Rapid degradation of dominant-negative Rab27 proteins in vivo precludes their use in transgenic mouse models
© Ramalho et al; licensee BioMed Central Ltd. 2002
Received: 12 July 2002
Accepted: 28 October 2002
Published: 28 October 2002
Transgenic mice have proven to be a powerful system to study normal and pathological gene functions. Here we describe an attempt to generate a transgenic mouse model for choroideremia (CHM), a slow-onset X-linked retinal degeneration caused by mutations in the Rab Escort Protein-1 (REP1) gene. REP1 is part of the Rab geranylgeranylation machinery, a modification that is essential for Rab function in membrane traffic. The loss of REP1 in CHM patients may trigger retinal degeneration through its effects on Rab proteins. We have previously reported that Rab27a is the Rab most affected in CHM lymphoblasts and hypothesised that the selective dysfunction of Rab27a (and possibly a few other Rab GTPases) plays an essential role in the retinal degenerative process.
To investigate this hypothesis, we generated several lines of dominant-negative, constitutively-active and wild-type Rab27a (and Rab27b) transgenic mice whose expression was driven either by the pigment cell-specific tyrosinase promoter or the ubiquitous β-actin promoter. High levels of mRNA and protein were observed in transgenic lines expressing wild-type or constitutively active Rab27a and Rab27b. However, only modest levels of transgenic protein were expressed. Pulse-chase experiments suggest that the dominant-negative proteins, but not the constitutively-active or wild type proteins, are rapidly degraded. Consistently, no significant phenotype was observed in our transgenic lines. Coat-colour was normal, indicating normal Rab27a activity. Retinal function as determined by fundoscopy, angiography, electroretinography and histology was also normal.
We suggest that the instability of the dominant-negative mutant Rab27 proteins in vivo precludes the use of this approach to generate mouse models of disease caused by Rab27 GTPases.
Membrane traffic in eukaryotic cells is mediated by vesicular carriers, which bud from a donor compartment, are targeted to, and fuse with the appropriate acceptor membrane. The Rab family of Ras-like GTPases is known to play a crucial role controlling these mechanisms [1, 2]. Among Rabs, the Rab27 subfamily consists of two isoforms, Rab27a (previously designated Ram)  and Rab27b (previously designated c25KG) .
In order to function in membrane traffic, Rabs require the covalent addition of one or two C20 geranylgeranyl groups [5, 6]. Geranylgeranylation serves as a membrane-anchoring device in Rab proteins . This reaction is complex and occurs in several steps. Newly synthesised Rab proteins first associate with Rab Escort Protein (REP) and form a stable 1:1 complex [7, 8]. The complex then serves as a substrate for Rab geranylgeranyl transferase to catalyse geranylgeranylation via thioether bonds to carboxy-terminal cysteine residues in Rabs [9, 10]. Alternatively, newly synthesised Rabs can associate with a pre-formed REP:Rab-geranylgeranyl-transferase complex . After geranylgeranylation, the REP:Rab complex is competent for delivery of the newly prenylated protein to cellular membranes by a process that has not been characterised [12, 13].
The human genome contains two related REP genes, REP1 and REP2 . Loss-of-function mutations in REP1 result in choroideremia, an X-linked slow-onset retinal degeneration that affects photoreceptors, retinal pigment epithelium and the choroid [8, 15]. We have speculated that the disease is caused by selective defects in Rab geranylgeranylation in the affected retinal cells and have identified Rab27a as one Rab that appears selectively unprenylated in choroideremia lymphoblasts . Thus, Rab27a, which is expressed at high levels in the retinal pigment epithelium and choroid, could play an important role in triggering the degenerative process in choroideremia [16, 17].
The rationale for the present study was to generate transgenic mice expressing dominant-negative mutant Rab27 proteins in order to interfere with the function of the wild type alleles and disrupt the Rab27 regulatory function. We found that dominant-negative Rab27 proteins are unstable and quickly degraded in vivo, and are thus unable to generate a dominant-interfering effect.
Construction of transgenic vectors
Mutations affecting the GTP/GDP cycle of Rab27a
expected biochemical properties
preferential binding of GDP
reduced GTPase activity
low affinity for GDP/GTP
Production of transgenic lines
Nomenclature for transgenic lines
transgenic line identification*
Transgene mRNA expression
Transgenic protein and mRNA expression.
transgenic protein expression
0.20 ± 0.02
0.29 ± 0.03
0.31 ± 0.01
0.29 ± 0.01
2.6 ± 0.2
1.8 ± 0.1
0.21 ± 0.03
1.7 ± 0.1
0.32 ± 0.06
0.61 ± 0.02
0.26 ± 0.02
0.95 ± 0.04
1.27 ± 0.08
2.2 ± 0.4
1.21 ± 0.03
0.6 ± 0.1
1.64 ± 0.06
Transgenic protein expression by immunoblotting
The results for the tyrosinase promoter-driven transgenes were less clear as the transgenic protein did not contain an epitope tag and could not be distinguished from the endogenous protein. We have not been able to detect differences in the levels of Rab27a in any of the twelve tissues described above (data not shown).
