chromosome were tested. Finally, a single BAC that hybridized to both ends of the chromosome was identified. We hypothesized that this BAC would contain the sequence harboring the breakpoint. Using FISH, we discovered that the breakpoint of the mutant Chr 4 is in the second intron of the Gnb1 gene. This disruption decreases by ∼50% the expression of both message and protein of the Gnb1 gene in heterozygotes before the onset of retinal degeneration. 
Methods
Animals
C57BL/6J (B6)+/+ and Rd4/+ mice were obtained from colonies bred from stock originated at the Jackson Laboratory (Bar Harbor, ME). Mice were reared under dim cyclic light and killed at appropriate postnatal days. The eyes were quickly enucleated after death and the retinas dissected rapidly and frozen on dry ice. All experiments were conducted in accordance with the Animal Care and Use Committee of UCLA and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. For the backcross, stock Rd4/+ mice were bred to Mus spretus mice to produce F1 mice. The Rd4/+ F1 mice were distinguished from +/+F1 mice by fundus observation with an ophthalmoscope. Rd4/+ F1 mice were crossed to B6 mice and the 284 N2 progeny phenotyped for Rd4 disease by fundus examination. The Rd4/+ phenotype was confirmed by ERG in a small cohort of these animals. ERG methodology has been described. 12 Figure 1shows the typical results obtained for the rod and cone responses of Rd4/+ mice at 3 and 6 weeks of postnatal life. 
Genotyping
After phenotyping, mice were killed, and DNA was isolated from liver tissue by standard procedures. Distal Chr 4 dinucleotide repeat markers polymorphic between the M. spretus and B6-Rd4/+ strains included D4Mit180, D4Mit42, D4Mit357, D4Mit253, D4Mit344, D4Mit209, and D4Mit51. Genotypes were determined by PCR and agarose gel electrophoresis. 
Fluorescence In Situ Hybridization Analysis
Metaphase chromosomes were prepared from primary cultures of mouse ear-clips. Slides of metaphase chromosomes were made using standard cytogenetic procedures. Fluorescence in situ hybridization (FISH) analysis was performed basically as described. 13 BACs were obtained from Invitrogen (Chicago, IL). Total BAC DNA was labeled with digoxigenin-11-dUTP (DIG-Nick Translation Mix; Roche Molecular Biochemicals, Indianapolis, IN) or biotin (Bio Nick Labeling System; Invitrogen). Signals were detected with anti DIG-FITC (Roche Molecular Biochemicals) or Texas red avidin (ID Laboratories Inc., London, Ontario, Canada). 
When small DNA fragments were used for FISH, several PCR products (0.5–1.0 kb) were combined and used as probes. Each mixture of PCR products covered approximately 2.0 kb of the genomic region. Primer sequences are shown in Table 1 . The probe was labeled with a DNA labeling system (Bio Prime; Invitrogen). Because small DNA fragments give a very low intensity signal, we amplified the signals by incubating the slides with 1:500 diluted FITC avidin D (Vector Laboratories Inc., Burlingame, CA) and then hybridizing them with 1:100 diluted biotinylated goat anti-avidin D (Vector Laboratories Inc.). Signals were detected by incubating again with 1:500 diluted FITC avidin D. The chromosomes were counterstained with 4′,6′-diamino-2-phenylindole (DAPI) and viewed on a microscope (Axiophot; Carl Zeiss Meditec, Inc., Dublin CA) equipped with appropriate filters and a charge-coupled device (CCD) camera for image analyses. 
Northern Hybridization
The cDNA clones used in this study were obtained from our mouse retinal libraries or prepared by RT-PCR using RNA from C57BL/6J retina. Total RNA was extracted from retinas of Rd4/+ and C57BL/6 mice (RNAzol B; Teltest, Inc., Friendswood, CA; or NucleoSpin RNA and Virus Purification Kits; BD-Clontech, Palo Alto, CA). Extracted RNA was separated by electrophoresis in 1.0% agarose gels containing 2.2 M formaldehyde, and transferred to Hybond-N+ membranes (GE Healthcare, Piscataway, NJ). cDNA probes were labeled with [α-32P]dCTP using the Rediprime II Random Prime Labeling System (GE Healthcare). After hybridization overnight in 0.25 M phosphate buffer, 1 mM EDTA, 7% SDS, and 1% bovine serum albumin at 65°C, the Northern blots were washed at a final stringency of 0.1× SSC, 0.1% SDS at 65°C and then exposed to x-ray film (GE Healthcare) for 2 to 16 hours at −80°C. The intensity of the signals was measured (ChemiImager 5500; Alpha Innotech Corp., San Leandro, CA). 
Real-Time Quantitative RT-PCR
The expression of mRNAs in C57BL/6J and Rd4/+ retinas was analyzed by real-time quantitative RT-PCR. Total RNA was isolated from C57BL/6J and Rd4/+ mouse retinas, as described earlier, and then treated with Turbo DNA-free (Ambion, Austin, TX) to remove the contaminating genomic DNA. Single-stranded cDNA was synthesized with reverse transcriptase (Improm-II; Promega, Madison, WI) and oligo(dT) primers. The resultant cDNA was amplified on the Mx3000 instrument (Stratagene, La Jolla, CA) using Brilliant SYBR Green QPCR master mix, which contains SureStart Taq DNA polymerase, SYBR Green-I dye, dNTPs, and the reference dye (Stratagene). The PCR reaction was run under the following conditions: denaturation at 95°C 10 minutes, followed by 40 cycles of 95°C for 30 seconds, 60°C for 1 minute, and 72°C for 30 seconds. Amplified products were incubated at 95°C for 1 minute and 55°C for 30 seconds to plot dissociation curves. Primers were chosen from exons separated by large introns, and the PCR quality and specificity was verified by dissociation curve analysis and gel electrophoresis of the amplified products. The primer sets used are shown in Table 2
Western Blot Analysis
Retinal homogenates were centrifuged at 1000g for 5 minutes. Proteins in the supernatants were separated by SDS-PAGE on a 12.5% T/3.0% C gel, 14 using a Tris/glycine buffer. They were blotted to a polyvinylidene difluoride (PVDF) membrane and incubated with a 1:4000 dilution of a Tβ1 antiserum (PA1-725, an antibody against a peptide corresponding to residues 8-25 of the Tβ1 sequence). Bands were visualized using alkaline phosphatase coupled to anti-rabbit secondary antibody and a chemiluminescence system (ECL; GE Healthcare). 
Sequence Analysis
The databases used for sequence analysis, BLAST and Map Viewer, can be found on the National Center for Biotechnology Information (NCBI) Web site (http://www.ncbi.nlm.nih.gov). The Primer 3 program was used to design primers (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3-www.cgi, provided in the public domain by the Whitehead Institute, Massachusetts Institute of Technology, Cambridge, MA). 
Results
At the outset of the study, we hypothesized that a gene disrupted at one of the inversion breakpoints was responsible for retinal disease in the Rd4/+ mouse. Initially, we performed FISH studies and determined that the proximal breakpoint of the inversion on Chr 4 was in the centromere (Fig. 2A) . Therefore, since protein-expressing genes are unlikely to be centromeric, we focused our studies on the distal breakpoint. 
Genetic Approximation of the Distal Breakpoint
