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  • Review Article
  • Published:

Emerging technologies for gene manipulation in Drosophila melanogaster

Nature Reviews Genetics volume 6, pages 167–178 (2005)Cite this article

An Erratum to this article was published on 01 April 2005

Key Points

  • Many genetic tools have been developed over the past 100 years to manipulate the genome of the fruitfly Drosophila melanogaster; despite the success of most of these methods, some limitations need to be overcome. The past 3 years have seen the introduction of several new technologies that allow flies to be manipulated more easily than any other multicellular organism.

  • The new methods include the ability to create molecularly designed deletions, improved genetic-mapping technologies, strategies for creating targeted mutations, new transgenic approaches and the ability to manipulate large fragments of DNA.

  • Two transposable elements, P-elements and piggyBacs, each having different properties and mobilization characteristics, are being used to generate insertions in every gene of the D. melanogaster genome.

  • Two P-elements, located at different positions in cis or in trans on the same homologous pair of chromosomes, can be used to generate transposon-induced deficiencies.

  • A P-element that contains an internal hobo transposable element allows nested deletions to be generated from a common starting point ('deletion-generator' technology).

  • Two transposons, each of which contains an FRT site, are used to create molecularly defined deletions through Flp-mediated excision of the intervening sequence.

  • SNPs and molecularly defined P-element insertions improve and accelerate gene-mapping efforts.

  • 'Ends-out replacement gene targeting' removes a desired genomic sequence through homologous recombination.

  • Target-induced local lesions in genomes (TILLING), a technique that was first applied in Arabidopsis thaliana, quickly and efficiently identifies single-nucleotide changes in specific genes and might allow the isolation of an allelic series for structure–function analysis.

  • The bacteriophage φC31 efficiently integrates DNA at defined loci in the fly genome.

  • Recombineering facilitates gap-repair-mediated subcloning and the subsequent mutagenesis of large DNA fragments.

Abstract

The popularity of Drosophila melanogaster as a model for understanding eukaryotic biology over the past 100 years has been accompanied by the development of numerous tools for manipulating the fruitfly genome. Here we review some recent technologies that will allow Drosophila melanogaster to be manipulated more easily than any other multicellular organism. These developments include the ability to create molecularly designed deletions, improved genetic mapping technologies, strategies for creating targeted mutations, new transgenic approaches and the means to clone and modify large fragments of DNA.

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Figure 1: Transposon-mediated gene-disruption screens.
Figure 2: Using transposons to create chromosomal aberrations.
Figure 3: Transposon-mediated mapping.
Figure 4: Homologous recombination.
Figure 5: The application of TILLING in Drosophila melanogaster.
Figure 6: Recombineering.

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Acknowledgements

We thank K. Cook, L. Cooley, K. Golic, M. González-Gaitán, S. Henikoff, P. Hiesinger, K. Matthews, N. Perrimon, J. Roote, S. Russell, E. Spana, A. Spradling and B. Till for communicating unpublished results. We are particularly grateful to E. Seto and P. Verstreken for critical readings of the manuscript and K. Cook for suggestions. We apologize to colleagues whose work could not be cited owing to space limitations. H.J.B. is supported by the Howard Hughes Medical Institute, the US National Institutes of Health and NASA. We also gratefully acknowledge the constructive comments of two anonymous reviewers.

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  1. Program in Developmental Biology, Baylor College of Medicine, Houston, 77030, Texas, USA

    Koen J. T. Venken & Hugo J. Bellen

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  1. Koen J. T. Venken
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Correspondence to Hugo J. Bellen.

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FURTHER INFORMATION

BACPAC Resource Center

Bloomington Drosophila Stock Center

DrosDel Drosophila Isogenic Deficiency Kit

Drosophila GeneTag database

Drosophila Genome Resource Center

Drosophila TILLING Project

Flybase Genome Browser

Flytrap — GFP Protein Trap database

FlyView — expression pattern of enhancer traps

Hugo Bellen's laboratory

Model Systems Genomics

NCI Frederick — Recombineering web site

P-screen database

Szeged Drosophila Stock Center

Glossary

P-ELEMENT

Transposable elements that are widely used to mutate and manipulate the genome of Drosophila melanogaster.

TRANSGENESIS

The process of introducing foreign DNA into a genome.

TRANSPOSON

A DNA element that can be mobilized within the genome; Drosophila melanogaster transposons are used for various applications, including insertional mutagenesis, gene tagging and as a carrier for transgenic DNA.

DEFICIENCY

A chromosomal aberration that is characterized by the deletion of a part of the genome.

IMPRECISE EXCISION

A P-element that is mobilized imprecisely can lead to removal of flanking genomic sequences, resulting in a local deletion.

RNA INTERFERENCE

(RNAi). A form of gene silencing in which dsRNA induces the degradation of the homologous endogenous mRNA transcripts, thereby mimicking the effect of the reduction, or loss, of gene activity.

ALLELIC SERIES

A series of alleles at the same genomic locus that can produce graded phenotypes and therefore can help to unravel different functions of the same gene.

TEMPERATURE-SENSITIVE MUTATIONS

Mutations that show a mutant phenotype at the restrictive temperature, but not at the permissive temperature. Most temperature-sensitive mutations in Drosophila melanogaster are heat-sensitive, but cold-sensitive mutations also occur.

