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Cloning and Expression of the hFEV Transcription Factor

. . . Serotonin (5-HT) A Critical Neurotransmitter. DepressionAnxietySleep disorderSchizophrenia. Reduced Serotonergic neurotransmission. . . Plays a perceived role in many psychological disorders . Fifth Ewing Variant (FEV) mRNABelongs to a family 85 amino acid ETS domain- activators and rep

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Cloning and Expression of the hFEV Transcription Factor

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    1. Cloning and Expression of the hFEV Transcription Factor Scholar: Di Wu Mentor: Dr. Abiye Iyo

    2. Serotonin (5-HT) A Critical Neurotransmitter Depression Anxiety Sleep disorder Schizophrenia

    3. Fifth Ewing Variant (FEV) mRNA Belongs to a family 85 amino acid ETS domain- activators and repressors Homologue of rodent Pet-1 gene ~96% similarity between hFEV and Pet-1 Both concentrated in the dorsal raphe nuclei Role in regulating serotonin-related genes Determinant of serotonin neuronal differentiation and development Potential role in psychiatric illness FEV Transcription Factor

    5. Methods To Isolate FEV cDNA from Rodent and Human Brains I. Rodent FEV cDNA Extract RNA from rodent brain Reverse transcription to get cDNA Perform PCR to get rodent FEV clone II. Human FEV cDNA Obtain human FEV clone (plasmid) PCR amplification using FEV specific primers Cut PCR product with the restriction enzymes - BamH1 and Xho1

    7. Methods cont’d Ligate the fragment into pGEX-4T-3 vector Transformation of E. coli cells PCR Screening using FEV primers Isolate the FEV plasmid III. Protein Expression Transform the plasmid into BL21 cells Grow up culture until OD of 0.6 Induce culture with IPTG (Isopropyl ß-D-1-thiogalactopyranoside) Aliquot 1 ml of cells (pre-induction, 1, and 2 hours post induction) Add SDS (sodium dodecyl sulfate) buffer, reducing agent, and deionized water to cells Run protein gel and stain with Coomassie Blue for detection

    8. Results

    9. III. Analyzed Transformants PCR screened No result on many occasions Enhanced Protocol Transformants expressed FEV- pGEX-4T-3

    10. Protein Expression Induced versus Uninduced cells

    11. Conclusions Cloned and expressed the FEV protein Future Studies Perform Western Blot analysis using FEV antibody/protein Analysis of FEV protein expression in both stressed rodents and our cohorts of depressed and suicide victims

    12. Acknowledgements Dr. Abiye Iyo, Ph.D. Niamh Kieran Dr. Ian Paul UMC Psychiatry and Neuroscience Department NSSP and Fellow Scholars

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