Abstract
BackgroundGenetically manipulated embryonic stem (ES) cell derived neurons (ESNs) provide a powerful system with which to study the consequences of gene manipulation in mature, synaptically connected neurons in vitro. Here we report a study of focal adhesion kinase (FAK), which has been implicated in synapse formation and regulation of ion channels, using the ESN system to circumvent the embryonic lethality of homozygous FAK mutant mice.ResultsMouse ES cells carrying homozygous null mutations (FAK-/-) were generated and differentiated in vitro into neurons. FAK-/- ESNs extended axons and dendrites and formed morphologically and electrophysiologically intact synapses. A detailed study of NMDA receptor gated currents and voltage sensitive calcium currents revealed no difference in their magnitude, or modulation by tyrosine kinases.ConclusionFAK does not have an obligatory role in neuronal differentiation, synapse formation or the expression of NMDA receptor or voltage-gated calcium currents under the conditions used in this study. The use of genetically modified ESNs has great potential for rapidly and effectively examining the consequences of neuronal gene manipulation and is complementary to mouse studies.
Highlights
Manipulated embryonic stem (ES) cell derived neurons (ESNs) provide a powerful system with which to study the consequences of gene manipulation in mature, synaptically connected neurons in vitro
Since neuronal focal adhesion kinase (FAK) appears to have functions other than adhesion/motility in neurons, we explored the possibilities, principally using the whole cell patch clamp technique, that FAK is instead involved in ion channel modulation and synapse formation
FAK expression in wild type and mutant cells Western blot analyses using FAK monoclonal antibody were performed on cellular extracts to examine FAK expression in wild type (WT) and FAK null ES cells
Summary
Manipulated embryonic stem (ES) cell derived neurons (ESNs) provide a powerful system with which to study the consequences of gene manipulation in mature, synaptically connected neurons in vitro. The tetracycline system has been used to transiently express genes in the brain [1] and the Cre/LoxP system used to delete genes in selected neuronal populations [2] One disadvantage to these techniques is that it can take 2 or more years for results, in part because of the breeding of mice. Any method that allows for a shorter duration and reduces the need for animals is potentially useful Toward this end, we have adopted an experimental strategy using genetically engineered embryonic stem (ES) cell derived neurons (ESNs). We have adopted an experimental strategy using genetically engineered embryonic stem (ES) cell derived neurons (ESNs) This system has the potential to greatly speed the functional analysis of genetic manipulations and importantly allows access to lethal mouse phenotypes
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