Sensitive detection of GFP utilizing tyramide signal amplification to overcome gene silencing.
The green fluorescent protein (GFP) is among the most commonly used expression markers in biology. GFP-tagged cells have played a particularly important role in studies of cell lineage. Sensitive detection of GFP is crucially important for such studies to be successful, and problems with detection may account for discrepancies in the literature regarding the possible fate choices of stem cells. Here we describe a very sensitive technique for visualization of GFP. Using it we can detect about 90% of cells of donor origin while we could only see about 50% of these cells when we employ the methods that are in general use in other laboratories. In addition, we provide evidence that some cells permanently silence GFP expression. In the case of the progeny of bone marrow stem cells, it appears that the more distantly related they are to their precursors, the more likely it is that they will turn off the lineage marker.
Toth
Zsuzsanna E
ZE
CSDB, NIH, NIDCR, Building 49, Room 5A-76, 49 Convent Drive, Bethesda, MD 20892, USA. ztoth@mail.nih.gov
Shahar
Tal
T
Leker
Ronen
R
Szalayova
Ildiko
I
Bratincsák
András
A
Key
Sharon
S
Lonyai
Anna
A
Németh
Krisztián
K
Mezey
Eva
E
D000818:Animals; D000906:Antibodies / Q000276:immunology; D002454:Cell Differentiation / Q000235:genetics; D019070:Cell Lineage / Q000235:genetics; D002469:Cell Separation / Q000379:methods; D005260:Female; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives* / Q000737:chemistry; D005455:Fluorescent Antibody Technique / Q000379:methods*; D005786:Gene Expression Regulation / Q000235:genetics; D020868:Gene Silencing / Q000502:physiology*; D017930:Genes, Reporter / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000378:metabolism*; D020244:Infarction, Middle Cerebral Artery / Q000235:genetics / Q000378:metabolism / Q000503:physiopathology; D051379:Mice; D008810:Mice, Inbred C57BL; D013194:Staining and Labeling / Q000379:methods*; D018183:Transplantation Chimera / Q000235:genetics; D014439:Tyramine / Q000031:analogs & derivatives* / Q000737:chemistry; D014998:Y Chromosome / Q000235:genetics
D000906:Antibodies; C473365:FITC-tyramide; D049452:Green Fluorescent Proteins; D016650:Fluorescein-5-isothiocyanate; D014439:Tyramine