Establishment, maintenance, and cloning of human primary cell strains

My laboratory is involved in characterizing the behavior of cultured fibroblasts established from skin biopsies of normal boys and those affected with Duchenne muscular dystrophy. We are also interested in the properties of fibroblasts obtained from female carriers of this X-linked disease who are generally clinically unaffected (1). Following the argument of the hypothesis for random X-chromosome inactivation proposed by Mary Lyon as a gene dosage compensation mechanism in all placental mammals, it can be predicted that the dermis of female carriers of Duchenne dystrophy will be populated by fibroblasts mosaic for the Duchenne genotype. By chance, some cells will be expressing the gene products of the unaffected X chromosome, whereas others will have inactivated the normal X chromosome and be expressing the Duchenne gene product. In theory, each carrier of Duchenne dystrophy should contain a roughly 1:1 ratio of normal and Duchenne-expressing fibroblasts, and techniques of cell cloning applied to cultures of carrier biopsy material should produce clones of cells exhibiting properties of either normal or Duchenne cell cultures in a similar ratio (2). In practice, things are not so simple, but the theory outlined above has led to the development of a routine cell-cloning method in my laboratory, which will be described here. Alternative methods do exist, which I have also tried and found to be quite successful, but more time-consuming.