Schematic drawings of rodent and human BDNF genes and the BAC transgenic construct used in this study. Rodent (A) and human (B) BDNF gene structures. Rodent BDNF gene consists of a number of 5' exons (I-VIII) spliced together with a common protein-coding sequence in exon IX (transcriptional start sites are indicated with arrows). BDNF transcription can also start from exon IX introducing a unique 5' UTR sequence. Hatched lines indicate sites of alternative splicing. Although the human BDNF gene has a similar structure and splicing pattern, it has additional exons Vh and VIIIh, longer and more complexly spliced 5'UTR of exon IX. Furthermore, human BDNF exons VIII and VIIIh are not used as 5'exons, but are always spliced with exon V. For detailed description see [14,16]. (C) Schematic drawing of the modified BAC construct used in this study containing the human BDNF locus. EGFP reporter gene was inserted in-frame with the BDNF coding region before the BDNF stop codon creating a fused BDNF-EGFP open reading frame within 168 kb of human BDNF locus. Arrows P1-3 indicate PCR primers used for analysis of transgene integration. (D) Slot-blot hybridization analysis of transgene copy number in hBDNF-EGFP transgenic founder mice (A4, C3, E1 and E4). BAC standard contains hBDNF-EGFP-BAC DNA in amounts equivalent to 1–3 copies of transgene in the blotted genomic DNA. WT- wild type mouse DNA. (E) PCR analysis of genomic DNA from transgenic mouse line C3 with primers detecting tandem integration of hBDNF-EGFP-BAC constructs. WT – wild type mouse DNA as a negative control; (+) – circular hBDNF-EGFP-BAC DNA as a positive control; (-) – PCR without DNA as a negative control.
Koppel et al. BMC Neuroscience 2009 10:68 doi:10.1186/1471-2202-10-68