Genetic Manipulation of Synapse Formation and Synapse Structure
Authors:
Stephan Maxeiner, Alison TanMentor:
Thomas Südhof, Professor of Molecular & Cellular Physiology; Professor (By courtesy) of Neurology & Neurological Sciences, Stanford University -- School of MedicineThe objective of this project is to study novel yet uncharacterized mutant mouse lines to provide insight into the function of two very different synaptic proteins. In the first case, we analyzed a mouse line in which the pre-synaptic scaffolding protein Ribeye can be conditionally deleted by means of Cre/loxP-recombination. Ribeye is a special splice form of CtBP2, which is exclusively found in retinal and inner ear ribbon synapses; both synapses are known to allow tonic release of neurotransmitters. Ribeye has an alternative exon 1 encoding the “A-domain” allowing for multimerization and a “B-domain” that it shares with CtBP2. Through techniques such as PCR genotyping, immunofluorescence analysis and immunoblotting, we are able to confirm and evaluate quantitative and qualitative differences in its expression in mice with different allelic combinations of the targeted gene locus. Among these, we established that the Knock-in of an A-domain fusion protein with GCamP3 is expressed properly and that the knockout is deficient of Ribeye. In the second part, we studied mice in which Latrophilin2 (Lphn2) can be conditionally deleted as well as a line in which Lphn2 is expressed as a fusion protein with the yellow fluorescent Venus. Lphn2 belongs to a gene family that has been shown to be a receptor for the black widow spider venom alpha-latrotoxin. Recently, Lphn3 has been linked to attention deficit hyperactivity disorder (ADHD). We determined by genotyping that parents, which are heterozygous for the constitutive knockout allele, do not give birth to knockout mice leading to the assumption that the Lphn2 knockout is lethal during embryonic development. The fusion protein of Lphn2 and Venus allows us now to screen for its expression in different adult tissues in order to understand its expression pattern for further analysis.