To determine the general region of the distal breakpoint, we conducted the backcross [Rd4/+ (B6) x Mus spretus (Ms)]F1 x B6. The progeny of this cross that were Rd4/+ should have been homozygous B6 and those that were +/+, heterozygous B6/Ms. Any mice not fitting these categories would be recombinants. It should be noted that because of the inversion, single recombinant Rd4 chromosomes would have either two centromeres or no centromeres; both formations are nonviable. Therefore, the only viable recombinants were rare double recombinants. The 284 mouse progeny were phenotyped by fundus examination and genotyped with a set of seven distal Chr 4 dinucleotide repeat markers (see the Methods section). Only three recombinants were found (Fig. 2B) , and each had a normal fundus but was homozygous B6 for some of the markers. The most informative progeny mouse had a normal fundus but was homozygous B6 at D4Mit209 and heterozygous at D4Mit51. This placed the breakpoint distal to D4Mit209 (Fig. 2C)
Identification of the Distal Breakpoint-Spanning BAC Clone
Using the NCBI Blast function (www.ncbi.nlm.nih.gov/genome/seq/MmBlast) we determined that D4Mit209 is situated at 152.302 Mb and that Chr 4 ends at 154.141 Mb. Therefore, we had less than 2 Mb to work with. FISH experiments using several BAC clones distal to D4Mit209 revealed that the critical region for Rd4 is distal to the BAC RP23-298e4 (Fig. 3A) . With this BAC as a starting point, we conducted several FISH experiments with additional BACs from a physical map of subtelomeric Chr 4 delineated by Li et al. 15 (Fig. 3A) . Dual-color FISH was performed using the BAC clones, RP23-298e4 and RP23-118e21, labeled with digoxigenin (green) and biotin (red), respectively (Fig. 3B) . For the normal Chr 4, both signals were localized on the telomeric end and the red signal of RP23-118e21 was immediately below the green signal of RP23-298e4 (Fig. 3B , top panel). This result corresponded to the physical order of these BAC clones (Fig. 3A) . For the Rd4 chromosome, the signal was split. Both BACs hybridized near the dark-staining (by DAPI) regions representative of centromeric DNA (Fig. 3B) , but at opposite ends (the dark-staining regions are derived from the split centromere; Fig. 3B , bottom). The characteristic small and large centromere fragments were distinguished with the bright-field microscope. The green-stained RP23-298e4 hybridized immediately adjacent to the large centromere fragment and the red-stained RP23-118e21 hybridized near to the small centromere fragment (Fig. 3B) . Therefore, the distal breakpoint had to be between these two BAC clones, with green RP23-298e4 proximal to the breakpoint (inverted) and red RP23-118e21 distal (not inverted; Fig. 3C ). With further analysis, we found that the single BAC RP23-183d11 (Fig. 3A)gave a split signal on the Rd4 Chr 4 (Fig. 3E)and a telomeric signal on the normal Chr 4 (Figs. 3D 3E) . This indicated that the distal breakpoint was within the chromosomal region complementary to RP23-183d11. 
Delineation of the Position of the Telomeric Breakpoint
A total of 193 kb of sequence of RP23-183d11 (AL627405) has been completed and is available from the NCBI database. Analysis of this complete sequence using the NCBI BLAST server and NCBI Map Viewer revealed the presence of three known genes (Gnb1, Cdc211, and Mmp23), four unknown genes (BC004012, NM-177186, NM-145124, and NM-178699), one unknown expressed sequence tag (EST; AK049013), and two predicted genes (LOC433812, LOC230991) within this BAC clone (Fig. 4)
To identify which gene was disrupted by the distal breakpoint, genomic DNA fragments of RP23-183d11 were prepared by PCR amplification and used as probes for FISH (Fig. 5A) . Primer sequences are listed in Table 1 . A minimum of 20 metaphases was analyzed for each FISH study. Probe 11-1 hybridized below the large centromere region of the Rd4 chromosome (Fig. 5B)and probe 11-4 below the small centromere region (Fig. 5C) , indicating that the breakpoint was in the interval between them. Only one known gene, Gnb1 has been mapped in this region and is oriented centromere to telomere (Fig. 4) . After using several probes (data not shown), we found that probe 11-2 hybridized below the large centromere region and 11-3 below the small centromere region (Figs. 5D 5E) . Probe 11-2 contains sequence entirely from proximal intron 2 while probe 11-3 contains sequence starting in distal intron 2 and extending into exon 3 of the Gnb1 gene. This localized the breakpoint to intron 2. 
To identify the breakpoint further, we performed Southern blot analysis, using restriction enzymes such as MaeIII, PstI, MboII, AccI, and HincII, which are known to digest mouse satellite sequences frequently. Probes derived from intron 2 sequence were used for this experiment. We always detected fragments from normal chromosome 4 but not from Rd4 chromosome 4. In the Rd4 chromosome, intron 2 fragments must be attached to the split distal and proximal centromere fragments and, therefore, unable to migrate in the gel. In addition, sequencing of these areas near the centromere is difficult because they are organized into large blocks of highly repetitive satellite-DNA. 16 Several groups have reported sequences of mouse satellites 17 18 19 yet whole centromere sequences have not been fully characterized because of these repetitive blocks. 
Developmental Expression Pattern of mRNA
Gnb1 spans at least 50 kb of genomic DNA and consists of 12 exons and 11 introns. The coding sequence starts in exon 3. 20 Our FISH results showed that in the Rd4/+ mouse the breakpoint is located in intron 2 of Gnb1. Therefore, its 5′-untranslated region (UTR) is interrupted and separated from the rest of the gene. Because of this interruption/separation, we hypothesized that expression of the Rd4 Gnb1 allele would be completely shut down. To test this, the developmental expression patterns of Gnb1 and other retina-specific mRNAs were measured by Northern blot analysis in normal and Rd4/+ retinas. There were no evident differences in expression levels of Gnat1 and Rho mRNAs between Rd4/+ and C57BL/6J retinas at postnatal day (P)8 and P11 (Fig. 6) . However, from P16, mRNAs for these two genes drastically decreased in Rd4/+ retinas, correlating with the photoreceptor loss that characterizes the Rd4/+ retina at this postnatal age. 11 In contrast, expression of Gnb1 mRNA in Rd4/+ relative to C57BL/6J control retinas was reduced by approximately 50% at P8 and P11 (Fig. 6) , suggesting haploinsufficiency due to the absence of expression of the Rd4 Gnb1 allele, but normal expression from the normal Gnb1 allele. These results were confirmed by real-time quantitative RT-PCR. The results show that the expression level of Gnb1 mRNA in Rd4/+ retina is 58% of wild-type at P8, and 36% at P11 (Fig. 7 , top). For the Gnat1 and Rho transcripts, there is no significant difference in expression at P8 (Fig. 7 , middle and bottom, respectively). Because the Rho primer pair used for real-time quantitative RT-PCR detects two transcripts (2.9 and 3.5 kb) of five, the relative quantity of Rho gene measured in adult retinas in these experiments is only 20- to 25-fold that obtained at P8. To examine for the possible presence of transcripts fused with the inverted Gnb1 promoter-exon 1 and 2 sequence, we used a cDNA probe comprised of exons 1 to 7 for Northern blot analysis. No other transcripts were observed from Rd4/+ retina after a 5-day exposure to x-ray film (data not shown). 
