Fig. 1.
Generation and validation of BDNFMet transgenic mice. (A) Schematic diagram of the strategy used to replace the coding region of the BDNF gene with BDNFMet. The entire coding region is in exon V. For the variant BDNF, a point mutation has been made (G196A) to change the valine in position 66 to a methionine. (B) Southern blots of representative embryonic stem cell clones for BDNFMet. Bgl II and Bam HI restriction enzyme digestion and 5′ external probe indicated in (A) were used to detect homologous replacement in the BDNF locus. The 5.6-kilobase (kb) WT and 7.4-kb rearranged variant DNA bands are indicated. (C) BDNF ELISA analyses of total BDNF levels from postnatal day 21 (P21) brain lysates from WT (+/+), heterozygous (+/Met), and homozygous (Met/Met) mice, as well as BDNF heterozygous KO mice (+/−) (**P < 0.01, Student’s t test). (D) Hippocampal-cortical neurons obtained from embryonic day 18 (E18) BDNF+/Met (+/Met), BDNFMet/Met (Met/Met), and WT (+/+) pups were cultured. After 72 hours, media were collected under depolarization (regulated) or basal (constitutive) secretion conditions as described previously (10). Media were then concentrated and analyzed by BDNF ELISA. (*P < 0.05, **P < 0.01, Student’s t test).