information in this way, which I strongly advocate, only requires
prior registration at the website.
The general principles underlying the successful expression and
detection of FPs within host cells were first established using GFP.
All involved modifications to the wild-type coding sequence and
the overwhelming majority of these experiments were first done
using bacteria and mammalian cells. These included: (1) optimization of FP light absorption to available light wavelengths (for GFP,
the single S65T mutation shifts the peak of the GFP absorption
spectrum to 488 nm, which coincides with the historically default
(argon ion) source of laser illumination for flow cytometers);
(2) altering the spectral properties of the FP to provide increased
and prolonged brightness under various redox and pH conditions;
(3) improving folding rates and maturation of fluorescence;
(4) decreasing FP aggregation; and (5) increasing total levels of
protein production, both through altering the codon usage within
the FP coding sequence to correspond to that of the recipient
organism, and using a translational initiation consensus appropriate
for the host. Specific to arabidopsis was the identification of a
cryptic intron within the wild-type GFP sequence, which is recognized and efficiently spliced. Engineered alterations to the cryptic
splice borders eliminates this problem [37], as does changing the
codon usage to that of plants or mammals.
Since 1994, there has been a dramatic and rapid identification
of different source FPs, and of sequence variants produced from
these FPs optimized for expression in mammalian cells. For work in
plants, the simplest strategy is to test in plants whether these variant
FP sequences are adequately expressed and detected [1]. Geldner
et al. [38], for example, described the transgenic expression in
Arabidopsis of various translational fusions of EYFP, mCherry,
mCerulean, and mTFP1 (a variant of the brown star polyp (Clavularia sp.) Cyan Fluorescent Protein cFP484), combining them
pair-wise. More recently, Marque `s-Bueno et al. [35] have described
the construction of a series of vectors and transgenic plants (the
SWELLines collection) in which markers are expressed in specific
root cell types. The collection includes two series expressing FPs:
the SAND lines, in which the cytoplasm is labeled with a 4xYFP
protein, and the RED TIDE lines, in which the nucleus is labeled
using a H2B-2xCherry chimeric protein, both in a cell type-specific
manner. Very recently, Machin et al. [36] described an updated
system, allowing the quantitative induction of expression of eGFP,
Venus, mKO2, and mCherry, that is spatially and temporally
regulated.
With some caveats, transgenic FP expression in vivo appears to
be nontoxic at levels that are useful for further scientific studies. FPs
can be targeted to essentially all subcellular locations, via translational fusions to topogenic motifs or entire proteins [38–42] and
references therein. Problems of toxicity involve (1) the adverse
Flow Cytometry and Sorting in Arabidopsis
259
prior registration at the website.
The general principles underlying the successful expression and
detection of FPs within host cells were first established using GFP.
All involved modifications to the wild-type coding sequence and
the overwhelming majority of these experiments were first done
using bacteria and mammalian cells. These included: (1) optimization of FP light absorption to available light wavelengths (for GFP,
the single S65T mutation shifts the peak of the GFP absorption
spectrum to 488 nm, which coincides with the historically default
(argon ion) source of laser illumination for flow cytometers);
(2) altering the spectral properties of the FP to provide increased
and prolonged brightness under various redox and pH conditions;
(3) improving folding rates and maturation of fluorescence;
(4) decreasing FP aggregation; and (5) increasing total levels of
protein production, both through altering the codon usage within
the FP coding sequence to correspond to that of the recipient
organism, and using a translational initiation consensus appropriate
for the host. Specific to arabidopsis was the identification of a
cryptic intron within the wild-type GFP sequence, which is recognized and efficiently spliced. Engineered alterations to the cryptic
splice borders eliminates this problem [37], as does changing the
codon usage to that of plants or mammals.
Since 1994, there has been a dramatic and rapid identification
of different source FPs, and of sequence variants produced from
these FPs optimized for expression in mammalian cells. For work in
plants, the simplest strategy is to test in plants whether these variant
FP sequences are adequately expressed and detected [1]. Geldner
et al. [38], for example, described the transgenic expression in
Arabidopsis of various translational fusions of EYFP, mCherry,
mCerulean, and mTFP1 (a variant of the brown star polyp (Clavularia sp.) Cyan Fluorescent Protein cFP484), combining them
pair-wise. More recently, Marque `s-Bueno et al. [35] have described
the construction of a series of vectors and transgenic plants (the
SWELLines collection) in which markers are expressed in specific
root cell types. The collection includes two series expressing FPs:
the SAND lines, in which the cytoplasm is labeled with a 4xYFP
protein, and the RED TIDE lines, in which the nucleus is labeled
using a H2B-2xCherry chimeric protein, both in a cell type-specific
manner. Very recently, Machin et al. [36] described an updated
system, allowing the quantitative induction of expression of eGFP,
Venus, mKO2, and mCherry, that is spatially and temporally
regulated.
With some caveats, transgenic FP expression in vivo appears to
be nontoxic at levels that are useful for further scientific studies. FPs
can be targeted to essentially all subcellular locations, via translational fusions to topogenic motifs or entire proteins [38–42] and
references therein. Problems of toxicity involve (1) the adverse
Flow Cytometry and Sorting in Arabidopsis
259