Turn-over of Rab27a mutants
Phenotypic characterisation: coat colour
To test the functionality of the transgenic Rab27a proteins in vivo, we produced mice where the transgenic protein was the only source of Rab27a. This was accomplished by crossing the transgenic lines twice with ashen homozygous mice to obtain homozygous ashen mice carrying the transgenes. The offspring were genotyped  and examined for coat colour. As expected, the wild-type Rab27a transgene was able to rescue the ashen mice coat colour nearly completely (Fig. 6C) while the dominant-negative Rab27aT23N transgene did not (Fig. 6D). Mice that were homozygous for the ashen mutation and carried the constitutively-active mutant Rab27aQ78L transgene (ash/ash, -/tgRab27aQ78L) partially rescued the coat colour defect (Fig. 6D). This effect was strengthened by transgene homozygosity. Transgenic ash/ash, tgRab27aQ78L/tgRab27aQ78L mice were almost entirely rescued to wild type coat colour (data not shown).
Phenotypic characterisation: vision
Despite the lack of a coat colour phenotype, we decided to study retinal function in the transgenic mice. These studies included fundoscopy, angiography, electroretinography and histology and were performed at different ages, from 1 month to over 1 year old.
Another important complementary method of eye examination is Ganzfeld electroretinography (ERG). In a Ganzfeld setup, the stimulus (usually a Xenon tube flash of less than 1 ms duration) passes through a diffusor that yields a relatively homogeneous distribution of light intensity on the inside of a bowl. The term 'Ganzfeld' meaning 'full field' denotes that the stimulus reaches practically all parts of the retina and its intensity is approximately equal across that area. For most applications, the ERG, which is an electric sum potential generated by retinal cells following exposure to light, is non-invasively measured at the corneal surface. Typically, the evoked response consists of an initial negative deflection (a-wave), followed by a large, positive component (b-wave). Superimposed on the ascending portion of the b-wave are the oscillatory potentials (OPs), a set of wavelets oscillating with approximately 4–5 times the frequency of the a- and b-wave. Finally, a prolonged positive component (c-wave) follows, which takes several seconds to develop.
At the outset, we wished to investigate the possible role of Rab27 proteins in human disease, in particular CHM. We decided to generate transgenic mice expressing dominant-negative forms of Rab27 for several reasons. Firstly, the strategy of generating Rab proteins defective in either GTP hydrolysis (constitutively-active) or GDP/GTP exchange (dominant-negative) to selectively alter the function of individual Rab proteins in cultured cells has been extensively used previously (for example [30, 31]). In some cases, these mutants have had profound effects on membrane trafficking such as with Rab5 , but in others such as Rab11a the effects were more subtle . Secondly, a transgenic approach to express the equivalent Rab5 dominant-negative mutant, Rab5N133I in immune cells reportedly resulted in the desired phenotype . Thirdly, this approach was the best model for studying the partial dysfunction of Rab27a observed previously since dominant-negative proteins would unlikely inactivate 100% of wild type activity [15, 16]. In addition, the dominant-negative approach could also result in inactivation of Rab27b activity, if not other related proteins. In contrast, a gene knock-out approach would result in complete absence of Rab27a while retaining full activity of the related protein Rab27b. Our recent finding that Rab27b shows functional redundancy with Rab27a further suggests that Rab27a-related Rabs may be involved in CHM . Fourthly, we observed that these same Rab27a mutations were effective in cell culture models .
While this work was in progress, there was a considerable advance in understanding the function of Rab27a and its role in disease. Loss-of-function mutations in Rab27a were observed in patients with Griscelli syndrome (online Mendelian Inheritance in Man (OMIM) # 214450, http://www.ncbi.nlm.nih.gov/Omim/) and in the corresponding mouse model, ashen [26, 27]. GS is a rare, lethal autosomal recessive disorder, characterised by pigment dilution of the hair (and to a lesser extent of skin) together with early-onset immune deficiency with episodes of hemophagocytic (uncontrolled T lymphocyte and macrophage activation) syndrome [35, 36]. The partial albinism in GS patients is due to clumping of melanin pigment in the hair shaft and accumulation of melanosomes around the nucleus within melanocytes [34–38]. The immune deficiency is due to loss of CTL killing activity [27, 39, 40]. If the Rab27a mutant proteins Rab27aT23N and Rab27aN133I acted as strong dominant-negative mutants, as they do in cell culture [34, 38], the transgenic lines generated should exhibit a phenotype resembling ashen mice, possibly with additional phenotypes due to inactivation of Rab27b as well. Unfortunately, no phenotype could be detected in the transgenic lines. Coat colour was not affected (Fig. 5) and CTL activity could not be studied given that the β-actin promoter used in this study does not express in these cells . We also performed detailed analysis of the retina in the transgenic lines, including fundoscopy, angiography, histology and ERG, and could not detect any significant pathological changes.
The lack of phenoytpe in the transgenic mice expressing dominant-negative mutant Rab27a and Rab27b could have been due to non-functional mutant proteins. However, this explanation seems unlikely as the same type of mutants are functional in cultured melanocytes [34, 38]. It has been proposed that one mechanism for dominant-negative action by these mutations in Ras-like proteins is by competition for GDP/GTP exchange factors, required for activation of the endogenous proteins . Thus, the absence of phenotype in these mice is probably due to inadequate levels of protein expression to elicit a dominant-negative effect. Indeed, out of more than twenty transgenic lines generated so far expressing dominant-negative mutant forms of Rab27a or Rab27b, only one (A27aT25/2) had transgenic protein levels close to the endogenous counterpart (Fig. 4B).
The low levels of dominant-negative transgenic protein are not due to poor expression of the transgenes as very high levels of mRNA was observed in many of the lines. In addition the wild-type and constitutively active proteins were present at high levels from similar constructs. The low levels of protein are probably due to more rapid turnover of the dominant-negative mutant proteins and we observed that the Rab27aT23N and Rab27aNI33I proteins have half-lives 4–8 times shorter than the wild type protein in tissue culture (Fig. 5). This fast turnover of the dominant-negative protein compared to the constitutively-active and wild-type proteins probably results from a combination of factors. The transgenic protein could be poorly prenylated, a possibility supported by the finding that only a small percentage of transgenic protein was membrane-associated (data not shown). Also, the mutation could reduce the stability of the tertiary structure of the mutant protein resulting in an increased rate of unfolding. Although the rate of turnover of Rab27b proteins was not determined, the low levels of dominant negative mutant protein in transgenic lines expressing high levels of mRNA suggests that Rab27bT23N and Rab27bNI33I also have high rates of turn-over. These observations in vivo contrast with the effects observed in cultured cells, where transient transfection leads to acute expression of very high levels of mutant proteins.
Our initial hypothesis that Rab27a plays a role in the retinal degenerative process of CHM remains untested given the lack of success of this approach. As we proposed originally, it is unlikely that Rab27a is the only dysfunctional Rab protein in this disorder and our inability to test retinal tissue from patients has hampered the identification of other Rabs that might be involved . Nevertheless, the availability of Rab27a knock-out mice (ashen) may allow us to determine whether Rab27a plays an important role in retinal physiology and further elucidate the pathogenesis of CHM.
The results presented here suggest that the use of dominant-negative proteins in in vivo models should be carefully considered. We report the generation of several transgenic lines expressing dominant-negative mutants of Rab27a and Rab27b where no phenotype could be elicited. The lack of a phenotype is likely due to the very modest levels of protein expression, probably caused by rapid degradation, despite very high levels of mRNA resulting from transcription driven by a strong promoter in the transgene.
All animals described here were maintained on 12-h light/12-h dark conditions at Imperial College, CBS Unit, London, UK, under Home Office Project Licence 70/5071.
Construction of mutant Rab27a transgenic vectors
Two general transgenic constructs were constructed, the first contained the pigment cell-specific tyrosinase promoter, Rab27a cDNA and the human growth hormone poly A signal and the second contained the strong ubiquitous chicken β-actin promoter and CMV-IE enhancer (PCAG), followed by an amino-terminal myc-epitope-tagged version of Rab27a or Rab27b, followed by rabbit β-globin poly(A) signal (Fig. 1). The construction of the first transgenic vector was initiated by subcloning a Bam HI – Not I fragment containing the human growth factor (hGH) termination sequence [41, 42] (a generous gift from David Russell, University of Texas Southwestern Medical Center, Dallas, USA) into pBS containing a 2.2 kb insert corresponding to mouse tyrosinase promoter (a generous gift from Paul Overbeek, Baylor College of Medicine, Houston, USA) [19, 20]. For the second transgenic construct, the promoter and poly A regions were from pCAGGS [22, 23]. The point mutations, Rab27aT23N, Rab27aN133I, Rab27aQ78L, Rab27bT23N and Rab27bN133I were generated by PCR mutagenesis using the rat Rab27a or the human Rab27b cDNA as a template. The myc-epitope was inserted in frame into the Rab27 cDNA by subcloning the Rab27 cDNA into pCMV7-MYC  or pBS-MYC. pBS-MYC was generated by cloning the Cla I/Sal I-fragment from pCMV7-MYC containing the myc-epitope into pBluescript SK. The fragments containing the myc-Rab27 cDNA were excised from pCMV7-MYC or pBS-MYC with Xba I and Bam HI or Xho I and Bam HI, respectively. After gel purification using QIAquick Gel Extraction Kit (Qiagen), the recessed 3' termini were filled with 0.5 U of Klenow fragment of Escherichia coli DNA polymerase I according to the manufacturer's instructions. The product was then subcloned into the blunted Xho I site of pCAGGS after Klenow fragment treatment (Fig. 1A).
Generation of the transgenic mice
A 3.4 kb Xho I – Not I fragment containing the Ptyr/Rab27a/hGH or a 3.4 kb Spe I – Bam HI fragment containing PCAG/myc-Rab27/β-globin were gel purified using QIAquick Gel Extraction Kit (Qiagen) as described by the manufacturer and eluted with 10 mM Tris-HCl (pH 8.5). DNA was then dissolved in sterile 0.1 mM EDTA, 10 mM Tris-HCl (pH 8.0) at about 2 ng/μl and microinjected into the pronuclei of one-cell stage embryos from a C57BL/6JxCBA background collected from super-ovulated female mice . Microinjected eggs were transferred at two-cell stage into the oviducts of pseudopregnant recipient females. Newborn mice were routinely screened for incorporation of the transgene by PCR and/or Southern blotting.
