GFP-PCNA as an S-phase marker in embryos during the first and subsequent cell cycles.
BACKGROUND
Proliferating cell nuclear antigen (PCNA) is a key component of the DNA replication machinery involved in the process of DNA elongation, recombination, methylation and repair. We have used PCNA fused with green fluorescent protein (GFP-PCNA) as a convenient tool to show the progress of S-phase in single embryos in vivo. Here we make a comparison between Hoechst 33342 and GFP-PCNA as in vivo event markers for DNA synthesis. Hoechst 33342 and DAPI (4,6-diamidino-2-phenylindole) have been used as a simple and rapid method for assessing membrane permeability and staining DNA in mammalian cells. However, it is difficult to use these dyes in living embryos during cell cycle progression studies over long periods of time as they are phototoxic. Moreover, though Hoechst staining reveals nuclear morphology, it gives no information about the progress of S-phase.


RESULTS
We have microinjected or expressed a GFP-PCNA chimera to develop a method which enables visualization of S-phase in sea urchin and Caenorhabditis elegans embryos during the first and subsequent embryonic cell cycles and in Drosophila stage 4 embryos during syncytial nuclear divisions. We find that nuclear accumulation of GFP-PCNA correlates with S-phase onset. Loss of the chimera from the nucleus occurs when the nuclear envelope breaks down at mitosis.


CONCLUSIONS
GFP-PCNA is a accurate and non-toxic marker of S-phase in embryos during early development.
Kisielewska
Jolanta
J
University of Newcastle upon Tyne, Institute of Cell and Molecular Biosciences, Medical School, Framlington Place, Newcastle upon Tyne NE2 4HH, UK.
Lu
Pin
P
Whitaker
Michael
M
D000818:Animals; D015415:Biomarkers / Q000032:analysis; D017173:Caenorhabditis elegans / Q000196:embryology; D004331:Drosophila melanogaster / Q000196:embryology; D004625:Embryo, Nonmammalian / Q000737:chemistry / Q000166:cytology*; D049452:Green Fluorescent Proteins / Q000032:analysis* / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D049408:Luminescent Agents; D018809:Proliferating Cell Nuclear Antigen / Q000032:analysis* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000032:analysis; D016196:S Phase; D012617:Sea Urchins / Q000196:embryology
D015415:Biomarkers; D049408:Luminescent Agents; D018809:Proliferating Cell Nuclear Antigen; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins