Genetic Engineering and Characterization of ZHTc6 and AF5 Cell Lines
Every year over 180,000 Americans are diagnosed with epilepsy, a chronic neurodegenerative disorder that is characterized by severe and unprovoked spontaneous seizures. Temporal lobe epilepsy (TLE) is particularly difficult to treat with conventional anti-epileptic drug therapy, and cell replacement therapy provides a promising alternative to temporal lobectomy (the removal of brain tissue containing the seizure focus). In preclinical trials, cells genetically engineered to secrete anticonvulsant molecules have been transplanted into the brain with some evidence of seizure suppression. One of these molecules, γ-aminobutyric acid (GABA), is an inhibitory amino acid that suppresses overly active neurons in seizure-susceptible regions of the brain. We transfect glutamic acid decarboxylase (GAD), an enzyme that converts glutamate to GABA, into cells that can be transplanted into animal models of epilepsy. The lab is currently working with a murine embryonic stem cell (ESC) line (ZHTc6), and has been developing protocols to promote their differentiation along a neural pathway; a neural cell line (AF5), derived from rat striatum, is used as a control. We show that both cell lines can be engineered to express reporter molecules (beta-galactosidase or green fluorescent protein) and GAD. High performance liquid chromatography illustrates that both cell lines produce GABA, and histochemistry and fluorescent microscopy show the detection of both cell lines in vitro. Our results suggest the possibility of developing therapeutic cells lines for TLE that can be differentiated prior to transplantation and identified in the host brain after transplantation?results relevant to all neurodegenerative disorders that are candidates for cell-based therapies.
Leith, Margaret, "Genetic Engineering and Characterization of ZHTc6 and AF5 Cell Lines" (2009). URC Student Scholarship.
Howard Hughes Medical Institute Undergraduate Science Education Grant