Screening and generation of transgenic lines
Mice were genotyped by PCR amplification and by Southern blot analysis using genomic DNA obtained from mouse-tail biopsy samples. The transgene was detected with sense oligonucleotide JR119 (5'-ATGGAACAAAAACTCATCTCAGAAGAGG) corresponding to the myc-tag sequence of the transgene or JR13 (5'-ACATGTGATAGTCACTCCAGGGGTTGCT) corresponding to tyrosinase promoter, and antisense oligonucleotide JR14 (5'-AGTTGAACTTCCCATCAGTGTACTGGTA) corresponding to the Rab27a coding sequence. Tail biopsies (~ 0.5 cm) were digested with 40 μg/ml of proteinase K in 500 μl of extraction buffer containing 100 mM Tris-HCl (pH 8.0), 200 mM NaCl, 5 mM EDTA and 0.2% SDS overnight at 55°C followed by phenol/chloroform extraction. After precipitation with isopropanol, DNA was washed with 80% ethanol and resuspended in 50 μl of 10 mM Tris-HCl (pH 8.5). For Southern blotting, approximately 20 μg of tail genomic DNA was digested with Kpn I overnight at 37°C. DNA was separated on a 0.8% TAE agarose gel overnight at 120 mA and blotted onto Hybond-N+ (Amersham) membrane. Membranes were prehybridised and hybridised with Rab27a or Rab27b probe, corresponding to the entire Rab27 coding sequence, radiolabelled with 20 μCi of [α-32P]dCTP by Random Oligopriming (Amersham). In order to generate stable transgenic lines, animals shown to be transgenic were subsequently mated to wild-type C57BL/6J mice; transgene-positive offspring (black colour mice) from these crosses were likewise bred. Transgenic lines were maintained as heterozygotes. Transgenic mice (Rab27T23N, Rab27aQ78L and Rab27aWT) were crossed with ashen (ash/ash) on a C57BL/6J background obtained by repeated backcrossing of ash/+ mice (over five generations) with wild-type C57BL/6J mice . The resulting heterozygous ashen (+/ash) mice carrying the transgene were intercrossed to generate mice that carried the transgene on the ash/ash background. These mice were visually evaluated for the rescue of the coat colour. The ashen mutation was screened for by PCR using the primers ASH1 (5'-ACCTGACAAATGAGCAAAGTTTCCTCAATG) and ASH2 (5'-GGAGCAGGGCAGGGCTGGGGAAACCACTCG) followed by restriction enzyme analysis with Taq I and Rsa I according to the procedure described previously .
RT-PCR was carried out on total RNA obtained from eyes and isolated using RNeasy (Qiagen). After total RNA isolation, samples were treated with 1 U RNase-free DNase for 45 min at 37°C to remove any transgenic DNA contamination. The DNase was inactivated by heating for 15 min at 70°C. cDNA synthesis and DNA amplification were carried out using 5 μg of total RNA. Labelling of the PCR amplification product was carried out by addition of 0.025 μCi of [α-33P]dATP. DNA was amplified using only 23 cycles (exponential phase). PCR amplification of Rab27a cDNA was performed with oligonucleotides JR62 (5'-GCATTGATTTCAGGGAAAAGAGAG) and JR63 (5'-TTCTCCACACACCGCTCCATCCGC). To ensure that both transgenic and endogenous mRNA were amplified equally, these oligonucleotides were designed to be homologous to both endogenous (mouse) and transgenic (rat) Rab27 cDNA. After amplification, the PCR products were digested with Eco RI or Sma I to distinguish the mouse endogenous from rat transgenic cDNA. In most cases, the transgene expression was much higher than the endogenous expression making quantification impossible. Therefore, the transgene expression level was determined in each line by quantitating the Rab27 (endogenous plus mutant mRNA) relative to Hprt cDNA using all four primers, 0.025 μCi of [α-33P] dATP and 23 cycles in PCR reaction. The oligonucleotides used for Hprt amplification were Hprt1 (5'-CCTGCTGGATTACATTAAAGCACTG) and Hprt2 (5'-GTCAAGGGCATATCCAACAACAAAC). Possible contamination of mRNA with transgenic DNA was excluded by control reactions without reverse transcriptase. The radiolabelled PCR products were separated on 2.5% agarose gels, stained with ethidium bromide, transferred onto 3 MM Whatman paper and quantified using a Cyclotron Storage Phosphor Screen (Packard).
