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TILLING — a high-throughput harvest for functional genomics

Nature Reviews Genetics volume 5, pages 145–150 (2004)Cite this article

Abstract

The availability of the whole genome sequence of many model organisms, combined with well-established chemical mutagenesis methods and cost-effective high-throughput DNA genotyping, allows mutations to be identified for virtually any gene. Recently dubbed TILLING (for targeting induced local lesions in genomes), this general method is gaining popularity. In this article, I discuss some of the TILLING methods that are available, the successes that have been reported for several organisms and the future outlook for such methods.

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Figure 1: The TILLING process.
Figure 2: The Cel1 endonuclease.
Figure 3: CODDLE analysis of the mouse Pax6 gene.
Figure 4: Generation of zebrafish germline chimaeras.

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Acknowledgements

I would like to thank M. Clark, R. Kettleborough, G. Wright and H.-Y. Hwang for their helpful comments on this manuscript, and acknowledge support from the Wellcome Trust.

Author information

Authors and Affiliations

  1. Vertebrate Development and Genetics (Team 31), Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1SA, UK

    Derek L. Stemple

Authors
  1. Derek L. Stemple
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The author declares no competing financial interests.

Related links

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DATABASES

Entrez

ADE2

ADH1

LocusLink

rag1

FURTHER INFORMATION

Arabidopsis TILLING project

CODDLE

dCAPs

PARSESNP

proWeb project

Wellcome Trust Sanger Institute Vertebrate Development and Genetics team

Glossary

ALLELIC SERIES

A series of different genotypes, or alleles, of a specific gene, that are often associated with different phenotypes.

BLASTOMERES

Cells of the early cleavage-stage embryo.

DENATURING HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

(dHPLC). A rapid chromatography method that can be used to distinguish heteroduplex from homoduplex DNA. It can detect single base differences between alleles.

HEDGEHOG

The Hedgehog proteins are a class of secreted cell–cell signalling molecule. The name derives from the appearance of embryonic Drosophila melanogaster mutants that lack hedgehog gene function. There are many vertebrate genes that encode Hedgehog homologues.

M2

Second generation mating of a specific mutagenized individual.

MATERNAL CONTRIBUTION

In many organisms, mRNA and proteins are stored in the egg before fertilization.

microRNAs

Short (22 bp), non-coding RNAs that are probably involved in gene regulation.

MORPHOLINO OLIGONUCLEOTIDE

A DNA analogue in which the bases are linked to a six-membered morpholine ring.

P-ELEMENTS

Transposable elements that are widely used for mutating and manipulating the Drosophila genome.

PHENOCOPY

The production of a phenotype, which closely resembles a phenotype that normally results from a specific gene mutation.

PHOSPHONOPEPTIDE NUCLEIC ACID

As with morpholino oligonucleotides, these have a modified backbone that is resistant to nuclease digestion, yet allows hybridization with complementary DNA or RNA molecules and can be used to interfere with protein synthesis of specific target mRNAs.

PRIMORDIAL GERM CELLS

Germline cells at all stages of development from the time when this lineage is formed until they arrive at the gonad and start differentiating into gametes.

SPERMATOGONIA

The mitotically dividing stem cells of the male germline, the descendants of which ultimately become mature sperm.

TRANSVERSION

A point mutation in which a purine base is substituted for a pyrimidine base and vice versa; for example, an A:T → C:G transversion.

WNT

The Wnt proteins are a class of secreted cell–cell signalling molecule. The name derives from a fusion of two original names. In Drosophila melanogaster, Wingless (like Hedgehog) is involved in patterning the early embryo. In vertebrates, Int-1 was the first member of this class of protein to be discovered, and was identified in a screen for viral cancer-causing insertions in mice.

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Stemple, D. TILLING — a high-throughput harvest for functional genomics. Nat Rev Genet 5, 145–150 (2004). https://doi.org/10.1038/nrg1273

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