Modular flexibility of dystrophin: implications for gene therapy of Duchenne muscular dystrophy.

Attempts to develop gene therapy for Duchenne muscular dystrophy (DMD) have been complicated by the enormous size of the dystrophin gene. We have performed a detailed functional analysis of dystrophin structural domains and show that multiple regions of the protein can be deleted in various combinations to generate highly functional mini- and micro-dystrophins. Studies in transgenic mdx mice, a model for DMD, reveal that a wide variety of functional characteristics of dystrophy are prevented by some of these truncated dystrophins. Muscles expressing the smallest dystrophins are fully protected against damage caused by muscle activity and are not morphologically different from normal muscle. Moreover, injection of adeno-associated viruses carrying micro-dystrophins into dystrophic muscles of immunocompetent mdx mice results in a striking reversal of histopathological features of this disease. These results demonstrate that the dystrophic pathology can be both prevented and reversed by gene therapy using micro-dystrophins.

Manipulating genes and gene copy number by bacterial artificial chromosomes transfection.

Bacterial artificial chromosomes (BACs) provide a well-characterized resource for studying the organization and activity of entire genes, replicons, and other large genomic loci. Protocols and parameters that influence the efficient transfection of these large DNA molecules into cells in culture were described here. By carefully optimizing the conditions for the formation of compact transfection complexes, BACs can be introduced into a variety of mammalian cells with reasonable efficiency. In addition, by cotransfection with a dihydrofolate reductase or hypoxanthine guanine phosphoribosyl transferase BAC, stable cell lines can be generated that carry 2-15 tandem chromosomal copies of the BAC of interest, thus providing a new avenue for studying gene dosage effects.

Parameters influencing high-efficiency transfection of bacterial artificial chromosomes into cultured mammalian cells.

Although bacterial artificial chromosomes (BACs) provide a well-characterized resource for the analysis of large chromosomal domains, low transfection rates have proven a significant limitation for their use in cell culture models. Using TP53 BAC clones that contain expression cassettes for enhanced green fluorescent protein or red fluorescent protein, we have examined conditions that promote BAC transfection in hamster, human, and mouse cell lines. Atomic force microscopy shows that BAC transfection efficiency correlates with the generation of small, highly condensed but dispersed lipid: BAC DNA transfection complexes. BAC DNA purity and concentration are critical for good transfection; debris from purification columns induces the formation of large aggregates that do not gain entry into the cell, and DNA concentrations must be optimized to promote intramolecular condensation rather than intermolecular linking, which also causes aggregation and diminished transfection efficiency. The expression of both markers and genes within BACs initially occurs at lower levels than observed with plasmids, requiring 3-5 days to evaluate the transfection results. We also show that BACs can be co-transfected with other BACs, which provides for increased experimental flexibility.