Immunoblotting was performed according to procedures described elsewhere  with some modifications using total, cytosol or membrane protein fractions obtained from several C57BL/6J wild-type or transgenic tissues. All the transgenic and non-transgenic mice used for immunoblotting studies were thoroughly perfused with phosphate-buffered saline (PBS) (pH 7.4) prior to dissection of the tissue samples that included eye (the lens was always removed from the ocular globe), spleen, liver, lung, kidney, skin, stomach, large intestine, small intestine, brain, testis and heart. Tissue samples were washed with PBS, and thoroughly homogenised using a Polytron homogeniser in 3–5 volumes of homogenisation buffer containing 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM phenyl-methylsulfonyl fluoride, 10 μg/ml leupeptin, 10 μg/ml aprotinin, 10 μg/ml pepstatin and 1 mM DTT. Homogenisation was followed by sonication for 5–10 seconds. To sediment unbroken cells and cell nuclei, the homogenates were centrifuged at 5,000 g for 30 min at 4°C. The postnuclear supernatant was then centrifuged at 100,000 g for 1 h at 4°C, resulting in the separation of cytosolic (supernatant) and membrane (pellet) protein fractions. The membrane fractions were resuspended into the same volume of homogenisation buffer containing 1% Triton X-100. The protein concentration was determined only in the total fraction by BCA method (Pierce) according to the manufacturer's instructions. For immunoblot analysis, samples containing 50 μg of protein were separated into soluble or membrane fractions and identical volumes were subjected to SDS gel electrophoresis. After being resuspended into loading buffer, protein samples were loaded onto 12.5% SDS-polyacrylamide electrophoresis gels, run at 35 mA and transferred to Immobilon-P polyvinylidene difluoride (Millipore) membrane in a minitrans-blot cell (Amersham) at room temperature in a buffer containing 25 mM Tris-HCl (pH 8.3), 192 mM glycine, and 10% methanol for 1 h and 30 min at 500 mA. To block non-specific binding sites, dried membranes were incubated in blocking solution containing 0.1% polyoxyethylenesorbitan monolaureate (Tween-20), 4% non-fat milk in PBS for 1 h at room temperature. Membranes were subsequently incubated with PBS/0.1% Tween-20 solution supplemented with the following preparation of antibodies: monoclonal 4B12 antibody (0.3 μg/ml) anti-rat Rab27a , monoclonal antibody anti-myc-tag (0.3 μg/ml) purchased from Oncogene (Cambridge, MA, USA) or polyclonal anti-calnexin antibody (1:5,000) purchased from StressGene (Victoria, BC, Canada). The mixture was incubated with the primary antibody with gentle agitation for 1 h at room temperature, rinsed and washed with PBS containing 0.1% Tween-20 for 15 min, 3 times. Membranes were then incubated with horseradish peroxidase-coupled secondary antibody (Dako). The blots were washed as described above and bands were visualised by chemiluminescence using SuperSignal West Pico Chemiluminescence Substrate (Pierce) according to the instructions supplied by the manufacturer. All blots were calibrated with prestained molecular weight markers (Bio-Rad Laboratories).
Cell culture, transfections and labelling
COS-7 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Transfections were performed using lipofectamine (Qiagen). Total amounts of transfected DNA were 1 μg/6-cm dish. For pulse-chase experiments, cells were washed with methionine/cysteine-free medium and subsequently incubated with 140 μCi of [35S]methionine/cysteine per 6-cm dish for 2 h. After this, cells were washed three times in PBS, and further maintained in normal Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. At indicated time points (0, 30 min, 3, 5, 8 and 22 h), cells were lysed by incubation in buffer containing 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM phenyl-methylsulfonyl fluoride, 10 μg/ml leupeptin, 10 μg/ml aprotinin, 10 μg/ml pepstatin, 1 mM DTT and 1% NP-40 for 20 min on ice followed by 5–10 s of sonication. After centrifugation at 14,000 rpm, equal amounts of radioactive lysates were incubated under agitation with 5 μg of 4B12 antibody for 1 h at RT, after lowering the concentration of NP-40 to 0.5% by dilution. Subsequently, 30 μl of protein G-Sepharose beads were added to the radioactive material and incubated overnight at 4°C, while rocking. Then beads were washed four times in PBS, denatured in SDS-containing sample buffer and loaded on a 12% SDS-polyacrylamide gel, transferred onto 3 MM Whatman paper and quantified using a Storm system (Amersham).
Fundus photographs of mice were taken using a small animal fundus camera (Kowa Genesis) according to John and co-workers . In order to have a better magnification and focal depth of the fundus, the camera was used in conjunction with an external 90 diopter condensing lens (Volk). This condensing lens was mounted between the camera and the mouse eye. The lens was placed about 5 cm below the camera. All the photographs were taken without anesthesia and with mice's vibrissae trimmed to avoid ocular clouding and obscuring photograph problems. Pupils were dilated with a drop of 1% Mydriacyl (Alcon Laboratories) 20–30 min before taking the photographs. For conventional photography of the fundus, Kodak 200 ASA slide film was used. The photographic flash on the power pack was set up for its highest level (position 6–7) for C57BL/6J mice as the highest intensity of light produced better results on pigmented mice. The mice were held beneath the external lens and the focusing was achieved by moving the mouse. To reduce the light reflection, a major problem of this technique, the position of the eye was very important. Accordingly, the procedure involved focusing through an off central position of the ocular globe.
