In vivo marking of single cells in chick embryos using photoactivation of GFP

Curr Protoc Cell Biol. 2005 Oct:Chapter 12:Unit 12.8. doi: 10.1002/0471143030.cb1208s28.

Abstract

Selective marking of a single cell within a living embryo is often difficult due to the inaccuracy and invasiveness of standard techniques. This unit describes a minimally invasive optical protocol that uses 405-nm laser light to photoactivate a variant of green fluorescent protein (PAGFP). This method takes advantage of the accessibility of the chick embryo to inject PAGFP into a region of interest and uses electroporation to deliver the construct into cells. This unit describes in detail how single and small groups of cells (n<10) that express PAGFP can be made visually distinguishable from the host population using the photoactivation process. Included is a means to maximize the fluorescence increase due to photoactivated GFP signal and to reduce photobleaching. Briefly outlined are previously developed chick culture and time-lapse imaging techniques to allow for the subsequent monitoring of photoactivated cell migratory behaviors. The technique has the potential to be a less-invasive, accurate tool for in vivo studies that involve following cell lineage and cell migration.

MeSH terms

  • Animals
  • Cell Lineage
  • Cells, Cultured
  • Chick Embryo / cytology*
  • Chick Embryo / embryology
  • Chick Embryo / metabolism
  • Cytological Techniques / methods
  • Electroporation
  • Embryology / methods*
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / pharmacokinetics
  • Green Fluorescent Proteins / radiation effects*
  • Lasers
  • Microscopy, Fluorescence / methods
  • Microscopy, Video / methods
  • Photochemistry

Substances

  • Green Fluorescent Proteins