BAC-based cellular model for screening regulators of BDNF gene transcription

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.

Dopamine cross‐reacts with adrenoreceptors in cortical astrocytes to induce BDNF expression, CREB signaling and morphological transformation

“…To test this hypothesis, we transfected cortical astrocytes with rat and human BDNF‐BAC constructs harboring about 207 and 169 kb of BDNF locus, respectively, and carrying a luciferase reporter instead of the BDNF ORF (Figure A). These BDNF‐BAC clones have previously been shown to recapitulate BDNF expression in transgenic mice (Koppel et al, ) and HeLa cell lines (Jaanson, Sepp, Aid‐Pavlidis, & Timmusk, ). Both BDNF‐BAC luciferase reporters were induced by catecholamine treatments, showing that both constructs carry distal regulatory regions necessary for catecholamine‐dependent activation of BDNF transcription (Figure F,G).…”

Screening inducers of neuronal BDNF gene transcription using primary cortical cell cultures from BDNF-luciferase transgenic mice

“…The most important aspect of our screening assay is that activation of Bdnf transcription could be evaluated in a primary neuronal culture. Previously, Jaanson et al ., (2014) developed a similar screening assay in stable HeLa cell 34 . They also used a BAC clone and replaced the coding region of the BDNF with the Renilla luciferase-EGFP gene.…”