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  • Review Article
  • Published:

Systematic genome-wide screens of gene function

Nature Reviews Genetics volume 5, pages 11–22 (2004)Cite this article

Key Points

  • High-throughput screens, in which each gene of an organism is systematically perturbed, are now routine in yeast and are becoming feasible in other organisms.

  • Before conducting a genome-wide screen, the list of gene sequences to be targeted must be compiled and a set of gene-perturbing reagents or strains must be constructed.

  • Gene-perturbing strategies include: homologous recombination and random insertional mutagenesis to delete or mutate genes at the DNA level, and RNA interference to reduce the mRNA levels of a gene.

  • Any observable phenotype can be screened using genome-wide collections of reagents or organisms, subject to practical limitations.

  • Examples of phenotypes that have been screened so far include cell growth and proliferation, classical morphological defects and reporter-gene activity.

  • Using automated microscopy and/or automated image analysis, visual phenotypes can also be screened in a high-throughput manner.

  • Genome-wide phenotypic screens generate large, high-quality data sets, which can be used immediately to identify genes that are involved in a particular process.

  • These genome-wide data sets can also be integrated with existing genome-wide data sets, such as transcriptional profiles and annotated databases, to provide further, broader insights.

  • Future screens will probably examine more complex phenotypes in increasingly physiological contexts.

Abstract

By using genome information to create tools for perturbing gene function, it is now possible to undertake systematic genome-wide functional screens that examine the contribution of every gene to a biological process. The directed nature of these experiments contrasts with traditional methods, in which random mutations are induced and the resulting mutants are screened for various phenotypes. The first genome-wide functional screens in Caenorhabditis elegans and Drosophila melanogaster have recently been published, and screens in human cells will soon follow. These high-throughput techniques promise the rapid annotation of genomes with high-quality information about the biological function of each gene.

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Figure 1: Steps involved in the design and implementation of a systematic genome-wide functional screen.
Figure 2: High-throughput formats for screening.
Figure 3: Examples of scorable phenotypes from various screens.

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Acknowledgements

The authors thank D. A. Guertin for helpful discussions, and M. Boutros and N. Perrimon for sharing unpublished work.

Author information

Authors and Affiliations

  1. MIT Department of Biology, Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, 02142, Massachusetts, USA

    Anne E. Carpenter & David M. Sabatini

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  1. Anne E. Carpenter
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Correspondence to David M. Sabatini.

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DATABASES

ColiBase

LacZ

Model organism databases

A. thaliana (TAIR)

C. elegans (WormBase)

D. melanogaster (FlyBase)

H. sapiens (Human genome resources)

M. musculus (Mouse genome informatics)

M. musculus (Mouse genome server)

S. cerevisiae (Saccharomyces genome database)

FURTHER INFORMATION

Open Microscopy Environment

Anne Carpenter's web site

David Sabatini's laboratory

Glossary

TRANSCRIPTIONAL PROFILING

The expression of thousands of genes can be measured simultaneously by spotting an array of DNA sequences on a glass slide and hybridizing a cell population's fluorescently labelled mRNA (or reverse-transcribed cDNA) to the slide. The fluorescence intensity of each spot corresponds to the prevalence in the cells of that nucleic acid species.

YEAST TWO-HYBRID ASSAY

One protein is fused to a transcriptional activation domain and the other to a DNA-binding domain, and both fusion proteins are introduced into yeast. Expression of a reporter gene with the appropriate DNA binding sites upstream of the promoter indicates that the two proteins physically interact.

RNA INTERFERENCE

(RNAi). The process by which the introduction or expression within cells of single- or double-stranded RNA leads to the degradation of the encoded mRNA and therefore to gene suppression.

LAMELLA

The dense, actin-rich structure that extends the leading edge of a migrating cell.

microRNAs

Tiny, noncoding RNAs that are probably involved in gene regulation.

HOMOLOGOUS RECOMBINATION

The process by which segments of DNA are exchanged between two DNA duplexes that share high sequence similarity.

MOLECULAR BAR CODES

Short, unique, engineered DNA sequences that are used as tags. For example, the bar code on each yeast deletion strain allows the identity of the strain to be determined by sequencing the code or by hybridizing DNA from the strain onto a microarray.

S2 CELLS

A cell line that is isolated from dissociated Drosophila melanogaster embryos. The cell line is phagocytic, which might contribute to its susceptibility to RNAi.

INTERFERON RESPONSE

A primitive antiviral mechanism that triggers sequence-nonspecific degradation of mRNA and downregulation of cellular protein synthesis.

DEGRON TAG

A degron (degradation) tag attached to a protein of interest specifically targets the protein for rapid proteolysis if the cells are grown at high temperature (37°) and in the presence of an overexpressed ubiquitin-associated protein that recognizes the tag.

PEPTIDE APTAMER INHIBITOR

Synthetic proteins that can bind and inhibit protein function.

TRAIL

A member of the tumour necrosis factor superfamily that preferentially induces apoptosis in tumour cells while leaving normal cells intact.

NON-HOMOLOGOUS END-JOINING

(NHEJ). One of two cellular DNA-repair pathways that are involved in the repair of double-strand breaks.

SYNTHETIC LETHAL

Synthetic interactions are identified if mutations in two separate genes produce a different phenotype from either gene alone, and indicate a functional association between the two genes. Two genes have a synthetic lethal relationship if mutants in either gene are viable but the double mutation is lethal.

CALCOFLUOR

A chemical that binds the chitin-rich bud scars that remain on the cell surface after cytokinesis.

SYNTHETIC DOSAGE LETHALITY

This type of genetic interaction is detected when overexpression of a gene is lethal only if another, normally nonlethal, mutation is present.

COULTER PARTICLE COUNTER

An instrument that measures the size of particles on the basis of changes in the electrical voltage as they pass through an orifice.

EPISTATIC TESTS

These can place genes in the same or different pathways and can establish the order of gene function if they are in a single, linear pathway. Gene A is epistatic to gene B if the phenotype that results from mutation of both genes matches the phenotype that results from gene A alone (and does not match that of gene B alone).

MITOTIC INDEX

The percentage of cells in the mitotic phase of the cell cycle.

FLUORESCENCE PLATE READERS

Instruments that read the fluorescence in each well of a multiwell plate.

FLUORESCENCE RESONANCE ENERGY TRANSFER

(FRET). A phenomenon by which the energy from an excited fluorophore is transferred to an acceptor molecule at short (<100 Å) distances, leading to decreased fluorescence of the donor and increased fluorescence of the acceptor. The efficiency of energy transfer depends strongly on the distance between the donor and acceptor molecules.

FLUORESCENCE ACTIVATED CELL SORTERS

(FACS). The separation of cells or chromosomes by their fluorescence and light-scattering properties, which are measured as the particles flow in a liquid stream as they pass through laser beams. The stream is then broken into droplets, and selected droplets are electrically charged and deflected into collection vessels as they pass through an electric field.

LENTIVIRUS

A type of retrovirus that can transduce overexpression and RNAi-inducing constructs to dividing and non-dividing mammalian cells.

CELL MICROARRAYS

An array of gene-perturbing reagents (such as plasmids plus a transfection reagent) that is spotted onto a glass slide. Cells that are are plated onto the slide and that land on a spot are affected by the reagent.

Z-FACTOR

A measurement that takes into account the dynamic range of the assay (how far apart the positive controls are from the negative controls), as well as data variability (how much variation is seen in the measurements of positive and negative controls).

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Carpenter, A., Sabatini, D. Systematic genome-wide screens of gene function. Nat Rev Genet 5, 11–22 (2004). https://doi.org/10.1038/nrg1248

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