The retinal angiography was performed using the general fundus photography procedure. The camera was set up for angiography through addition of an emission barrier filter specific for fluorescein emission and the power pack was set up for fluorescein angiography by changing the excitation light to fluorescein excitation. For this type of photography Kodak black and white Tmax 400 ASA professional film was used. The photographic flash on the power pack was set up for its highest level (position 6–7). Mice were intraperitoneally injected with 20% injectable sodium fluorescein (Faure) at a dose of 10 μl per 5–6 g body weight . Photographs were taken at several intervals, starting at 30 s post-injection.
ERGs were obtained according to previously reported procedures . Briefly, before anesthesia with ketamine (66.7 mg/kg), xylazine (11.7 mg/kg), and atropine (1 mg/kg), the pupils of dark-adapted mice were dilated. The ERG equipment consisted of a Ganzfeld bowl, a DC amplifier, and a PC-based control and recording unit (Multiliner Vision, Jaeger/Toennies, Hoechberg, Germany). Band-pass filter cut-off frequencies were 0.1 and 3000 Hz. Single flash recordings were obtained both under dark-adapted (scotopic) and light-adapted (photopic) conditions. Light adaptation before the photopic session was performed with a background illumination of 30 cd/m2 for 10 minutes. Single flash stimulus intensities were increased from 10-4 cd*s/m2 to 25 cd*s/m2, divided into ten steps of 0.5 and 1 log cd*s/m2. Ten responses were averaged with an inter-stimulus interval (ISI) of either 5s or 17s (for 1, 3, 10, 25 cd*s/m2).
Eyes were fixed in 4% paraformaldehyde, 5% glutaraldehyde and 0.1 M cacodylate buffer for 1 h. Subsequently, the eyes were cut in half and the anterior segment was removed. Fixed eyes were washed three times for 10 min at room temperature with PBS. Specimen were embedded in paraffin, sectioned to 3–5 μm thickness and stained with hematoxylin and eosin according to standard procedures.
List of abbreviations
Rab Escort Protein
polymerase chain reaction
Hypoxanthine phosphoribosyl transferase.
We thank Amanda McGuigan and Duarte Barral for help with the animal experiments, Lorraine Lawrence for histological preparations and, Paul Overbeek and David Russell for providing cDNA clones. This work was supported by the Medical Research Council, the Foundation Fighting Blindness, the German research council (DFG Se837/1-2) and an anonymous donor.
- Zerial M, McBride H: Rab proteins as membrane organizers. Nat Rev Mol Cell Biol. 2001, 2: 107-17. 10.1038/35052055.View ArticlePubMedGoogle Scholar
- Pfeffer SR: Rab GTPases: specifying and deciphering organelle identity and function. Trends Cell Biol. 2001, 11: 487-91. 10.1016/S0962-8924(01)02147-X.View ArticlePubMedGoogle Scholar
- Nagata K, Satoh T, Itoh H, Kozasa T, Okano Y, Doi T, Kaziro Y, Nozawa Y: The ram: a novel low molecular weight GTP-binding protein cDNA from a rat megakaryocyte library. FEBS Lett. 1990, 275: 29-32. 10.1016/0014-5793(90)81431-M.View ArticlePubMedGoogle Scholar
- Nagata K, Itoh H, Katada T, Takenaka K, Ui M, Kaziro Y, Nozawa Y: Purification, identification, and characterization of two GTP-binding proteins with molecular weights of 25,000 and 21,000 in human platelet cytosol. One is the rap1/smg21/Krev-1 protein and the other is a novel GTP-binding protein. J Biol Chem. 1989, 264: 17000-5.PubMedGoogle Scholar
- Seabra MC: Membrane association and targeting of prenylated Ras-like GTPases. Cell Signal. 1998, 10: 167-72. 10.1016/S0898-6568(97)00120-4.View ArticlePubMedGoogle Scholar
- Pereira-Leal JB, Hume AN, Seabra MC: Prenylation of Rab GTPases: molecular mechanisms and involvement in genetic disease. FEBS Lett. 2001, 498: 197-200. 10.1016/S0014-5793(01)02483-8.View ArticlePubMedGoogle Scholar
- Seabra MC: Nucleotide dependence of Rab geranylgeranylation. Rab escort protein interacts preferentially with GDP-bound Rab. J Biol Chem. 1996, 271: 14398-404.View ArticlePubMedGoogle Scholar
- Seabra MC, Brown MS, Slaughter CA, Sudhof TC, Goldstein JL: Purification of component A of Rab geranylgeranyl transferase: possible identity with the choroideremia gene product. Cell. 1992, 70: 1049-57.View ArticlePubMedGoogle Scholar
