Figure 1.

A) Construct Diagrams. Promoters were switched into the backbone by replacement of the CMV promoter between the first NotI and AgeI sites. The Des construct was created using primers against human genomic DNA (forward [F] Des enhancer primer containing NotI) ATA AGA ATG CGG CCG CAC CCA TGC CTC CTC AGG TA, (reverse [R] Des enhancer primer containing XhoI) CCG CTC GAG GGT GGG GCC TCA AGT TTA T, ([F] Des promoter primer containing XhoI) CCG CTC GAG ATA ACC AGG GCT GAA AGA, ([R] Des promoter primer containing AgeI) TGTA CCG GTG ACG GCG CGG GCG AGG CT. The α-MHC construct was created by amplifying human genomic DNA: ([F] containing NotI) ATA AGA ATG CGG CCG CCC AGT TGT TCA ACT CAC CCT TCA and ([R] containing AgeI) TGT ACC GGT GGG TTG GAG AAA TCT CTG ACA GCT. The MLC-2 construct was created by replacing the backbone with the previously described rat MLC-2 promoter[20]. ([F] containing NotI) ATA AGA ATG CGG CCG CGA CCC AGA GCA CAG AGC ATC GT ([R] containing AgeI) TGT ACC GGT GAA TTC AAG GAG CCT GCT. The cTnC construct was created by amplifying human genomic DNA: ([F] containing Not1) ATA AGA ATG CGG CCG CCA GCC TGA GAT CAC TGG GAC CAG A ([R] containing Age1) TGT ACC GGT CCA TGC TGG CGG CTC ACA GGA. 5 × 10¹⁰vg/mouse was administered (n = 6 per promoter group) [23]. Tissue lysates were assayed using the Galacto-Star chemiluminescence reporter gene assay system (Tropix, Inc., Bedford, MA, USA). Protein concentrations were determined using the Bio-Rad DC protein assay kit (Bio-Rad, Hercules, CA, USA). B) β-galactosidase (β-gal) expression levels show that CMV provides the greatest amount of expression in the heart followed by Des and α-MHC. C) β-gal levels in skeletal muscle including the diaphragm were highest in mice that were administered the Des construct. (Di, diaphragm; Qu, quadriceps; So, soleus; ED, extensor digitorum longus; TA, tibialis anterior; Ga, gastrocnemius) D) Evaluation by β-gal assay of non-heart, non-skeletal muscle tissues revealed highest expression levels in brain and lung from mice injected with the Des construct. (Ht, heart; Br, brain; Lu, lung; Li, liver; Sp, spleen; Ki, kidney; SI, small intestine) E) and F) β-gal levels and biodistribution profiles from α-MHC and Des construct injected mice (respectively).