Abstract
BackgroundBrain derived neurotrophic factor (BDNF) belongs to a family of structurally related proteins called neurotrophins that have been shown to regulate survival and growth of neurons in the developing central and peripheral nervous system and also to take part in synaptic plasticity related processes in adulthood. Since BDNF is associated with several nervous system disorders it would be beneficial to have cellular reporter system for studying its expression regulation.MethodsUsing modified bacterial artificial chromosome (BAC), we generated several transgenic cell lines expressing humanised Renilla luciferase (hRluc)-EGFP fusion reporter gene under the control of rat BDNF gene regulatory sequences (rBDNF-hRluc-EGFP) in HeLa background. To see if the hRluc-EGFP reporter was regulated in response to known regulators of BDNF expression we treated cell lines with substances known to regulate BDNF and also overexpressed transcription factors known to regulate BDNF gene in established cell lines.ResultsrBDNF-hRluc-EGFP cell lines had high transgene copy numbers when assayed with qPCR and FISH analysis showed that transgene was maintained episomally in all cell lines. Luciferase activity in transgenic cell lines was induced in response to ionomycin-mediated rise of intracellular calcium levels, treatment with HDAC inhibitors and by over-expression of transcription factors known to increase BDNF expression, indicating that transcription of the transgenic reporter is regulated similarly to the endogenous BDNF gene.ConclusionsGenerated rBDNF-hRluc-EGFP BAC cell lines respond to known modulators of BDNF expression and could be used for screening of compounds/small molecules or transcription factors altering BDNF expression.
Highlights
Brain derived neurotrophic factor (BDNF) belongs to a family of structurally related proteins called neurotrophins that have been shown to regulate survival and growth of neurons in the developing central and peripheral nervous system and to take part in synaptic plasticity related processes in adulthood
Generation of rBDNF-humanised Renilla luciferase (hRluc)-EGFP HeLa stable cell lines HeLa cell line was chosen for generation of rBDNFhRluc-EGFP cell lines because its relatively carefree growth conditions and fast growth are good properties for transgenic cell line
Endogenous human BDNF gene was expressed in HeLa cell line showing that signaling pathways regulating BDNF expression were active in HeLa cells
Summary
Brain derived neurotrophic factor (BDNF) belongs to a family of structurally related proteins called neurotrophins that have been shown to regulate survival and growth of neurons in the developing central and peripheral nervous system and to take part in synaptic plasticity related processes in adulthood. BDNF gene has complex transcriptional regulation with different untranslated 5′ exons spliced to a common protein coding 3′ exon. Due to the presence of many promoters and resulting large number of transcripts with the same protein coding sequence but alternating 5′ and 3′ untranslated regions, BDNF expression is temporally and spatially controlled in different tissues [11,12,29], developmental stages [30] and within different cell compartments [31,32,33]. Transcripts of the antisense BDNF gene have been shown to form dsRNA duplexes with BDNF transcripts [12] and regulate BDNF levels [35] in vivo
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