... One of the main strengths of yeast as a genetic tool is the relative ease with which recessive mutations can be recognized and characterized at its haploid life stage (28), a trait absent from the commonly studied higher eukaryotes. The approach described here will allow similar types of screens for the human genome.
The discovery of RNA interference has enabled targeted reduction of gene expression in diploid cultured mammalian and insect cells, which opened the door to large-scale screens. At the same time, limitations of this approach are increasingly apparent, such as the induction of off-target effects that complicate genome-wide screens in particular (29, 30) and the inability to completely switch off gene expression. When similar small interfering RNA screens are conducted independently in mammalian cells, the lack of concordance between them is an additional complicating factor (31, 32). Finally, mammals are rather robust in their tolerance to partial loss of gene function: Haploinsufficiency appears to be the exception rather than the rule, because inactivation of one gene copy, as in heterozygous knockout mice, rarely leads to severe phenotypes.
Although we have focused on host-pathogen biology, similar screens could in principle be applied to any phenotype that can be recognized in a population of mutant cells, such as modulation of a genetically encoded reporter. In the future, haploid genetic screens could be used to generate comprehensive compendia of host factors that are used by different pathogens and may yield new strategies to combat infectious disease.
The key component of this system is near-haploid KBM7
cells, which was isolated in 1999
Exp Cell Res. 1999 Nov 1;252(2):273-80.
Isolation and characterization of a near-haploid human cell line.
Kotecki M, Reddy PS, Cochran BH (To whom correspondence and reprint requests should be addressed. Fax: 617-636-6745. E-mail: cochran@opal.tufts.edu.)
Department of Physiology, Tufts University School of Medicine, 136 Harrison Avenue, Boston, Massachusetts, 02111, USA.
Mammalian somatic cells are usually diploid. Occasional rare human tumors have been shown to have a hypodiploid karyotype. We have isolated a near-haploid subclone (P1-55) from a heterogeneous human leukemia cell line, KBM-7. These near-haploid cells have approximately half the human diploid DNA content and have a haploid karyotype except for a disomy of chromosome 8 (25, XY, +8, Ph(+)). This cell line maintains a majority of cells with a near-haploid karyotype for at least 12 weeks in culture. By serial subcloning, we have isolated near-haploid subclones that maintain ploidy for at least 8 months in culture. Near-haploid cells can also be efficiently isolated from mixed ploidy cultures by size selection. The availability of this human near-haploid cell line should facilitate the genetic analysis of cultured human cells. Copyright 1999 Academic Press.
Several experimental details
* Retroviral gene trap vectors containing an inactivated 3’ LTR, a strong adenoviral (Ad40) splice-acceptor site, either GFP or the puromycin resistance gene (PURO) and the SV40 polyadenylation signal were constructed as follows.
* KBM7 cells were maintained in IMDM supplemented with 10% FBS and antibiotics.
* Gene trap virus was produced by transfection of 293T cells in T175 dishes with either pGT-GFP or pGT-PURO combined with retroviral packaging plasmids. The virus-containing supernatant was concentrated using ultracentrifugation for 1.5 h at 25,000 r.p.m. in a Beckman SW28 rotor.
* Batches of mutant KBM7 cells were typically prepared by infection of one 24-well tissue culture dish containing 1.5 million cells per well using spin infection for 45 minutes at 2,000 rpm.
* Prior to infection cells were enriched for a near-haploid karyotype using either subcloning or flow-cytometric sorting for cells with a small size, if needed.
* Cells infected with the gene trap virus containing the puromycin resistance gene were selected 2 days after infection using 0.5 ug/ml puromycin. After selection, cells were expanded and frozen down for further screens. The GFP gene trap infected cells were directly used for screens or first selected using FACS sorting for GFP-expressing cells.
* Screens were started at least 6 days after gene trap infection.
* In general, mutagenized KBM7 cells were resuspended in medium containing the appropriate concentration of screening agent and plated at 20,000 cells per well in a 96-well tissue culture plate. One or more 96-well plates were used per genetic screen. The cells were incubated for two to three weeks after which resistant cells formed clearly visible colonies. Because typically less than one colony was present per well, most of the picked colonies were clonal and used to map insertion sites.
* Hits in screens were identified using the following three criteria: 1) insertion in a gene needs to occur in the correct orientation or into an exon 2) Genes should be mutated by multiple independent gene trap mutation insertion events 3) Mutant cells can be complemented by expression of a wild-type cDNA version of the mutated gene, thereby proving that the gene trap insertion in the affected locus is directly responsible for the phenotype of interest.
* The host sequences flanking the proviral insertion site were determined using an inverse PCR protocol. Genomic DNA was isolated from 5 million cells using the QiaAmp DNA mini kit (Qiagen) and 4 ug was digested with NlaIII or MseI. After spin column purification (Qiagen), 1 ug digested DNA was ligated using T4 DNA ligase in a volume of 300 ul. The reaction mix was purified using spin columns and used in a PCR reaction with primers annealing to internal sequences in the gene trap vector (5’-CTGCAGCATCGTTCTGTGTT-3’ and 5’-TCTCCAAATCTCGGTGGAAC-3’). The resulting PCR products that include the
flanking sequence were sequenced using (5’-CTCGGTGGAACCTCCAAAT-3’).
Here is the paper
Science. 2009 Nov 27;326(5957):1231-5.
Haploid genetic screens in human cells identify host factors used by pathogens..
Carette JE, Guimaraes CP, Varadarajan M, Park AS, Wuethrich I, Godarova A, Kotecki M, Cochran BH, Spooner E, Ploegh HL, Brummelkamp TR.
Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, MA 02142, USA.
Loss-of-function genetic screens in model organisms have elucidated numerous biological processes, but the diploid genome of mammalian cells has precluded large-scale gene disruption. We used insertional mutagenesis to develop a screening method to generate null alleles in a human cell line haploid for all chromosomes except chromosome 8. Using this approach, we identified host factors essential for infection with influenza and genes encoding important elements of the biosynthetic pathway of diphthamide, which are required for the cytotoxic effects of diphtheria toxin and exotoxin A. We also identified genes needed for the action of cytolethal distending toxin, including a cell-surface protein that interacts with the toxin. This approach has both conceptual and practical parallels with genetic approaches in haploid yeast.
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