Generation of conditional human HSP60 transgenic mice. (a) G-Lox-HSP60 and EGFP-Cre Tg vectors. The CAGGS promoter was used to drive both Tg vectors. Placed between two LoxP sites, the GFP cDNA with 3 stop codons (SC) and the SV40 polyA sequence was used to inhibit the expression of the downstream transgene. After the second LoxP site, a full-length human HSP60 cDNA with endogenous SC and a portion of the last exon was inserted in front of ATG of the DsRedT1 cDNA sequence. After the LoxP sites were rejoined using the Cre DNA recombinase, the HSP60/DsRedT1 transcript was expressed; however, only HSP60 translated into proteins. (b) (A) Analysis of four possible littermate genotypes using PCR on tail DNA. (I) PCR amplification using the primer pair complementary to human HSP60 but not to mouse HSP60, (II) amplification of EGFP-Cre, and (III) the reaction to identify double transgenic (H+/C+) mice by amplifying the abridged sequence from CAGGS promoter to human HSP60. (B) RT-PCR results of human HSP60 mRNA expression in the neonatal mouse heart. HSP60 (m, h) indicates the PCR reaction to amplify HSP60 mRNA of both mouse and human origions. GAPDH amplification served as the internal control. MDA-MB231 human breast tumor cells mRNA were used as the positive control for human HSP60. (C) Western blotting for HSP60 and EGFP proteins in neonatal hearts. Anti-HSP60 antibodies recognize both mouse and human HSP60.