Single Cell Knock-out of the Fragile X Mental Retardation 1 Gene in the Mice Cortex
Physical and Biological Sciences
STEM Diversity Programs Summer Research Experience (SRE) 2016/ BIOL 186
The Fragile X syndrome (FXS), the most common form of inherited mental retardation, is a genetic disorder caused by inactivation of the Fragile X Mental Retardation 1 (FMR1) gene. The FMR1 gene encodes the Fragile X Mental Retardation Protein (FMRP), which is highly expressed in neurons and is necessary for normal brain function. The main goal of this project is to examine how the deletion of FMRP affects the structural dynamics of synapses in mouse cortical neurons in vivo, and whether the effect is cell-autonomous. We used the CRE-DOG (Cre recombinase dependent on GFP) system to sparsely knockout FMRP in cortical neurons. Specifically, we first crossed the thy1-GFP-M line mice with mice containing a floxed Fmr1 gene (Fmr1fl). Then we injected adeno-associated viruses (AAVs) encoding CRE-DOG and FLEX-tdTomato into the mouse cortex. The thy1-GFP-M line labels a sparse population of layer 5 pyramidal neurons in the cortex; the CRE-DOG system targets GFP-positive neurons for full Cre reconstitution so that the Fmr1 gene is knocked out in these Cre+ cells (marked by co-expression of tdTomato). We are using immunohistochemical staining to confirm that our strategy selectively knocks out FMRP, and using transcranial in vivo two-photon microscopy to investigate the synaptic dynamics in individual neurons lacking FMRP.
