Microinjection techniques for studying mitosis in the Drosophila melanogaster syncytial embryo

Ingrid Brust-Mascher, Jonathan M. Scholey

Research output: Contribution to journalArticle

17 Citations (Scopus)

Abstract

This protocol describes the use of the Drosophila melanogaster syncytial embryo for studying mitosis. Drosophila has useful genetics with a sequenced genome, and it can be easily maintained and manipulated. Many mitotic mutants exist, and transgenic flies expressing functional fluorescently (e.g. GFP) - tagged mitotic proteins have been and are being generated. Targeted gene expression is possible using the GAL4/UAS system. The Drosophila early embryo carries out multiple mitoses very rapidly (cell cycle duration, ≈10 min). It is well suited for imaging mitosis, because during cycles 10-13, nuclei divide rapidly and synchronously without intervening cytokinesis at the surface of the embryo in a single monolayer just underneath the cortex. These rapidly dividing nuclei probably use the same mitotic machinery as other cells, but they are optimized for speed; the checkpoint is generally believed to not be stringent, allowing the study of mitotic proteins whose absence would cause cell cycle arrest in cells with a strong checkpoint. Embryos expressing GFP labeled proteins or microinjected with fluorescently labeled proteins can be easily imaged to follow live dynamics (Fig. 1). In addition, embryos can be microinjected with function-blocking antibodies or inhibitors of specific proteins to study the effect of the loss or perturbation of their function. These reagents can diffuse throughout the embryo, reaching many spindles to produce a gradient of concentration of inhibitor, which in turn results in a gradient of defects comparable to an allelic series of mutants. Ideally, if the target protein is fluorescently labeled, the gradient of inhibition can be directly visualized. It is assumed that the strongest phenotype is comparable to the null phenotype, although it is hard to formally exclude the possibility that the antibodies may have dominant effects in rare instances, so rigorous controls and cautious interpretation must be applied. Further away from the injection site, protein function is only partially lost allowing other functions of the target protein to become evident.

Original languageEnglish (US)
Article numbere1382
JournalJournal of Visualized Experiments
Issue number31
DOIs
StatePublished - Sep 2009

Fingerprint

Microinjections
Drosophila melanogaster
Mitosis
Embryonic Structures
Proteins
Antibodies
Drosophila
Cells
Phenotype
Blocking Antibodies
Cytokinesis
Cell Cycle Checkpoints
Gene expression
Diptera
Machinery
Monolayers
Cell Cycle
Genes
Genome
Gene Expression

Keywords

  • Developmental Biology
  • Drosophila melanogaster syncytial embryo
  • Issue 31
  • Microinjection
  • Mitosis
  • Protein inhibition

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Chemical Engineering(all)
  • Immunology and Microbiology(all)
  • Neuroscience(all)

Cite this

Microinjection techniques for studying mitosis in the Drosophila melanogaster syncytial embryo. / Brust-Mascher, Ingrid; Scholey, Jonathan M.

In: Journal of Visualized Experiments, No. 31, e1382, 09.2009.

Research output: Contribution to journalArticle

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