Stable expression of Anthozoa fluorescent proteins in mammalian cells.
BACKGROUND
Fluorescent proteins have become invaluable reporters in many areas of cellular and developmental biology. An enhanced version of the Aequorea victoria green fluorescent protein (AvEGFP) is the most widely used fluorescent protein. For a variety of reasons, it is useful to have alternative fluorescent proteins to AvEGFP.


METHODS
The cDNA sequences for enhanced variants of the Anemonia cyan fluorescent protein (AmCyan1), as well as the Zoanthus green (ZsGreen1) and yellow (ZsYellow1) fluorescent proteins, were cloned downstream of a constitutive cytomegalovirus (CMV) promoter within a retroviral expression vector. NIH3T3, HEK293, SW620, and WM35 cells were transduced with recombinant retroviruses at a low multiplicity of infection (MOI) to bias for single-copy integration. Both unselected and stably selected cells transduced with the retroviral expression constructs were characterized. Expression of each fluorescent protein in cells was detected using flow cytometry and fluorescence microscopy with filter sets typically used for AvEGFP/fluorescein isothiocyanate (FITC) detection and was compared with the expression of AvEGFP. In addition, a fluorescence plate reader with several excitation and emission filter sets was used for detection.


RESULTS
Expression of each protein was observable by fluorescence microscopy. Under given conditions of flow cytometry, the ZsGreen1 mean fluorescence was approximately 3-fold, 10-fold, and 50-fold greater than that of AvEGFP, ZsYellow1, and AmCyan1, respectively. AmCyan1, ZsGreen1, and AvEGFP were detected by a fluorescence plate reader.


CONCLUSIONS
We determined that fluorescent proteins from Anthozoa species are detectable using a standard flow cytometer and fluorescence microscope. All of the mammalian cell lines tested expressed detectable levels of fluorescent proteins from stable integrated provirus. In cell lines where the AvEGFP protein is toxic or poorly expressed, these Anthozoa fluorescent proteins may serve as alternative fluorescent reporters.
Richards
Burt
B
Deltagen Proteomics, Inc., 320 Wakara Way, Salt Lake City, UT 84108, USA.
Zharkikh
Ludmilla
L
Hsu
Forrest
F
Dunn
Christine
C
Kamb
Alexander
A
Teng
David H-F
DH
D016475:3T3 Cells; D000818:Animals; D037421:Anthozoa / Q000502:physiology*; D005434:Flow Cytometry / Q000379:methods; D005455:Fluorescent Antibody Technique; D005456:Fluorescent Dyes / Q000032:analysis / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006801:Humans; D007202:Indicators and Reagents; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D008545:Melanoma; D051379:Mice; D008856:Microscopy, Fluorescence; D012190:Retroviridae / Q000235:genetics; D014407:Tumor Cells, Cultured
D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins