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

Ramalho JS, Anders R, Jaissle GB, Seeliger MW, Huxley C, Seabra MC - BMC Cell Biol. (2002)

Bottom Line: 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.

Affiliation: Cell and Molecular Biology, Division of Biomedical Sciences, Faculty of Medicine, Imperial College, Sir Alexander Fleming Building, Exhibition Road, London, SW7 2AZ, UK. ramalho@imagem.ibili.uc.pt

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.

Fluorescein fundus angiograms of Rab27aT23N transgenic mouse retina. Angiograms of six monthsold wild-type (C57BL/6J littermate control) (A and B) or Rab27aT23N 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.
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Figure 8: Fluorescein fundus angiograms of Rab27aT23N transgenic mouse retina. Angiograms of six monthsold wild-type (C57BL/6J littermate control) (A and B) or Rab27aT23N 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.

Mentions: The retinal vasculature was examined by fluorescein angiography, i.e., fundus photography combined with intravenous fluorescein injection [29]. Neovascularisation that is often present in retinal degeneration is easily identified. It is also possible to detect defects in the RPE, since the melanin in the RPE can attenuate the fluorescence from the choroidal circulation. Figure 8 depicts representative examples of fluorescein angiography from Rab27aT23N transgenic mice (A27aT25/2 line) and wild-type litter mates. The retinal vessels are clearly visualised 30 s after injection. We observed no narrowing vessels, no abnormal leakage of fluorescein dye from the retinal capillaries and no hyperfluorescence of the retinal pigment epithelium.

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

Ramalho JS, Anders R, Jaissle GB, Seeliger MW, Huxley C, Seabra MC - BMC Cell Biol. (2002)

Fluorescein fundus angiograms of Rab27aT23N transgenic mouse retina. Angiograms of six monthsold wild-type (C57BL/6J littermate control) (A and B) or Rab27aT23N 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.
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Figure 8: Fluorescein fundus angiograms of Rab27aT23N transgenic mouse retina. Angiograms of six monthsold wild-type (C57BL/6J littermate control) (A and B) or Rab27aT23N 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.
Mentions: The retinal vasculature was examined by fluorescein angiography, i.e., fundus photography combined with intravenous fluorescein injection [29]. Neovascularisation that is often present in retinal degeneration is easily identified. It is also possible to detect defects in the RPE, since the melanin in the RPE can attenuate the fluorescence from the choroidal circulation. Figure 8 depicts representative examples of fluorescein angiography from Rab27aT23N transgenic mice (A27aT25/2 line) and wild-type litter mates. The retinal vessels are clearly visualised 30 s after injection. We observed no narrowing vessels, no abnormal leakage of fluorescein dye from the retinal capillaries and no hyperfluorescence of the retinal pigment epithelium.

Bottom Line: 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.

Affiliation: Cell and Molecular Biology, Division of Biomedical Sciences, Faculty of Medicine, Imperial College, Sir Alexander Fleming Building, Exhibition Road, London, SW7 2AZ, UK. ramalho@imagem.ibili.uc.pt

ABSTRACT

Background:

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.

View Similar Images In: Results  - Collection
View Article: Pubmed Central - HTML -  PubMed
Show All Figures - Show MeSH
getmorefigures.php?pmc=137576&rFormat=json&query=null&req=5