A NEAR-HAPLOID HUMAN CELL LINE FOR FUNCTIONAL GENOMICS

确定基因功能的最佳方法之一是对基因进行遗传破坏，并检查由此产生的表型。对于哺乳动物来说，传统的方法是在胚胎干细胞中进行同源重组，然后将胚胎移植到养母体内，再进行后代的杂交。基因敲除也被用于体细胞，但这是一项费力且耗时的任务，需要按顺序靶向基因的两个拷贝。这些操作在哺乳动物系统中比在酵母菌等低等生物中需要更长时间的原因主要是由于哺乳动物细胞是二倍体。为了规避这些问题，我们分离并鉴定了一个接近单倍体的人类细胞系。这个细胞系除了8条染色体外，其余的染色体都是单体的。使用单倍体细胞系进行遗传分析的优点是，由于第二个野生型等位基因的表达，每个通常是隐性的突变在突变后立即表现出表型，而无需进一步的操作。在这里，我们建议利用细胞系的近单倍体特性来加快基因敲除的分离和体细胞突变体的选择。利用基因诱捕载体的逆转录病毒插入诱变技术将构建一个前病毒插入文库，以插入该细胞系中大多数表达的基因。使用组合策略，我们将产生有序的DNA池和细胞克隆，这将允许在短时间内以相对较少的努力鉴定在这些细胞中表达的几乎任何人类基因的基因敲除。此外，基因敲除的细胞文库可用于体细胞突变体的选择和突变基因的快速恢复，适用于该细胞系的任何遗传选择。为了评估敲除文库在这方面的使用，我们将使用该文库来选择在TPA、TNF- α或柔红霉素存在下导致细胞生长停滞或细胞凋亡失败的基因。使用逆转录病毒序列作为探针，将分离出导致这些突变的基因。这种资源的可用性将大大加快对大量人类基因的功能分析。

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.