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  • Review Article
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The art and design of genetic screens: mammalian culture cells

Nature Reviews Genetics volume 5, pages 179–189 (2004)Cite this article

Key Points

  • Various cellular read-outs, such as apoptosis, senescence, tissue-specific differentiation and oncogenic transformation, some of which are especially relevant for human diseases, can accurately be observed in mammalian cell lines and can best be determined in genetic screens.

  • Screens in cell lines take advantage of the Human Genome Project, with its complete annotation of all expressed genes.

  • Many reporter genes and different read-out cell systems have been used to dissect the genetic circuitries of cell culture cells for defined biochemical changes rather than gross morphological alterations.

  • Mainly forward-genetic screens were used in cell culture cells, which rely on the mutagenesis of the organisms (usually through DNA transfection), the recognition of the altered phenotype or biochemical changes and the determination of the causative DNA element.

  • Reverse-genetic screens in cell lines have also been performed, which usually comprise the random mutagenesis of known genes and their investigation in vitro.

  • Even more sophisticated genetic approaches, such as synthetic lethal screens and complementation screens, have been completed in cell lines.

  • Examples of successful screening in cell culture include the isolation of such diverse genes as oncogenes, secreted growth factors, membrane-bound receptors, transporters, transcription activators and various signal-transducing molecules.

  • The set-up of the functional assay is the pivotal step for the success of genetic screens in mammalian cells. It must be tailored to the targeted function and requires rigorous testing of the experimental setting, such as the signal background and the verification of positive controls.

  • Genome-wide genetic screens in cell lines require the massive parallel investigation of single genes, which can be accomplished by either miniaturization or automation.

  • Promising developments in the screening of cell culture cells also include high-content screening, which, rather than focusing on single biochemical changes in the cell, uses a range of powerful optical read-outs for multiple cellular targets in parallel.

Abstract

Given the wealth of sequence information from the Human Genome Project, many open reading frames urgently require assignment of function. Whereas genetic model organisms, such as yeast, Drosophila and Caenorhabditis elegans, have successfully been used in genetic screens for a long time, mammalian culture cells have only recently emerged as a suitable screening system to elucidate gene function. Diverse cellular activities, such as apoptosis, senescence, tissue-specific differentiation and oncogenic transformation, can be studied in cell culture. There is a plethora of functional assays that can provide a link between genes and physiology. The number of genetic elements to be tested necessitates the use of miniaturization strategies or robotic instrumentation for effective screens that use mammalian cell lines.

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Figure 1: Possible fates of mammalian cells in vitro.
Figure 2: Examples of reporter plasmids.
Figure 3: Two alternative approches to genome-wide screens in mammalian cells.

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Acknowledgements

I thank H. Hermeking and A. Gewies for comments on the manuscript. The work in my laboratory was supported by Roche, the Deutsche Forschungsgemeinschaft and Bayrisches Staatsministerium. I apologize to all colleagues whose work was not cited owing to space limitations.

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  1. Max-Planck-Institute for Biochemistry, Am Klopferspitz 18a, 82152 Martinsried, Germany, and Imperial College London, Hammersmith Campus, Du Cane Road, London, W12 0NN, UK

    Stefan Grimm

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DATABASES

LocusLink

cybL/SDHC

fusin

KAI1

NET1

p21

XRCC1

OMIM

CD4

HIV

Wilms' tumour

FURTHER INFORMATION

Encyclopedia of Life Sciences

cell culture: basic procedures

genetic engineering: reporter plasmids

Human Genome Resources

Imperial College

Glossary

SENESCENCE

State in which normal cells irreversibly stop dividing.

PRIMARY CELLS

Culture cells with limited lifespan that are taken directly from an organism.

SOMATIC-CELL GENETICS

Area of genetics that deals with diploid, somatic cells and not germ-line cells.

EPISOMAL

In the context of transient transfection, it refers to a plasmid that is extra chromosomal.

FLUORESCENCE ACTIVATED CELL SORTING

(FACS). A method whereby dissociated and individual living cells are sorted, in a liquid stream, according to the intensity of fluorescence that they emit as they pass through a laser beam.

SPLICE DONOR SITES

DNA sequences that mediate splicing at the 5′ end of an intron.

NORMALIZED LIBRARY

Ensemble of genes with equal numerical representation.

SPLICING ACCEPTOR SITES

DNA elements that mediate splicing at the 3′ end of an intron.

RIBOZYMES

RNAs with catalytic activity.

COMPLEMENTATION

Restoration of the wild type by non-allelic genes that act in trans.

MICROCELLS

Vesicles formed after treatment of cells with colchemid and cytochalasin b. They contain large DNA fragments or single chromosomes that are surrounded by a nuclear and a plasma membrane.

POLYETHYLENE GLYCOL

Highly hydrated polymer that can bring cell membranes into near molecular contact by making the water between them thermodynamically unfavourable.

NEPHROBLASTOMA

Tumour of the kidney.

METASTASIS SUPPRESSOR GENES

Genes that, when inactivated, lead to metastasis formation rather than conferring a growth advantage (as with tumour-suppressor genes).

LIPOFECTION

A technique for transfecting cells that uses lipids or lipid-related carrier molecules.

FLUORESCENT RESONANCE ENERGY TRANSFER EFFECT

(FRET effect). A technique that is used to detect the closeness of fluorescent molecules.

MAMMALIAN TWO-HYBRID ASSAY

A technique that is used to detect protein–protein interactions in mammalian cells; it corresponds to the yeast two-hybrid assay, except that mammalian expression vectors are used. An intact transcription complex is generated if two fusion proteins interact: one that contains a DNA-binding domain and another that contains a transactivation domain.

COLONY-FORMATION ACTIVITY

Activity to sustain the growth of mammalian cells, as measured by the number of surviving cell colonies in vitro.

CONTACT INHIBITION

Inhibition of cell division following membrane contact of neighbouring cells, characteristic of untransformed cells.

FOCUS FORMATION

Generation of groups of transformed cells with distinctive morphology in cell cultures.

SUBTRACTION HYBRIDIZATION

A technique that is used to specifically enrich the DNA species that are present in one sample by hybridizing with nucleic acids of another sample and removing the associated double-stranded molecules.

DNA SHUFFLING

An in vitro technique that uses the artificial recombination between genes to accelerate the mutation rate to select for pre-defined qualities.

MONOPOLAR SPINDLE

Aberrant mitotic spindle arrangement that shows a monoastral microtubule array (instead of the normal bipolar spindle).

HIRTH EXTRACTION

Gentle lysis of cells that releases extrachromosomal plasmids in the supernatant.

PACKAGING CELLS

Cell lines that express one or more viral proteins and, in combination with additionally transfected components, release in trans reconstituted infectious particles.

INTRACELLULAR SECOND MESSENGER

Signalling molecule in cells the concentration of which changes on the binding of an extracellular ligand to a plasma membrane-bound receptor.

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Grimm, S. The art and design of genetic screens: mammalian culture cells. Nat Rev Genet 5, 179–189 (2004). https://doi.org/10.1038/nrg1291

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