Fig. 1. Replacement of the endogenous NT3-coding region with BDNF. (A) Diagram of the replacement vector and strategy used to ‘knock-in’ BDNF into the NT3 locus. Probes external to the targeting vector were the same as previously described (Tessarollo et al., 1994). Successful targeting of the locus with the replacement vector is revealed by a change of the wild-type-specific 6.5 kb ScaI DNA restriction fragment (wt) to an 8.5 kb ScaI fragment (mt). Removal of the neo selectable marker cassette by CRE-mediated recombination of the targeted allele is indicated by the switch of the 8.5 kb ScaI fragment (mt) to a 7.0 kb fragment (mt (cre)). X, XbaI; S, ScaI; B, BamHI; E, EcoRI; Sm, SmaI. (B) Southern blot analysis of DNA from wild-type embryonic stem (ES) cells (+/+), a heterozygous B/N ES cell clone (+/mt), and a heterozygous B/N ES cell clone following Cre-mediated recombination (+/mt (cre)). The ScaI restriction enzyme digestion and the (NT3) 5' probe indicated in A were used to detect the rearrangements in the murine NT3 locus. The BDNF-coding region (BDNF) and the neo (neo) probes were hybridized to parallel blots. Bacteriophage lambda DNA cut with HindIII was used as the size markers. (C) To ensure that the recombinant vector could produce biologically active BDNF, we introduced the NT3/BDNF chimeric genomic sequence into the pMEX vector which drives expression from an MSV-LTR promoter (Martin-Zanca et al., 1989). The supernatant of NIH 3T3 cells transfected with the pMEXneo-BDNF expression vector was harvested and tested on TrkB-expressing PC12 cells for the presence of BDNF activity. The presence of BDNF in the media was revealed by neurite outgrowth (bottom right). Unconditioned media (top left) and conditioned media by NIH 3T3 cells transfected with the pMEXneo (top right) did not induce neurite extension. Treatment of the PC12 cells with 1ng/ml recombinant BDNF was used as positive control (bottom left).