- Anant JS, Desnoyers L, Machius M, Demeler B, Hansen JC, Westover KD, Deisenhofer J, Seabra MC: Mechanism of Rab geranylgeranylation: formation of the catalytic ternary complex. Biochemistry. 1998, 37: 12559-68. 10.1021/bi980881a.View ArticlePubMedGoogle Scholar
- Seabra MC, Goldstein JL, Sudhof TC, Brown MS: Rab geranylgeranyl transferase. A multisubunit enzyme that prenylates GTP-binding proteins terminating in Cys-X-Cys or Cys-Cys. J Biol Chem. 1992, 267: 14497-503.PubMedGoogle Scholar
- Thoma NH, Iakovenko A, Goody RS, Alexandrov K: Phosphoisoprenoids modulate association of Rab geranylgeranyltransferase with REP-1. J Biol Chem. 2001, 276: 48637-43. 10.1074/jbc.M108241200.View ArticlePubMedGoogle Scholar
- Alexandrov K, Horiuchi H, Steele-Mortimer O, Seabra MC, Zerial M: Rab escort protein-1 is a multifunctional protein that accompanies newly prenylated rab proteins to their target membranes. Embo J. 1994, 13: 5262-73.PubMed CentralPubMedGoogle Scholar
- Wilson AL, Erdman RA, Maltese WA: Association of Rab1B with GDP-dissociation inhibitor (GDI) is required for recycling but not initial membrane targeting of the Rab protein. J Biol Chem. 1996, 271: 10932-40. 10.1074/jbc.271.18.10932.View ArticlePubMedGoogle Scholar
- Cremers FP, Armstrong SA, Seabra MC, Brown MS, Goldstein JL: REP-2, a Rab escort protein encoded by the choroideremia-like gene. J Biol Chem. 1994, 269: 2111-7.PubMedGoogle Scholar
- Seabra MC: New insights into the pathogenesis of choroideremia: a tale of two REPs. Ophthalmic Genet. 1996, 17: 43-6.View ArticlePubMedGoogle Scholar
- Seabra MC, Ho YK, Anant JS: Deficient geranylgeranylation of Ram/Rab27 in choroideremia. J Biol Chem. 1995, 270: 24420-7. 10.1074/jbc.270.41.24420.View ArticlePubMedGoogle Scholar
- Seabra MC, Brown MS, Goldstein JL: Retinal degeneration in choroideremia: deficiency of rab geranylgeranyl transferase. Science. 1993, 259: 377-81.View ArticlePubMedGoogle Scholar
- Feig LA: Tools of the trade: use of dominant-inhibitory mutants of Ras-family GTPases. Nat Cell Biol. 1999, 1: E25-7. 10.1038/10018.View ArticlePubMedGoogle Scholar
- Kluppel M, Beermann F, Ruppert S, Schmid E, Hummler E, Schutz G: The mouse tyrosinase promoter is sufficient for expression in melanocytes and in the pigmented epithelium of the retina. Proc Natl Acad Sci U S A. 1991, 88: 3777-81.PubMed CentralView ArticlePubMedGoogle Scholar
- Beermann F, Schmid E, Schutz G: Expression of the mouse tyrosinase gene during embryonic development: recapitulation of the temporal regulation in transgenic mice. Proc Natl Acad Sci U S A. 1992, 89: 2809-13.PubMed CentralView ArticlePubMedGoogle Scholar
- Hogan B, Bedington R, Costantini F, Lacy E: Manipulating the Mouse Embryo, A Laboratory Manual:. Cold Spring Harbor Laboratory Press;. 1994Google Scholar
- Fregien N, Davidson N: Activating elements in the promoter region of the chicken beta-actin gene. Gene. 1986, 48: 1-11. 10.1016/0378-1119(86)90346-X.View ArticlePubMedGoogle Scholar
- Niwa H, Yamamura K, Miyazaki J: Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene. 1991, 108: 193-9. 10.1016/0378-1119(91)90434-D.View ArticlePubMedGoogle Scholar
- Hammer RE, Krumlauf R, Camper SA, Brinster RL, Tilghman SM: Diversity of alpha-fetoprotein gene expression in mice is generated by a combination of separate enhancer elements. Science. 1987, 235: 53-8.View ArticlePubMedGoogle Scholar
- Overbeek PA, Lai SP, Van Quill KR, Westphal H: Tissue-specific expression in transgenic mice of a fused gene containing RSV terminal sequences. Science. 1986, 231: 1574-7.View ArticlePubMedGoogle Scholar
- Wilson SM, Yip R, Swing DA, O'Sullivan TN, Zhang Y, Novak EK, Swank RT, Russell LB, Copeland NG, Jenkins NA: A mutation in Rab27a causes the vesicle transport defects observed in ashen mice. Proc Natl Acad Sci U S A. 2000, 97: 7933-8. 10.1073/pnas.140212797.PubMed CentralView ArticlePubMedGoogle Scholar
- Menasche G, Pastural E, Feldmann J, Certain S, Ersoy F, Dupuis S, Wulffraat N, Bianchi D, Fischer A, Le Deist F: Mutations in RAB27A cause Griscelli syndrome associated with haemophagocytic syndrome. Nat Genet. 2000, 25: 173-6. 10.1038/76024.View ArticlePubMedGoogle Scholar
