A NEAR-HAPLOID HUMAN CELL LINE FOR FUNCTIONAL GENOMICS

确定基因功能的最好方法之一是从遗传上破坏基因并检查产生的表型。 对于哺乳动物来说，这通常是通过胚胎干细胞中的同源重组，然后将胚胎转移到寄养母体中并杂交后代来完成的。基因敲除也已经在体细胞中被工程化，但这是一项费力且耗时的任务，需要依次靶向基因的两个拷贝。 这些操作在哺乳动物系统中比在低等生物如酵母中花费更长时间的原因主要是由于哺乳动物细胞是二倍体的事实。为了规避这些问题，我们已经分离并表征了近单倍体人细胞系。该细胞系除8条染色体外，其余染色体均为单体。具有用于遗传分析的单倍体细胞系的优点在于，由于第二野生型等位基因的表达而通常为隐性的每个突变在突变后立即表型表达而无需进一步操作。在这里，我们建议利用近单倍体性质的细胞系，以加快基因敲除的分离和体细胞突变体的选择。使用基因捕获载体的逆转录病毒插入诱变将用于构建插入该细胞系中大多数表达基因的前病毒文库。使用组合策略，我们将产生有序的DNA和细胞克隆池，这将允许在相对较少的努力下在短时间内鉴定在这些细胞中表达的几乎任何人类基因中的基因敲除。此外，具有基因敲除的细胞文库可用于选择体细胞突变体和突变基因的快速回收，用于适用于该细胞系的任何遗传选择。为了评估敲除文库用于此目的的用途，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失败或经历细胞凋亡的基因。使用逆转录病毒序列作为探针，负责这些突变的基因将被分离。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.