A NEAR-HAPLOID HUMAN CELL LINE FOR FUNCTIONAL GENOMICS

确定基因功能的最好方法之一是从基因上扰乱基因，并检查由此产生的表型。对于哺乳动物来说，这传统上是通过胚胎干细胞的同源重组，然后将胚胎移植到寄养母亲和后代杂交来完成的。基因敲除也已经在体细胞中进行了工程，但这是一项既费力又耗时的任务，需要按顺序瞄准基因的两个副本。在哺乳动物系统中，这些操作比在酵母等低等生物中需要更长时间的原因主要是因为哺乳动物细胞是二倍体。为了绕过这些问题，我们分离并鉴定了一个接近单倍体的人类细胞系。这种细胞系对除8条染色体以外的每条染色体都是单体。具有单倍体细胞系用于遗传分析的优点是，通常由于第二个野生型等位基因的表达而导致的每个突变在突变时立即表现出表型，而不需要进一步的操作。在这里，我们建议利用细胞系的接近单倍体的性质来加快基因敲除的分离和体细胞突变体的选择。基因陷阱载体的逆转录病毒插入突变将被用来构建该细胞系中大多数表达基因的前病毒插入文库。使用组合策略，我们将产生有序的DNA池和细胞克隆，这将允许在较短的时间内识别在这些细胞中表达的几乎任何人类基因的基因敲除，而所做的工作相对较少。此外，具有基因敲除的细胞库可用于体细胞突变体的选择和突变基因的快速恢复，以适用于该细胞系的任何遗传选择。为了评估基因敲除文库的用途，我们将使用这个文库来筛选在TPA、TNF-α或柔红霉素存在的情况下导致细胞生长停滞或发生凋亡的基因。利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。这一资源的可获得性应该会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.