Comparison of mRNA Expression of Genes Mapped to the RP23-183d11 BAC
To examine whether genes that are close to the distal breakpoint are affected in their expression, we compared the mRNA level of five of such genes, BC004012, NM177186, Cdc211, Mmp23, and NM145124 (see Fig. 4 ) using real-time quantitative RT-PCR and RNA from C57BL/6J and Rd4/+ retinas at two postnatal ages: P8 and P12 to 13 (Fig. 8) . The expression pattern of Gnb1 was consistent with that observed in Figure 6 . For all five genes tested, the levels of mRNA were not lower in the Rd4 mouse at P8 and P12 to P13 compared with those in the normal mouse. In fact, for three of the genes, BC004012, NM177186, and NM145124, the mRNA levels were clearly higher in Rd4/+ than in normal mice at P8 and then decreased at P12 to P13, correlating with photoreceptor cell death. No significant difference was detected in the other two genes, Cdc211 and Mmp23 between Rd4/+ and C57BL/6J at either age. These results show that the Gnb1 expression pattern is completely different from that of these nearby genes, indicating that the presence of the distal breakpoint does not bring about any deleterious changes in their expression. 
Expression of Tβ1 Protein
Levels of Tβ1 protein were analyzed by Western blot. Tβ1 protein was expressed at a lower level in Rd4/+ retina than in C57BL/6J retina at P8, P10, and P12 and continued to decrease with age (Fig. 9) , consistent with the Gnb1 mRNA expression pattern. 
Discussion
In this study, we have demonstrated that the distal breakpoint of the inverted Rd4 Chr 4 is localized in the second intron of the Gnb1 gene, whereas the proximal breakpoint lies within the centromere. The centromere plays an important role in the proper segregation of chromosomes during cell division. However, several investigators have found that centromere breaks and disrupted centromeres are stable in normal human samples both in mitosis and in meiosis, suggesting that these breaks neither impair the centromeric function nor have clinical effects. 21 22 23 Hence, the centromere DNA disruption observed in the Rd4 chromosome probably does not contribute, in this case, to the observed retinal degeneration. 
In addition, we have shown that the disruption of the Gnb1 gene by the distal breakpoint reduces mRNA and protein expression in Rd4/+ retina. This is not surprising, because the gene is disrupted in intron 2, before the coding region that starts in exon 3, and transcription would result in a very small mRNA fragment that would not be translated. Therefore, no protein would be made from the disrupted allele. This finding, along with the fact that Gnb1 encodes the transducin β1-subunit, a protein involved in phototransduction and in the normal maintenance of photoreceptors, makes this disrupted gene the most likely cause of Rd4 retinal degeneration. The locus of the Rd4 breakpoint in the Gnb1 gene at distal mouse Chr 4 is homologous to human chromosome 1, region p36, where GNB1 maps. Recently, a locus for Leber’s congenital amaurosis (LCA9; OMIM 608553; Online Mendelian Inheritance in Man; http://www.ncbi.nlm.nih.gov/Omim/ provided in the public domain by the NCBI, Bethesda, MD) has been identified between markers D1S1612 and D1S228 on 1p36. 24 The phenotype of LCA, which is characterized by severe retinal degeneration at birth or early onset, is similar to that of the Rd4 mouse. LCA, however, is recessively inherited, whereas Rd4 is inherited dominantly. In addition, a contig map from the NCBI database reveals that human Gnb1 maps approximately 6.1 Mb distal to the LCA locus. Therefore, Gnb1 is not a candidate gene for LCA9. 
Several investigators have reported that translocation breakpoints are found to map outside the putative gene and that the gene defect is caused by a position effect of the breakpoint. 25 For example, PAX6 haploinsufficiency at human chromosome 11, region q13, has been shown to cause aniridia, which is a congenital malformation characterized by severe hypoplasia of the iris. In two patients with aniridia, the translocation breakpoints mapped at 100 and 125 kb downstream of PAX6. 25 26 We investigated the possibility that the distal breakpoint provides a position effect on five other genes present in the BAC that includes the Rd4 gene. We showed that the mRNA expression patterns of these genes are different from that of Gnb1. None showed decreased expression at P8 and P11 like Gnb1. These results indicate that there is no position effect of the distal breakpoint, at least on these five genes. 
Gnat1 / mice show a mild retinal degeneration, having a slightly thinner ONL (the outer nuclear layer of the retina; the nuclei of the photoreceptor rods and cones) compared with wild-type at 4 weeks of age and a thinner photoreceptor layer with one row of nuclei in the ONL at 13 weeks (normal is 10–12 rows of nuclei). 27 Gnat1 / retina expresses the Tβ1 protein at a level that is close to normal. In Gnat1 / + mice, there is a small reduction of Tα protein content but retinal morphology is almost normal. Other groups have constructed Rho / mice 28 29 and have shown that at P15 Rho / retinas contain a normal number of nuclei in the ONL and normal-appearing inner segments, but that outer segments are absent. Although the ONL and inner segments become thinner with age, there is still one row of nuclei at P90. Rho / + mice have shorter outer segments than normal in early life, but no significant degeneration is observed with age in these retinas. In contrast to these knockout mice, the Rd4/+ mouse has a severe, progressive, retinal degeneration that begins at P10 and results in complete loss of photoreceptor cells by 6 weeks of age. 11 In Rd4/+ retina, expression of Gnb1 mRNA is reduced to ∼50% that of control retina at an early stage, P8, whereas no obvious reductions of Gnat1 and Rho mRNAs are detected at the same age. The complete absence of Tα in the Gnat / mouse causes only a mild retinal degeneration, whereas there is a severe phenotype associated with heterozygosity in the Rd4/+ mouse. This suggests that Gnb1 may be essential not only for phototransduction but also for survival of photoreceptor cells. For example, the decreased levels of Tβ may impair the trafficking of other proteins or protein complexes contributing to the demise of the visual cells. Therefore, the association of a defective Gnb1 gene with Rd4 disease opens two avenues of study. The first, of course, is to screen patients with retinal degeneration for mutations in the human GNB1 gene, and the second is to use the Rd4 mouse to study the role that Tβ1 plays in the maintenance of photoreceptors. 
 
