In vivo marking of single cells in chick embryos using photoactivation of GFP.
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.
Stark
D A
DA
Stowers Institute for Medical Research, Kansas City, Missouri, USA.
Kulesa
P M
PM
D000818:Animals; D019070:Cell Lineage; D002478:Cells, Cultured; D002642:Chick Embryo / Q000166:cytology* / Q000196:embryology / Q000378:metabolism; D003584:Cytological Techniques / Q000379:methods; D018274:Electroporation; D004626:Embryology / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000493:pharmacokinetics / Q000528:radiation effects*; D007834:Lasers; D008856:Microscopy, Fluorescence / Q000379:methods; D018715:Microscopy, Video / Q000379:methods; D010777:Photochemistry
D049452:Green Fluorescent Proteins