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. 2008 Nov;5(11):955-7.
doi: 10.1038/nmeth.1264. Epub 2008 Oct 26.

A noncytotoxic DsRed variant for whole-cell labeling

Rita L Strack  1 , Daniel E Strongin, Dibyendu Bhattacharyya, Wen Tao, Allison Berman, Hal E Broxmeyer, Robert J Keenan, Benjamin S Glick
Affiliations

A noncytotoxic DsRed variant for whole-cell labeling

Rita L Strack et al. Nat Methods. 2008 Nov.

Abstract

A common application of fluorescent proteins is to label whole cells, but many RFPs are cytotoxic when used with standard high-level expression systems. We engineered a rapidly maturing tetrameric fluorescent protein called DsRed-Express2 that has minimal cytotoxicity. DsRed-Express2 exhibits strong and stable expression in bacterial and mammalian cells, and it outperforms other available RFPs with regard to photostability and phototoxicity.

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Figures

Figure 1
Figure 1
Overcoming the cytotoxicity of red fluorescent proteins. (a) Many red fluorescent proteins are cytotoxic. HeLa cells were transiently transfected for constitutive high-level expression of the indicated fluorescent proteins. At daily time points, three wells for each fluorescent protein were analyzed by flow cytometry to measure the average signal from the viable fluorescent cells. The strongest signal obtained for a given fluorescent protein was defined as 100 units. Bars represent s.e.m. For clarity, the results are shown in four separate panels. (b) Eliminating the higher-order aggregation of DsRed-Express. For each indicated fluorescent protein, percent fluorescence in the pellet from a bacterial lysate is plotted as mean and s.e.m. for eight independent replicates. The signal with mEGFP represents background in the assay. (c) The new fluorescent proteins are minimally cytotoxic in bacteria. E. coli cells were transformed with plasmids for constitutive expression of DsRed-Express, DsRed-Express2, or DsRed-Max, and were grown overnight on adjacent sectors of a Petri plate. Equally sized representative areas are shown. Scale bar, 1 mm. (d) The new fluorescent proteins are minimally cytotoxic in mammalian cells. As in (a), HeLa cells were transiently transfected for expression of DsRed-Express2 or DsRed-Max.
Figure 2
Figure 2
Fluorescent protein cytotoxicity after lentiviral transduction. (a) HeLa cells were transduced with lentiviruses encoding the indicated fluorescent proteins, or with a control lentivirus lacking a fluorescent protein gene. At 3 and 10 days after transduction, three wells for each fluorescent protein were analyzed by flow cytometry. Fluorescence from viable cells was detected using a 488-nm laser and a FITC filter set (mEGFP) or a 543-nm laser and a PE filter set (red fluorescent proteins). Plotted are the average fluorescence signals relative to the control lentivirus background, with s.e.m. (b) The data in (a) were used to determine the percentage of viable cells that were fluorescent. (c) In a separate experiment, cells were lentivirally transduced to express the indicated fluorescent proteins. At 3 days after transduction, fluorescent cells were sorted and grown in culture, in parallel with unsorted cells that had been transduced with a control lentivirus lacking a fluorescent protein gene. The number of cells was counted by microscopy at daily intervals. Bars indicate s.e.m.
Figure 3
Figure 3
Robust growth of murine bone marrow hematopoietic stem and progenitor cells expressing DsRed-Express2. (a) Flow cytometry of retrovirally transduced cells. Mononuclear bone marrow cells were transduced with retroviral vectors encoding DsRed-Express, DsRed-Express2, or EGFP, and fluorescent cells were sorted after 87 h. Red and green fluorescence signals were detected using PE and FITC filter sets, respectively. The lines represent gates defined by analyzing untransduced cells. (b) Sorted cells from (a) were cultured under conditions favoring preservation and growth of hematopoietic stem cells, and the cultures were analyzed by flow cytometry after 3, 6, and 10 days. Bars represent mean and s.d. Green and red indicate fluorescent cell number, and yellow indicates non-fluorescent cell number. The decrease in total cell number at day 10 is not fluorescent protein-related, but reflects senescence that is routinely observed under in vitro conditions.
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