Figure 1.
 
ERG recordings from a representative 3-week-old C57BL/6J mouse (left), and 3-and 6-week old Rd4/+ mice (middle and right, respectively). (A) Rod-mediated ERGs recorded to blue flashes of light of increasing intensity. (B) Cone-mediated ERGs recorded to white flashes of increasing intensity.
Figure 1.
 
ERG recordings from a representative 3-week-old C57BL/6J mouse (left), and 3-and 6-week old Rd4/+ mice (middle and right, respectively). (A) Rod-mediated ERGs recorded to blue flashes of light of increasing intensity. (B) Cone-mediated ERGs recorded to white flashes of increasing intensity.
Table 1.
 
Primers Used to Prepare Probes for FISH Analysis
Table 1.
 
Primers Used to Prepare Probes for FISH Analysis
Probe Primer 5′–3′
11-1 Forward-1 TTC CTG CTT CTC TGG TTT CC
Reverse-1 GGG CTC CAC AAA GAC TCA TC
Forward-2 CCC CGT GGG TAC TCT GTC T
Reverse-2 AGC TTA GGA CCC CTT TCC AC
11-2 Forward-1 AGC CCT GAC AAT TAG CTC CA
Reverse-1 CTT GCT GCC CAC TCT TTC TC
Forward-2 TTG GAT TTT CAG TCA GGA ATC A
Reverse-2 TCC TCA AAA CGA GCA AAA GG
11–3 Forward-1 TCT GAG TTG TTC TTT CTG TGC TG
Reverse-1 GCT TAG TGA GAC GCT CAA GGA
Forward-2 TTG TGT GTG GAT AAC AGA GTG AGA
Reverse-2 TCT GCT CTC TAT GGT GGC CTA
Forward-3 GCA CCA CCT CTA TTG GCA TT
Reverse-3 TCT CAC TCT GTT ATC CAC ACA CAA
11–4 Forward-1 GAT GAG CCT CCT GCT TTG TC
Reverse-1 CGA TTC AAG GTT GGA ATG GT
Forward-2 TGA TCC TGT TCT GTG GCA TT
Reverse-2 GTG GAC AGT GAG GGG ACA G
Forward-3 CTG GAA GAA CAG CCT TTT GC
Reverse-3 CAT CAC ACC AGA GTC CCT CA
Table 2.
 
Primers Used for Real-Time Quantitative RT-PCR
Table 2.
 
Primers Used for Real-Time Quantitative RT-PCR
Gene Primer 5′–3′
Gnb1 Forward TGA TGC TTC AGC CAA GCT CT
Reverse ACC TGC TCT GTC AGC TTT GA
Gnat1 Forward GAC GAC GAA GTG AAC CGA AT
Reverse GGT GAC AGC GTC AAA GAC AA
Rho Forward TCG TCA TCT TCT TCT GCT ATG G
Reverse AGC GTG GTG AGC ATA CAG TT
BC004012 Forward TCT GCA CTT TCC GTG AAG AC
Reverse ATT TTC ACT GAG GCC GTT GT
NM-177186 Forward TGG ATA TCC CTG TGA TTG GAA
Reverse TGT GTC ATC TTC TGG GTT TCG
Cdc211 Forward AAT GGC GAG AGA ACA TTC CA
Reverse TGT CTT GGA GGT CTG ACA ACA
Mmp23 Forward GGC AGC TCA GGG AAA TGT AG
Reverse GGT TTG TGA GCC AAA CAC CT
NM-145124 Forward CGC GTC TGC TTT GAC TAT GA
Reverse ACA CGT TCG TGG TTC CAT CT
Figure 2.
 
Genetic localization of the distal breakpoint of the Rd4 chromosome. (A) FISH analysis of chromosomes from Rd4/+ mouse probed with a distal (left) and a proximal (right) BAC. (B) Alleles of the N2 progeny of the backcross [Rd4/+(B6) x M. Spretus (Ms)]F1 x B6. Gold boxes: homozygous C57Bl/6J; cyan boxes: C57Bl/6J/Ms. N, normal phenotype; Rd4, affected phenotype. (C) Physical map of the subtelomeric region of Chr 4. The breakpoint is distal to D4Mit209.
Figure 2.
 
