The primary focus of Dr. Ashlock's work is to further the field of gene transfer so that large DNA molecules can be efficiently delivered intact into mammalian cells. Ultimately, her team would like to introduce extensive stretches of DNA including full length genes and their regulatory elements so that genes can be more easily identified and analyzed functionally. Presently, the transfer of large DNA molecules is possible, but it is difficult to deliver genes intact into mammalian cells and demonstrate that the gene of interest functions. The team has used the cystic fibrosis transmembrane conductance regulator gene [CFTR; 230 kilobases (kb)] as a model gene in the form of yeast artificial chromosomes (YACs) ranging form 330-610 kb to begin to address these issues. They have modified the CFTR YACs to contain marker genes and using the standard technique of yeast spheroplast fusion and transfection of purified YAC DNA, they have delivered CFTR to mammalian cells and demonstrated that the CFTR contained within the YACs are functional. They have also introduced mutations into the modified YACs so that the CFTR contained in these YACs can be distinguished from the endogenous CFTR in the host cell. In addition, they are further modifying the YACs so that reporter genes can be used to study regulation of CFTR expression, especially the role of distant flanking sequences and/or elements contained in introns in controlling CFTR expression. They are using the functional analyses employed in these studies in our investigation of optimal delivery systems for large DNA molecules.

They have used also YAC gene transfer as a method of facilitating the identification of human and murine disease genes by complementation of a cellular phenotype. Using YAC complementation cloning, we rapidly narrowed the candidate region for the gene responsible for the disease, Niemann-Pick Type-C (NP-C) and thereby aided in the eventual identification of NPC1.

With regard to new vector systems, they are developing and testing human artificial chromosome constructs for the purposes of: (1) understanding chromosome structure and function and (2) delivering and expressing full length genes in host cells in a more physiologic manner than that obtained using delivery systems in which the introduced DNA integrates into the host chromosomes. They are currently attempting to enhance the efficiency and specificity of the delivery of such high molecular weight DNA into human cells using strategies which utilize receptor mediated endocytosis.