The Cloning of the Fanconi's Anemia Gene

The aim of this project was to pinpoint the location and ultimately the gene code of one of at least four possible genes of Fanconi's anemia, a human "DNA repair" disorder. In the first part of the study, microcell-mediated chromosome transfer, an established procedure, was used to transfer a normal tagged human chromosome #20 (the reputed location of the FA-A gene, carried in an A9 mouse cell line) into FA-group A lymphoblasts and fibroblasts at which point they would be tested to see if the uptake of this chromosome corrects two prominent manifestations of the FA defect, namely its inherent chromosomal instability and its hypersensitivity to DNA cross-linking agents. Success in this venture was elusive. In the second part of the study, the first steps of a proven method of gene cloning was tried involving first, the transfection of a cDNA library, carried in an Epstein-Barr virus (EBV) vector into an FA-group B cell line, and then pulling out the FA-B gene from these cells by treating them with DNA cross-linking agents and selecting for ones which have lost this hypersensitivity. Three different methods of transfection, electroporation, lipofection and endocytosis-fection, were tested to discover which was the most efficient at transferring plasmid DNA into the FA group B cells. This group, which is one of the most sensitive of the four known FA cell lines, was transfected, in transient assays, most efficiently via endocytosis-fection. However, the attempt to select for long term maintenance of plasmid DNA was unsuccessful for FA-B cells using any of the transfection methods. However, successful maintenance of the cDNA-containing plasmid by FA-A lymphoblasts was obtained. Modifications are being made to circumvent the problem with the former cell line while work continues to build on the success with the latter cell line.