Genetic localization of the distal breakpoint of the Rd4 chromosome. (A) FISH analysis of chromosomes from Rd4/+ mouse probed with a distal (left) and a proximal (right) BAC. (B) Alleles of the N2 progeny of the backcross [Rd4/+(B6) x M. Spretus (Ms)]F1 x B6. Gold boxes: homozygous C57Bl/6J; cyan boxes: C57Bl/6J/Ms. N, normal phenotype; Rd4, affected phenotype. (C) Physical map of the subtelomeric region of Chr 4. The breakpoint is distal to D4Mit209.
Figure 3.
 
Characterization of the distal breakpoint region. (A) Physical map of the critical region for Rd4 (not in scale). Markers are indicated above the diagram. BAC clones are shown as solid bars with RP23 addresses. (B) Top: dual-color FISH was performed with BAC clones, RP23-298e4 (green) and RP23-118e21 (red). Bottom: The FISH image was processed to show the split centromeres (dark-stained regions), which allow identification of the Rd4 allele. (C) The ideogram with the expected localization of the signals obtained using RP23-298e4 (green) and RP23-118e21 (red). (D) BAC clone RP23-183d11 labeled with FITC was hybridized to a chromosome spread prepared from a normal C57BL/6J mouse. (E) Hybridization with RP23-183d11 to a chromosome spread from a Rd4/+ mouse demonstrates split signals on Rd4 allele. N, normal allele; In4, inverted allele.
Figure 3.
 
Characterization of the distal breakpoint region. (A) Physical map of the critical region for Rd4 (not in scale). Markers are indicated above the diagram. BAC clones are shown as solid bars with RP23 addresses. (B) Top: dual-color FISH was performed with BAC clones, RP23-298e4 (green) and RP23-118e21 (red). Bottom: The FISH image was processed to show the split centromeres (dark-stained regions), which allow identification of the Rd4 allele. (C) The ideogram with the expected localization of the signals obtained using RP23-298e4 (green) and RP23-118e21 (red). (D) BAC clone RP23-183d11 labeled with FITC was hybridized to a chromosome spread prepared from a normal C57BL/6J mouse. (E) Hybridization with RP23-183d11 to a chromosome spread from a Rd4/+ mouse demonstrates split signals on Rd4 allele. N, normal allele; In4, inverted allele.
Figure 4.
 
Genes and cDNA clones mapped on the BAC RP23-183d11, which spans the region of the distal breakpoint. Arrows: orientation of each gene.
Figure 4.
 
Genes and cDNA clones mapped on the BAC RP23-183d11, which spans the region of the distal breakpoint. Arrows: orientation of each gene.
Figure 5.
 
Localization by FISH of the distal breakpoint, using small genomic DNA fragments. (A) Top horizontal lines: BAC clones with RP23 addresses. Bottom line with vertical boxes: genomic structure of Gnb1. Red bars: small genomic fragments used as probes in FISH experiments. Fragments 11-1 and 11-2 are proximal to the breakpoint, because they hybridize to an inverted chromosomal region (B, D). Fragments 11-3 and 11-4 are distal to the breakpoint, because they hybridize to chromosomal regions that do not invert (C, E). The breakpoint region was narrowed down to the 10-kb region between fragments 11-2 and 11-3.
Figure 5.
 
Localization by FISH of the distal breakpoint, using small genomic DNA fragments. (A) Top horizontal lines: BAC clones with RP23 addresses. Bottom line with vertical boxes: genomic structure of Gnb1. Red bars: small genomic fragments used as probes in FISH experiments. Fragments 11-1 and 11-2 are proximal to the breakpoint, because they hybridize to an inverted chromosomal region (B, D). Fragments 11-3 and 11-4 are distal to the breakpoint, because they hybridize to chromosomal regions that do not invert (C, E). The breakpoint region was narrowed down to the 10-kb region between fragments 11-2 and 11-3.
Figure 6.
 
Expression of Gnb1, Gnat1, and Rho mRNAs during retinal development. Shown is a Northern blot of retinal RNA isolated at various ages of postnatal development. All lanes contain 5 μg of total RNA. β-Actin was used to normalize for RNA quantity per lane. Note that at P11 there was more RNA from Rd4/+ loaded than from C57BL/6J, as shown by the intensity of the β-actin signal of the same samples.
Figure 6.
 
Expression of Gnb1, Gnat1, and Rho mRNAs during retinal development. Shown is a Northern blot of retinal RNA isolated at various ages of postnatal development. All lanes contain 5 μg of total RNA. β-Actin was used to normalize for RNA quantity per lane. Note that at P11 there was more RNA from Rd4/+ loaded than from C57BL/6J, as shown by the intensity of the β-actin signal of the same samples.
Figure 7.
 
Expression levels of Gnb1, Gnat1, and Rho mRNAs at various postnatal ages determined by quantitative RT-PCR. mRNA expression was quantified relative to the expression of the corresponding gene in C57BL/6J at P8. The expression value was calculated as the mean of three independent quantitative RT-PCR experiments performed in duplicate. Error bars, ±SEM. For the Rho gene, only two transcripts (2.9 and 3.5 kb) were detected by the primer pair used.
Figure 7.
 
Expression levels of Gnb1, Gnat1, and Rho mRNAs at various postnatal ages determined by quantitative RT-PCR. mRNA expression was quantified relative to the expression of the corresponding gene in C57BL/6J at P8. The expression value was calculated as the mean of three independent quantitative RT-PCR experiments performed in duplicate. Error bars, ±SEM. For the Rho gene, only two transcripts (2.9 and 3.5 kb) were detected by the primer pair used.
Figure 8.
 
Comparative expression of genes mapped on the BAC RP23-183d11 at P8 and P12 to P13. mRNA expression was quantified relative to the expression of the corresponding gene in C57BL/6J at P8. The mRNA levels were calculated as the mean of three independent quantitative RT-PCR experiments performed in duplicate. Error bars, ±SEM.
Figure 8.
 
