Abstract
The isolation of haploid cell lines has recently allowed the power of forward genetic screens to be applied to mammalian cells. The interest in applying this powerful genetic approach to a mammalian system is only tempered by the limited utility of these screens, if confined to lethal phenotypes. Here we expand the scope of these approaches beyond live/dead screens and show that selection for a cell surface phenotype via fluorescence-activated cell sorting can identify the key molecules in an intracellular pathway, in this case MHC class I antigen presentation. Non-lethal haploid genetic screens are widely applicable to identify genes involved in essentially any cellular pathway.
Highlights
Forward genetic analysis – the concept of identifying gene function from mutants with a discernible phenotype – has a proven track record of elucidating gene function
Near-haploid KBM7 cells express high cell surface major histocompatibility complex class I (MHC-I) and HLA typing showed them to be HLA-A2, HLA-B60 and HLA-Cw10. These cells were mutagenised with a gene-trap retrovirus [1] to create a library of knockout cells, which were selected by fluorescence-activated cell sorting (FACS) for the very small population of cells (,0.02%) expressing low cell surface MHC-I (Fig. 1A)
To identify the disrupted genes responsible for the defect in MHC-I expression, the retroviral integration sites in all sets of selected cells were amplified using splinkerette-PCR and sequenced using 454 pyrosequencing [8,9]. Together this analysis revealed retroviral insertions in four genes known to be involved in the MHC-I antigen presentation pathway: 9 independent retroviral integrations were found in the gene encoding b2m, 8 in tapasin (TAPBP), 2 in TAP2 and 1 in the HLA-A2 gene itself (Fig. 1C)
Summary
Forward genetic analysis – the concept of identifying gene function from mutants with a discernible phenotype – has a proven track record of elucidating gene function. Three groups recently circumvented this problem by pioneering forward genetic screens in haploid cells. Carette and colleagues [1] initially demonstrated the power of this approach by performing forward genetic screens in the near-haploid human KBM7 chronic myeloid leukemia cell line [2]. By creating a library of knockout KBM7 cells using a gene-trap retrovirus and screening for mutant cells resistant to a range of lethal insults, including bacterial toxins and cytotoxic viruses, they identified host genes required for toxin or viral killing [1,3,4]. Two independent groups derived haploid murine embryonic stem cells lines and demonstrated the ability to perform forward genetic screens to identify genes required for sensitivity to toxins [5,6]
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