One of the best ways to determine the function of the gene is to genetically disrupt the gene and examine the resulting phenotype. For mammals this has traditionally been done by a homologous recombination in embryonic stems cells followed by embryonic transfer into a foster mother and interbreeding of progeny. Gene knockouts have also been engineered in somatic cells, but this is a laborious and time- consuming task that requires targeting both copies of the gene sequentially. The reason these manipulations take longer in mammalian systems then in lower organisms such as yeast is principally due the fact that mammalian cells are diploid. In order to circumvent these problems, we have isolated and characterized a near haploid human cell line. This cell line is monosomic for every chromosome except 8. The advantage of having a haploid cell line for genetic analysis is that every mutation that would normally be recessive by virtue of expression of the second wild type allele is immediately phenotypically expressed upon mutation without further manipulation. Here we propose to take advantage of near-haploid nature of the cell line to expedite the isolation of gene knockouts and the selection of somatic cell mutants. Retroviral insertional mutagenesis with gene trap vectors will be used to construct a library of proviral insertions into the majority of expressed genes in this cell line. Using a combinatorial strategy, we will produce ordered pools of DNA and cell clones that will allow identification of gene knockouts in almost any human gene expressed in these cells in a short period of time with relatively little effort. In addition, libraries of cells with gene knockouts can be used for the selection of somatic cell mutants and rapid recovery of the mutated gene for any genetic selection applicable to this cell line. To evaluate the use of the knockout library for this purpose, we will use this library to select for genes that cause failure to cell growth arrest or undergo apoptosis in the presence of TPA, TNF- alpha, or daunorubicin. Using retroviral sequences as probes, the genes responsible for these mutations will be isolated. The availability of this resource should greatly speed the functional analysis of large numbers of human genes.