Comparative expression of genes mapped on the BAC RP23-183d11 at P8 and P12 to P13. mRNA expression was quantified relative to the expression of the corresponding gene in C57BL/6J at P8. The mRNA levels were calculated as the mean of three independent quantitative RT-PCR experiments performed in duplicate. Error bars, ±SEM.
Figure 9.
 
Comparison of transducin β1-subunit expression in Rd4/+ and C57BL/6J retinas. Each lane of the Western blot contains 25 μg protein. A polyclonal antibody specific for Tβ1-subunit was used at a 1:4000 dilution.
Figure 9.
 
Comparison of transducin β1-subunit expression in Rd4/+ and C57BL/6J retinas. Each lane of the Western blot contains 25 μg protein. A polyclonal antibody specific for Tβ1-subunit was used at a 1:4000 dilution.
The authors thank Elika Decker and Cathy Do for technical assistance. 
BowesC, LiT, DancigerM, BaxterLC, AppleburyML, FarberDB. Retinal degeneration in the rd mouse is caused by a defect in the β subunit of rod cGMP-phosphodiesterase. Nature. 1990;347:677–680. [CrossRef] [PubMed]
BowesC, LiT, FrankelWN, et al. Localization of a retroviral element within the rd gene coding for the β subunit of cGMP-phosphodiesterase. Proc Natl Acad Sci USA. 1993;90:2955–2959. [CrossRef] [PubMed]
PittlerSJ, BaehrW. Identification of a nonsense mutation in the rod photoreceptor cGMP phosphodiesterase β subunit gene of the rd mouse. Proc Natl Acad Sci USA. 1991;88:8322–8326. [CrossRef] [PubMed]
McLaughlinME, SandbergMA, BersonEL, DryjaTP. Recessive mutations in the gene encoding the β-subunit of rod phosphodiesterase in patients with retinitis pigmentosa. Nat Genet. 1993;4:130–134. [CrossRef] [PubMed]
DancigerM, BlaneyJ, GaoY, et al. Mutations in the PDE6B gene in autosomal recessive retinitis pigmentosa. Genomics. 1995;30:1–7. [CrossRef] [PubMed]
BayesM, GiordanoM, BalcellsS, et al. Homozygous tandem duplication within the gene encoding the β-subunit of rod phosphodiesterase as a cause of autosomal recessive retinitis pigmentosa. Hum Mutat. 1995;5:228–234. [CrossRef] [PubMed]
AkhmedovNB, PirievNI, ChangB, et al. A deletion in a photoreceptor-specific nuclear receptor mRNA causes retinal degeneration in the rd7 mouse. Proc Natl Acad Sci USA. 2000;97:5551–5556. [CrossRef] [PubMed]
HaiderNB, JacobsonSG, CideciyanAV, et al. Mutation of a nuclear receptor gene, NR2E3, causes enhanced S cone syndrome, a disorder of retinal cell fate. Nat Genet. 2000;24:127–131. [CrossRef] [PubMed]
HaiderNB, NaggertJK, NishinaPM. Excess cone cell proliferation due to lack of a functional NR2E3 causes retinal dysplasia and degeneration in rd7/rd7 mice. Hum Mol Genet. 2001;10:1619–1626. [CrossRef] [PubMed]
MilamAH, RoseL, CideciyanAV, et al. The nuclear receptor NR2E3 plays a role in human retinal photoreceptor differentiation and degeneration. Proc Natl Acad Sci USA. 2002;99:473–478. [CrossRef] [PubMed]
RoderickTH, ChangB, HawesNL, HeckenlivelyJR. A new dominant retinal degeneration (Rd4) associated with a chromosomal inversion in the mouse. Genomics. 1997;42:393–396. [CrossRef] [PubMed]
NusinowitzS, NguyenL, RaduR, KashaniZ, FarberD, DancigerM. Electroretinographic evidence for altered phototransduction gain and slowed recovery from photobleaches in albino mice with a MET450 variant in RPE65. Exp Eye Res. 2003;77:627–638. [CrossRef] [PubMed]
KitamuraE, KuemerleBA, ChernovaOB, CowellJK. Molecular characterization of the breakpoint region associated with a constitutional t(2;15)(q34;q26) in a patient with multiple myeloma. Cancer Genet Cytogenet. 2001;129:112–119. [CrossRef] [PubMed]
LaemmliUK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227:680–685. [CrossRef] [PubMed]
LiX, BachmanovAA, LiS, et al. Genetic, physical, and comparative map of the subtelomeric region of mouse Chromosome 4. Mamm Genome. 2002;13:5–19. [CrossRef] [PubMed]
LambJC, BirchlerJA. The role of DNA sequence in centromere formation. Genome Biol. 2003;4:214. [CrossRef] [PubMed]
HorzW, AltenburgerW. Nucleotide sequence of mouse satellite DNA. Nucleic Acids Res. 1981;9:683–696. [CrossRef] [PubMed]
ManuelidisL. Consensus sequence of mouse satellite DNA indicates it is derived from tandem 116 basepair repeats. FEBS Lett. 1981;129:25–28. [CrossRef] [PubMed]
WongAK, RattnerJB. Sequence organization and cytological localization of the minor satellite of mouse. Nucleic Acids Res. 1988;16:11645–11661. [CrossRef] [PubMed]
KitanakaJ, WangXB, KitanakaN, HembreeCM, UhlGR. Genomic organization of the murine G protein beta subunit genes and related processed pseudogenes. DNA Seq. 2001;12:345–354. [PubMed]
WangJC, MamunesP, KouSY, SchmidtJ, MaoR, HsuWT. Centromeric DNA break in a 10;16 reciprocal translocation associated with trisomy 16 confined placental mosaicism and maternal uniparental disomy for chromosome 16. Am J Med Genet. 1998;80:418–422. [CrossRef] [PubMed]
TumerZ, BergA, MikkelsenM. Analysis of a whole arm translocation between chromosomes 18 and 20 using fluorescence in situ hybridization: detection of a break in the centromeric alpha-satellite sequences. Hum Genet. 1995;95:299–302. [PubMed]
CantuES, KhanTA, PaiGS. Fluorescence in situ hybridization (FISH) of a whole-arm translocation involving chromosomes 18 and 20 with alpha-satellite DNA probes: detection of a centromeric DNA break?. Am J Med Genet. 1992;44:340–344. [CrossRef] [PubMed]
KeenTJ, MohamedMD, McKibbinM, et al. Identification of a locus (LCA9) for Leber’s congenital amaurosis on chromosome 1p36. Eur J Hum Genet. 2003;11:420–423. [CrossRef] [PubMed]
KleinjanDJ, van HeyningenV. Position effect in human genetic disease. Hum Mol Genet. 1998;7:1611–1618. [CrossRef] [PubMed]
FantesJ, RedekerB, BreenM, et al. Aniridia-associated cytogenetic rearrangements suggest that a position effect may cause the mutant phenotype. Hum Mol Genet. 1995;4:415–422. [CrossRef] [PubMed]
CalvertPD, KrasnoperovaNV, LyubarskyAL, et al. Phototransduction in transgenic mice after targeted deletion of the rod transducin alpha-subunit. Proc Natl Acad Sci USA. 2000;97:13913–13918. [CrossRef] [PubMed]
HumphriesMM, RancourtD, FarrarGJ, et al. Retinopathy induced in mice by targeted disruption of the rhodopsin gene. Nat Genet. 1997;15:216–219. [CrossRef] [PubMed]
LemJ, KrasnoperovaNV, CalvertPD, et al. Morphological, physiological, and biochemical changes in rhodopsin knockout mice. Proc Natl Acad Sci USA. 1999;96:736–741. [CrossRef] [PubMed]
Figure 1.
 
