UASt regulatory sequences drive the expression of a Rab7 mutation (V162M) that has been associated with Charcot-Marie-Tooth 2B in the human ortholog (Hsap\RAB7A). The construct is tagged at the N-terminal with Venus.
G23994549A
V162M | Rab7-PA; V162M | Rab7-PB; V162M | Rab7-PC
V162M
Carried in construct; Inserted at PBac{y+-attP-3B}VK00002 landing site on 2L. Site of nucleotide substitution in mutant inferred by FlyBase based on reported amino acid change.
The Charcot-Marie-Tooth 2B disease is a genetically dominant disorder as all patients contain one wild-type copy and one mutant copy of the human Hsap\RAB7A gene. It has been proposed that the mutant forms of Hsap\RAB7A that are seen in Charcot-Marie-Tooth 2B disease patients are gain of function mutations which cause the adult onset neurodegeneration characteristic of the disease. However, studies in a Drosophila model suggest that the neurodegeneration seen in Charcot-Marie-Tooth 2B disease may be caused by loss of normal Hsap\RAB7A function in the patients rather than by the presence of the mutant protein: mutant flies that completely lack function of the Drosophila Rab7 gene in the eye show adult onset neurodegeneration, while flies overexpressing mutant forms of Rab7 which have been identified in Charcot-Marie-Tooth 2B disease patients (the flies express either a mutant form of Hsap\RAB7A or express the equivalent mutations in the Drosophila Rab7 ortholog) do not show neurodegeneration.
viable (with Rab7Gal4-KO), with Scer\GAL4Rab7-Gal4-KO
Flies expressing two copies of Rab7V162M.Scer\UAS.T:Avic\GFP-YFP.Venus under the control of Scer\GAL4Rab7.T:Disc\RFP are viable.
Flies expressing one copy of Rab7V162M.Scer\UAS.T:Avic\GFP-YFP.Venus under the control of one copy of Scer\GAL4Rab7.T:Disc\RFP (in a Rab7GAL4.T:Disc\RFP/+ mutant background) are viable and do not display any obvious behavioural defects or altered lifespan. Electroretinogram recordings from the larval neuromuscular junction are indistinguishable from wild type. The frequency and amplitudes of spontaneous single vesicle release events are normal, as are evoked neurotransmission events. As in wild type, no photoreceptor defects, either morphological or functional, are seen in adult flies that have been exposed to 10 days of constant light stimulation.
Expression of Rab7V162M.Scer\UAS.T:Avic\GFP-YFP.Venus under the control of Scer\GAL4Rab7.T:Disc\RFP rescues the lethality seen in homozygous Rab7GAL4.T:Disc\RFP mutant flies.
Expression of Rab7V162M.Scer\UAS.T:Avic\GFP-YFP.Venus under the control of Scer\GAL4Rab7.T:Disc\RFP rescues the progressive synaptic and neuronal degeneration seen in homozygous Rab7GAL4.T:Disc\RFP mutant eye clones after 10 days of constant light stimulation.
Expression of Rab7V162M.Scer\UAS.T:Avic\GFP-YFP.Venus under the control of Scer\GAL4Rab7.T:Disc\RFP rescues the dominant defects in synaptic function and rhabdomere structure seen in Rab7GAL4.T:Disc\RFP/+ mutant adults after 5 days of constant light stimulation. A similar level of rescue is seen whether the transgene expresses 0.5x, 1x or >10x the amount of endogenous Rab7 protein.
