Gene Targeting in Human Somatic Cells

Abstract

Somatic gene targeting in human cells has two general applications of importance and wide interest. One is the inactivation of genes (“knockouts”), a process utilized to delineate the loss-of-function phenotype(s) of a particular gene. The second application is the process of gene therapy, which involves correcting a pre-existing mutated allele(s) of a gene back to wild-type in order to ameliorate some pathological phenotype associated with the mutation. Both of these processes require a form of DNA double-strand break repair known as homologous recombination. Although bacteria and lower eukaryotes utilize homologous recombination almost exclusively, a competing process, known as nonhomologous end joining, predominates in higher eukaryotes and was presumed to prevent the use of gene targeting in human somatic cells in culture. A series of molecular and technical advances developed in the 1990s disproved this notion, but still resulted in a process that was cumbersome, labor intensive, highly inefficient, and slow. Within the past 5 years, the use of new gene delivery vectors such as recombinant adeno-associated virus and the identification of cell lines such as Nalm-6 that appear to undergo high rates of gene targeting have significantly brightened the outlook for this field and resulted in a gene delivery system that facilitates both gene knockouts and gene therapy modifications at robust levels. Thus, gene targeting in human somatic cells in culture has become not only feasible, but also relatively facile, and it harbingers a golden age for directed mutagenesis.