ERG recordings from a representative 3-week-old C57BL/6J mouse (left), and 3-and 6-week old Rd4/+ mice (middle and right, respectively). (A) Rod-mediated ERGs recorded to blue flashes of light of increasing intensity. (B) Cone-mediated ERGs recorded to white flashes of increasing intensity.
Figure 1.
 
ERG recordings from a representative 3-week-old C57BL/6J mouse (left), and 3-and 6-week old Rd4/+ mice (middle and right, respectively). (A) Rod-mediated ERGs recorded to blue flashes of light of increasing intensity. (B) Cone-mediated ERGs recorded to white flashes of increasing intensity.
Figure 2.
 
Genetic localization of the distal breakpoint of the Rd4 chromosome. (A) FISH analysis of chromosomes from Rd4/+ mouse probed with a distal (left) and a proximal (right) BAC. (B) Alleles of the N2 progeny of the backcross [Rd4/+(B6) x M. Spretus (Ms)]F1 x B6. Gold boxes: homozygous C57Bl/6J; cyan boxes: C57Bl/6J/Ms. N, normal phenotype; Rd4, affected phenotype. (C) Physical map of the subtelomeric region of Chr 4. The breakpoint is distal to D4Mit209.
Figure 2.
 
Genetic localization of the distal breakpoint of the Rd4 chromosome. (A) FISH analysis of chromosomes from Rd4/+ mouse probed with a distal (left) and a proximal (right) BAC. (B) Alleles of the N2 progeny of the backcross [Rd4/+(B6) x M. Spretus (Ms)]F1 x B6. Gold boxes: homozygous C57Bl/6J; cyan boxes: C57Bl/6J/Ms. N, normal phenotype; Rd4, affected phenotype. (C) Physical map of the subtelomeric region of Chr 4. The breakpoint is distal to D4Mit209.
Figure 3.
 
Characterization of the distal breakpoint region. (A) Physical map of the critical region for Rd4 (not in scale). Markers are indicated above the diagram. BAC clones are shown as solid bars with RP23 addresses. (B) Top: dual-color FISH was performed with BAC clones, RP23-298e4 (green) and RP23-118e21 (red). Bottom: The FISH image was processed to show the split centromeres (dark-stained regions), which allow identification of the Rd4 allele. (C) The ideogram with the expected localization of the signals obtained using RP23-298e4 (green) and RP23-118e21 (red). (D) BAC clone RP23-183d11 labeled with FITC was hybridized to a chromosome spread prepared from a normal C57BL/6J mouse. (E) Hybridization with RP23-183d11 to a chromosome spread from a Rd4/+ mouse demonstrates split signals on Rd4 allele. N, normal allele; In4, inverted allele.
Figure 3.
 
Characterization of the distal breakpoint region. (A) Physical map of the critical region for Rd4 (not in scale). Markers are indicated above the diagram. BAC clones are shown as solid bars with RP23 addresses. (B) Top: dual-color FISH was performed with BAC clones, RP23-298e4 (green) and RP23-118e21 (red). Bottom: The FISH image was processed to show the split centromeres (dark-stained regions), which allow identification of the Rd4 allele. (C) The ideogram with the expected localization of the signals obtained using RP23-298e4 (green) and RP23-118e21 (red). (D) BAC clone RP23-183d11 labeled with FITC was hybridized to a chromosome spread prepared from a normal C57BL/6J mouse. (E) Hybridization with RP23-183d11 to a chromosome spread from a Rd4/+ mouse demonstrates split signals on Rd4 allele. N, normal allele; In4, inverted allele.
Figure 4.
 
Genes and cDNA clones mapped on the BAC RP23-183d11, which spans the region of the distal breakpoint. Arrows: orientation of each gene.
Figure 4.
 
Genes and cDNA clones mapped on the BAC RP23-183d11, which spans the region of the distal breakpoint. Arrows: orientation of each gene.
Figure 5.
 
Localization by FISH of the distal breakpoint, using small genomic DNA fragments. (A) Top horizontal lines: BAC clones with RP23 addresses. Bottom line with vertical boxes: genomic structure of Gnb1. Red bars: small genomic fragments used as probes in FISH experiments. Fragments 11-1 and 11-2 are proximal to the breakpoint, because they hybridize to an inverted chromosomal region (B, D). Fragments 11-3 and 11-4 are distal to the breakpoint, because they hybridize to chromosomal regions that do not invert (C, E). The breakpoint region was narrowed down to the 10-kb region between fragments 11-2 and 11-3.
Figure 5.
 
