Rapid degradation of dominant-negative Rab27 proteins in vivo precludes their use in transgenic mouse models

Abstract

Background: 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.

Results: 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 beta-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.

Conclusions: 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.

Figure 1

Organisation of transgenic constructs. Panel A depicts the PTyr/Rab27a/hGH constructs and Panel B depicts the pCAG/myc-Rab27/β-globin constructs generated as described under "Methods". Point mutations are indicated by an asterisk.

Figure 2

Screening of transgenic mice. Southern analysis was performed using mouse genomic DNA extracted from the tail of potential Rab27a^T23N and Rab27a^N133I transgenics. Each well contains genomic DNA extracted from an individual mouse tail biopsy and electrophoresed on an agarose gel as described under "Methods". The band around 3.4 kb suggests the presence of an array of transgenes.

Figure 3

Detection of Rab27a mRNA. (Panel A) Schematic depiction of transgenic (rat) versus endogenous (mouse) Rab27a coding sequence. EcoRI or SmaI restriction sites as well as resulting fragment sizes are indicated. (Panel B) Comparison of transgenic versus endogenous Rab27a expression in the eye by RT-PCR and restriction digestion of PCR products. Rab27a mRNA from wild-type and independent mutant transgenic lines (Rab27a^T23N and Rab27a^N133I) amplified by RT-PCR was digested with EcoRI or SmaI and electrophoresed as described under "Methods". Rat Rab27a cDNA was used as a control. (Panel C) Determination of Rab27a expression (transgenic and endogenous) in eyes by RT-PCR (+). The oligonucleotides and conditions were as described under "Methods". The PCR products were analysed on an agarose gel stained with ethidium bromide. Reactions performed without reverse transcriptase are indicated (-). The arrows on the left-hand side indicate the positions of DNA marker sizes. Hprt expression was used as an internal control.

Figure 4

Expression of transgenic Rab27a proteins in mouse tissues. Protein extracts obtained from eyes, spleen, liver, skin, lung, kidney, stomach, large intestine, small intestine, brain, testis and heart (50 μg each) were subjected to SDS-PAGE and probed with monoclonal anti-myc-tag antibody (9E10) and/or a specific anti-Rab27a monoclonal antibody (4B12). Calnexin was used as a loading control and detected using a specific polyclonal antibody. A protein extract of HeLa cells transfected with myc-Rab27a^T23N was used as a positive control. (Panel A) Tissues from the A27aT25/2 line carrying Rab27a^T23N. (Panel B) Tissues from the A27aQ24 line carrying Rab27a^Q78L.

Figure 5

Half-lives of wild-type, Rab27a^T23N, Rab27a^Q78L and Rab27a^N133I mutant proteins. COS-7 cells transfected with either pCAGGS myc-Rab27a^WT (A), pCAGGS myc-Rab27a^T23N (B), pCAGGS myc-Rab27a^Q78L (C), or co-transfected with pCAGGS myc-Rab27a^WT and pCAGGS myc-Rab27a^T23N (D) were labelled with [³⁵S]methionine/cysteine for 2 h and subsequently chased in Dulbecco's modified Eagle's medium for the indicated times. Rab27a protein was immunoprecipitated from cell extracts, collected with protein G-Sepharose beads, separated on a 12% SDS-polyacrylamide gel and autoradiographed as described under "Methods". (Panel E) Quantification of radiolabelled samples obtained above.

Figure 6

Photographs of representative transgenic and mutant mice. +/+, wild type C57BL/6J mouse; ash/ash, homozygous Rab27a^ashen mutant mouse; -/tg, heterozygous transgenic of the indicated line.

Figure 7

Photographs of the fundus of the Rab27a^T23N transgenic mouse. Wild-type (C57BL/6J control littermate) (A) and Rab27a^T23N transgenic mouse (A27aT25/2 line) (B) were photographed at 6 months of age as described under "Methods". The venules (v) are twice the diameter of the arterioles (a). The optical nerve head is indicated by an arrow. Note that the two white dots are result of light reflection on the lens between the camera and the mouse's eye.

Figure 8

Fluorescein fundus angiograms of Rab27a^T23N transgenic mouse retina. Angiograms of six monthsold wild-type (C57BL/6J littermate control) (A and B) or Rab27a^T23N transgenic mouse (A27aT25/2 line) (C and D) taken one minute after dye injection were obtained as described under "Methods". The venules (v) are twice the diameter of the arterioles (a). The arrow denotes the optical nerve.

Figure 9

Electrophysiological characteristics of the A27aT25/2 transgenic mouse line. (Panel A) Dark-adapted (scotopic) intensity series of a six month-old wild-type control (left column) and a A27aT25/2 transgenic mouse (right column). Calibration marks: Vertical 100 μV/div.; horizontal 40 ms/div. Stimulus intensities increased from top to bottom from 10^-4 to 25 cd*s/m². (Panel B) Amplitude vs. intensity plot for the A27aT25/2 transgenic line in comparison to wild type mice. The crosses indicate the median, the boxes the 25%- to 75%-quantiles, and the whiskers the 5%- to 95%-quantiles. The upper (95% quantile) and lower (5% quantile) red line indicate the normal range based on wild type data.

Figure 10

Histochemical staining of Rab27a^T23N transgenic mouse retina. Transverse paraffin sections (3 μm) from 10 month-old C57BL/6J (A) or Rab27a^T23N transgenic mouse (line A27aT25/2) (B) are shown. Magnification is approximately ×250. The abbreviations used are: sclera (SC), choroid (CH), retinal pigment epithelium (RPE), photoreceptor segments of rods and cones (ROS), nuclei of rods and cones (ONL), outer synaptic layer (OSL), neuron nuclear layer (INL), inner synaptic layer (ISL), ganglion cell layer (GCL), slender glial (Muller) cell processes (M), ganglion cells (G) and inner limiting membrane (IM).