BACTERIOPHAGE

A virus that infects and replicates in bacteria. Lytic bacteriophages kill the host cell, whereas so-called temperate phages can establish a stable relationship in which the bacteriophage genome is stably maintained within that of the host.

GENE TRAP

A DNA construct that contains a reporter-gene sequence downstream of a splice-acceptor site and that can integrate into random chromosomal locations. A gene trap sometimes also contains a splice donor site that is downstream of the marker. Integration of the gene trap into an intron allows the expression of a new mRNA that contains one or more upstream exons followed by the reporter gene.

INTEIN

'Protein introns' that autonomously splice themselves out of proteins (post-translationally), thereby generating a functional protein.

JUMP-STARTER ELEMENT

A transposable element that is inserted into the genome and that is used to create more insertions at other sites.

COMPARATIVE GENOMIC HYBRIDIZATION MICROARRAY

A high-density microarray that contains overlapping DNA clones of genomic sequences or overlapping oligonucleotides that encompass a particular genomic region. This allows chromosomal imbalances to be identified at a high resolution, and can even be used to determine exact chromosomal breakpoints.

HAPLOINSUFFICIENCY

The requirement for a diploid organism to have both functional copies of a gene or locus to produce a wild-type phenotype.

YELLOW+ (y+)

The wild-type allele of a fly gene that confers dark body colour; by contrast, yellow− flies have a yellowish body colour. One copy of yellow+ is often used as a dominant genetic marker in fly trangenesis.

WHITE+ (w+)

The wild-type allele of a fly gene that confers red eyes; white− flies have white eyes. One copy of white+ is often used as a dominant genetic marker in fly trangenesis.

INVERSE PCR

A method for cloning DNA that flanks a known sequence. Genomic DNA is digested and ligated into circles, and is then subjected to PCR. The PCR primers are designed to be complementary to the known sequence, but point outwards from this sequence, allowing the unknown flanking DNA to be amplified.

MEIOTIC RECOMBINATION MAPPING

Uses the recombination rate between a wild-type chromosome and a chromosome that is marked with recessive markers to obtain a recombination distance, or map unit, between two genes, mutations or markers.

MALE RECOMBINATION MAPPING

Meiotic recombination does not occur in Drosophila melanogaster males, so P-element-mediated recombination between homologous chromosomes can be used to determine the position of the mutation relative to the position of P-element insertions.

DEFICIENCY MAPPING

Uses chromosomes that have different sections deleted to locate the position of a gene of interest. Without the deficiency, the normal functional gene usually masks the effect of (that is, complements) the defective or foreign copy that we wish to identify.

GENOTYPING

Comparative methodologies are used to obtain information about the sequence of a certain genomic region between different strains of the same species or between wild-type and mutagenized strains.

BALANCER CHROMOSOMES

Chromosomes that carry lethal mutations, dominant markers and multiple inversions and that are used in trans for a chromosome that carries a lethal or sterile mutation. They are used as genetic tools because they allow lethal mutations to be propagated indefinitely.

RARE-CUTTING RESTRICTION ENZYME

Restriction enzymes that cleave a 16–18 bp recognition site. Most of these sites are not present in the host genome and can therefore be used for genome manipulations.

ZINC FINGER

A protein domain in which cysteine or cysteine–histidine residues coordinate a zinc ion. Zinc fingers are often used in DNA recognition and also in protein–protein interactions.

ISOGENIZED FLY STOCK

A starting strain of animals used for mutagenesis that contains identical pairs of chromosomes. Isogenization avoids the presence of pre-existing lethal and visible mutations.

TRANSITION

A mutation between two pyrimidines (T–C) or two purines (A–G).

ENHANCER DETECTION

A method that allows the identification of genes on the basis of their expression pattern. Engineered insertion elements carry a reporter-gene construct that is under the control of a minimal promoter that can respond to cis-acting regulatory elements near the insertion site.

PHENOTYPIC RESCUE

The ability of a DNA construct that contains the wild-type DNA sequence to rescue the phenotype of an identified genomic mutation.

POSITION EFFECT

The effect of the local chromosomal environment on the levels or patterns of transgene expression, possibly owing to local chromatin configuration or nearby cis-acting regulatory elements.

BACTERIAL ARTIFICIAL CHROMOSOME

(BAC). A single-copy cloning vector that is derived from the F-factor of Escherichia coli. BACs can contain large genomic fragments. Drosophila melanogaster BACs carry an average insert size of 163 kb. Mapping positions of D. melanogaster BACs can be seen on the Flybase Genome Browser (see Online links box).

P1 ARTIFICIAL CHROMOSOME

(PAC). A single-copy cloning vector that is derived from the F-factor of Escherichia coli. PACs can contain large genomic fragments. Drosophila melanogaster PACs carry an average insert size of 80 kb. The library represents a ∼6-fold coverage of the genome.

SATURATION

The stage in mutagenesis at which mutations in new genes cannot be obtained by further mutagenesis. This occurs when at least one mutation in every gene has been obtained.

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Venken, K., Bellen, H. Emerging technologies for gene manipulation in Drosophila melanogaster. Nat Rev Genet 6, 167–178 (2005). https://doi.org/10.1038/nrg1553

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