Localization by FISH of the distal breakpoint, using small genomic DNA fragments. (A) Top horizontal lines: BAC clones with RP23 addresses. Bottom line with vertical boxes: genomic structure of Gnb1. Red bars: small genomic fragments used as probes in FISH experiments. Fragments 11-1 and 11-2 are proximal to the breakpoint, because they hybridize to an inverted chromosomal region (B, D). Fragments 11-3 and 11-4 are distal to the breakpoint, because they hybridize to chromosomal regions that do not invert (C, E). The breakpoint region was narrowed down to the 10-kb region between fragments 11-2 and 11-3.
Figure 6.
 
Expression of Gnb1, Gnat1, and Rho mRNAs during retinal development. Shown is a Northern blot of retinal RNA isolated at various ages of postnatal development. All lanes contain 5 μg of total RNA. β-Actin was used to normalize for RNA quantity per lane. Note that at P11 there was more RNA from Rd4/+ loaded than from C57BL/6J, as shown by the intensity of the β-actin signal of the same samples.
Figure 6.
 
Expression of Gnb1, Gnat1, and Rho mRNAs during retinal development. Shown is a Northern blot of retinal RNA isolated at various ages of postnatal development. All lanes contain 5 μg of total RNA. β-Actin was used to normalize for RNA quantity per lane. Note that at P11 there was more RNA from Rd4/+ loaded than from C57BL/6J, as shown by the intensity of the β-actin signal of the same samples.
Figure 7.
 
Expression levels of Gnb1, Gnat1, and Rho mRNAs at various postnatal ages determined by quantitative RT-PCR. mRNA expression was quantified relative to the expression of the corresponding gene in C57BL/6J at P8. The expression value was calculated as the mean of three independent quantitative RT-PCR experiments performed in duplicate. Error bars, ±SEM. For the Rho gene, only two transcripts (2.9 and 3.5 kb) were detected by the primer pair used.
Figure 7.
 
Expression levels of Gnb1, Gnat1, and Rho mRNAs at various postnatal ages determined by quantitative RT-PCR. mRNA expression was quantified relative to the expression of the corresponding gene in C57BL/6J at P8. The expression value was calculated as the mean of three independent quantitative RT-PCR experiments performed in duplicate. Error bars, ±SEM. For the Rho gene, only two transcripts (2.9 and 3.5 kb) were detected by the primer pair used.
Figure 8.
 
Comparative expression of genes mapped on the BAC RP23-183d11 at P8 and P12 to P13. mRNA expression was quantified relative to the expression of the corresponding gene in C57BL/6J at P8. The mRNA levels were calculated as the mean of three independent quantitative RT-PCR experiments performed in duplicate. Error bars, ±SEM.
Figure 8.
 
Comparative expression of genes mapped on the BAC RP23-183d11 at P8 and P12 to P13. mRNA expression was quantified relative to the expression of the corresponding gene in C57BL/6J at P8. The mRNA levels were calculated as the mean of three independent quantitative RT-PCR experiments performed in duplicate. Error bars, ±SEM.
Figure 9.
 
Comparison of transducin β1-subunit expression in Rd4/+ and C57BL/6J retinas. Each lane of the Western blot contains 25 μg protein. A polyclonal antibody specific for Tβ1-subunit was used at a 1:4000 dilution.
Figure 9.
 
Comparison of transducin β1-subunit expression in Rd4/+ and C57BL/6J retinas. Each lane of the Western blot contains 25 μg protein. A polyclonal antibody specific for Tβ1-subunit was used at a 1:4000 dilution.
Table 1.
 
Primers Used to Prepare Probes for FISH Analysis
Table 1.
 
Primers Used to Prepare Probes for FISH Analysis
Probe Primer 5′–3′
11-1 Forward-1 TTC CTG CTT CTC TGG TTT CC
Reverse-1 GGG CTC CAC AAA GAC TCA TC
Forward-2 CCC CGT GGG TAC TCT GTC T
Reverse-2 AGC TTA GGA CCC CTT TCC AC
11-2 Forward-1 AGC CCT GAC AAT TAG CTC CA
Reverse-1 CTT GCT GCC CAC TCT TTC TC
Forward-2 TTG GAT TTT CAG TCA GGA ATC A
Reverse-2 TCC TCA AAA CGA GCA AAA GG
11–3 Forward-1 TCT GAG TTG TTC TTT CTG TGC TG
Reverse-1 GCT TAG TGA GAC GCT CAA GGA
Forward-2 TTG TGT GTG GAT AAC AGA GTG AGA
Reverse-2 TCT GCT CTC TAT GGT GGC CTA
Forward-3 GCA CCA CCT CTA TTG GCA TT
Reverse-3 TCT CAC TCT GTT ATC CAC ACA CAA
11–4 Forward-1 GAT GAG CCT CCT GCT TTG TC
Reverse-1 CGA TTC AAG GTT GGA ATG GT
Forward-2 TGA TCC TGT TCT GTG GCA TT
Reverse-2 GTG GAC AGT GAG GGG ACA G
Forward-3 CTG GAA GAA CAG CCT TTT GC
Reverse-3 CAT CAC ACC AGA GTC CCT CA
Table 2.
 
Primers Used for Real-Time Quantitative RT-PCR
Table 2.
 
Primers Used for Real-Time Quantitative RT-PCR
Gene Primer 5′–3′
Gnb1 Forward TGA TGC TTC AGC CAA GCT CT
Reverse ACC TGC TCT GTC AGC TTT GA
Gnat1 Forward GAC GAC GAA GTG AAC CGA AT
Reverse GGT GAC AGC GTC AAA GAC AA
Rho Forward TCG TCA TCT TCT TCT GCT ATG G
Reverse AGC GTG GTG AGC ATA CAG TT
BC004012 Forward TCT GCA CTT TCC GTG AAG AC
Reverse ATT TTC ACT GAG GCC GTT GT
NM-177186 Forward TGG ATA TCC CTG TGA TTG GAA
Reverse TGT GTC ATC TTC TGG GTT TCG
Cdc211 Forward AAT GGC GAG AGA ACA TTC CA
Reverse TGT CTT GGA GGT CTG ACA ACA
Mmp23 Forward GGC AGC TCA GGG AAA TGT AG
Reverse GGT TTG TGA GCC AAA CAC CT
NM-145124 Forward CGC GTC TGC TTT GAC TAT GA
Reverse ACA CGT TCG TGG TTC CAT CT
×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×