- Barral DC, Ramalho JS, Anders R, Hume AN, Knapton HJ, Tolmachova T, Collinson LM, Goulding D, Authi KS, Seabra MC: Functional redundancy of Rab27 proteins and the pathogenesis of Griscelli syndrome. J Clin Invest. 2002, 110: 247-251. 10.1172/JCI200215058.PubMed CentralView ArticlePubMedGoogle Scholar
- Hawes NL, Smith RS, Chang B, Davisson M, Heckenlively JR, John SW: Mouse fundus photography and angiography: a catalogue of normal and mutant phenotypes. Mol Vis. 1999, 5: 22-PubMedGoogle Scholar
- van der Sluijs P, Hull M, Webster P, Male P, Goud B, Mellman I: The small GTP-binding protein rab4 controls an early sorting event on the endocytic pathway. Cell. 1992, 70: 729-40.View ArticlePubMedGoogle Scholar
- Bucci C, Parton RG, Mather IH, Stunnenberg H, Simons K, Hoflack B, Zerial M: The small GTPase rab5 functions as a regulatory factor in the early endocytic pathway. Cell. 1992, 70: 715-28.View ArticlePubMedGoogle Scholar
- Ullrich O, Reinsch S, Urbe S, Zerial M, Parton RG: Rab11 regulates recycling through the pericentriolar recycling endosome. J Cell Biol. 1996, 135: 913-24.View ArticlePubMedGoogle Scholar
- Andre P, Boretto J, Hueber AO, Regnier-Vigouroux A, Gorvel JP, Ferrier P, Chavrier P: A dominant-negative mutant of the Rab5 GTPase enhances T cell signaling by interfering with TCR down-modulation in transgenic mice. J Immunol. 1997, 159: 5253-63.PubMedGoogle Scholar
- Hume AN, Collinson LM, Rapak A, Gomes AQ, Hopkins CR, Seabra MC: Rab27a regulates the peripheral distribution of melanosomes in melanocytes. J Cell Biol. 2001, 152: 795-808. 10.1083/jcb.152.4.795.PubMed CentralView ArticlePubMedGoogle Scholar
- Griscelli C, Durandy A, Guy-Grand D, Daguillard F, Herzog C, Prunieras M: A syndrome associating partial albinism and immunodeficiency. Am J Med. 1978, 65: 691-702.View ArticlePubMedGoogle Scholar
- Klein C, Philippe N, Le Deist F, Fraitag S, Prost C, Durandy A, Fischer A, Griscelli C: Partial albinism with immunodeficiency (Griscelli syndrome). J Pediatr. 1994, 125: 886-95.View ArticlePubMedGoogle Scholar
- Bahadoran P, Aberdam E, Mantoux F, Busca R, Bille K, Yalman N, de Saint-Basile G, Casaroli-Marano R, Ortonne JP, Ballotti R: Rab27a: A key to melanosome transport in human melanocytes. J Cell Biol. 2001, 152: 843-50. 10.1083/jcb.152.4.843.PubMed CentralView ArticlePubMedGoogle Scholar
- Wu X, Rao K, Bowers MB, Copeland NG, Jenkins NA, Hammer JA: Rab27a enables myosin Va-dependent melanosome capture by recruiting the myosin to the organelle. J Cell Sci. 2001, 114: 1091-100.PubMedGoogle Scholar
- Stinchcombe JC, Barral DC, Mules EH, Booth S, Hume AN, Machesky LM, Seabra MC, Griffiths GM: Rab27a is required for regulated secretion in cytotoxic T lymphocytes. J Cell Biol. 2001, 152: 825-34. 10.1083/jcb.152.4.825.PubMed CentralView ArticlePubMedGoogle Scholar
- Haddad EK, Wu X, Hammer JA, Henkart PA: Defective granule exocytosis in Rab27a-deficient lymphocytes from Ashen mice. J Cell Biol. 2001, 152: 835-42. 10.1083/jcb.152.4.835.PubMed CentralView ArticlePubMedGoogle Scholar
- Hofmann SL, Russell DW, Brown MS, Goldstein JL, Hammer RE: Overexpression of low density lipoprotein (LDL) receptor eliminates LDL from plasma in transgenic mice. Science. 1988, 239: 1277-81.View ArticlePubMedGoogle Scholar
- Palmiter RD, Norstedt G, Gelinas RE, Hammer RE, Brinster RL: Metallothionein-human GH fusion genes stimulate growth of mice. Science. 1983, 222: 809-14.View ArticlePubMedGoogle Scholar
- Strom M, Hume AN, Tarafder AK, Barkagianni E, Seabra MC: A family of Rab27-binding proteins: Melanophilin links Rab27a and myosin Va function in melanosome transport. J Biol Chem. 2002, 277: 25423-30. 10.1074/jbc.M202574200.View ArticlePubMedGoogle Scholar
- Okamoto N, Tobe T, Hackett SF, Ozaki H, Vinores MA, LaRochelle W, Zack DJ, Campochiaro PA: Transgenic mice with increased expression of vascular endothelial growth factor in the retina: a new model of intraretinal and subretinal neovascularization. Am J Pathol. 1997, 151: 281-91.PubMed CentralPubMedGoogle Scholar
- Seeliger MW, Grimm C, Stahlberg F, Friedburg C, Jaissle G, Zrenner E, Guo H, Reme CE, Humphries P, Hofmann F: New views on RPE65 deficiency: the rod system is the source of vision in a mouse model of Leber congenital amaurosis. Nat Genet. 2001, 29: 70-4. 10.1038/ng712.View ArticlePubMedGoogle Scholar
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