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In vitro modification of full-length genes (100-250 kb) carried in bacterial associated chromosome vectors (BAC). Mutations responsible for human neurologic disease are placed into the genomic DNA sequence using homologous recombination in E. coli. Epitope tags are added. Transcriptional and translational stop cassettes flanked by loxP sequences are added to enable cell-type targeted expression of the mutant disease gene. Transgenic mice are created using the modified full-length gene. This method is currently being used to recreate inherited forms of childhood absence and temporal lobe epilepsy.
Cell-type specific gene manipulations in using Cre recombinase and loxP target sequences methods in transgenic mice.
Targeted gene manipulation in delimited cell-types within specific neuronal circuitry. Cre recombinase gene is placed behind promoters that express selectively in the thalamocortical neuron, the thalamic reticular neuron, cortical interneuron subtypes, or cortical pyramidal neurons. A thalamocortical neuron-specific Cre transgenic mouse has been created using the promoter for the delayed rectifier potassium channel Kv3.2.
Embryonic stem cell homologous recombination. To enable cell-type specific deletion of a disease gene, we engineer loxP sequences into introns of a gene and insert these into the mouse genome using homologous recombination. This method has already been applied to the T-type Ca2+ channel Cav3.1 (alpha1G), which plays a critical role in absence epilepsy, pain, and sleep.
In vitro brain slice electrophysiology. We use acute brain slice whole-cell current clamp recording methods to investigate the regulation of action potential firing and synaptic transmission. Using whole-cell and single channel voltage clamp methods, we study the regulation and gating of individual ion channels and electrogenic transporters. We use double patch clamp recording from neurons coupled by electrical and chemical synapses. These studies are performed in neurons and glia within their native neural circuitry to define the cellular mechanisms underlying neurologic disorders.
In-utero electroporation of cDNA constructs into the embryonic brain for studying the effects of this molecular intervention on brain circuit development and function in the postnatal and adult brain.
Primary neuronal cell culture (mouse and human) combined with transient transfection of cDNA and morphometric and electrophysiologic analysis of the properties of the formed neuronal circuits.
Neurolucida reconstruction and analysis of neuronal dendritic arbor and spines after filling neurons with markers using the patch electrophysiology pipette.
Seizure recordings using implanted electrodes.
Behavioral analysis (vocalization, social interaction, grooming, learning and memory tasks, motor learning tasks, etc...)
Proteomics analysis including isolation of protein complexes combined with mass spectrometry and western blot.
Simultaneous recordings from multiple neurons within a brain slice.
In vivo multiple single unit recordings from awake behaving mice (Neuralynx system).