Expression of reef coral fluorescent proteins in the central nervous system of transgenic mice

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Mol. Cell. Neurosci. 30 (2005) 291 – 303

Expression of reef coral fluorescent proteins in the central nervous

system of transgenic mice

Petra G. Hirrlinger,a,e,1 Anja Scheller,a,e,1 Christian Braun,a Markus Quintela-Schneider,b

Babette Fuss,c Johannes Hirrlinger,a,d and Frank Kirchhoff a,d,*

aDepartment of Neurogenetics, Max Planck Institute of Experimental Medicine, Gottingen, GermanybDepartment of Molecular Neurobiology, Max Planck Institute of Experimental Medicine, Gottingen, GermanycDepartment of Anatomy and Neurobiology, Virginia Commonwealth University, Richmond, VA 23284, USAdDFG Research Center for Molecular Physiology of the Brain (CMPB), Gottingen, GermanyeFreie Universitat Berlin, Section of Biology, Chemistry and Pharmacy, Berlin, Germany

Received 25 April 2005; revised 2 August 2005; accepted 22 August 2005

Reef coral fluorescent proteins (RCFPs) are bright fluorescent proteins

(FPs) covering a wide spectral range. We used various RCFP genes to

transgenically color different cell populations in the brain. The mouse

Thy1.2 promoter was used to target expression of HcRed1 in neurons,

the human glial fibrillary acidic protein (GFAP) promoter to label

astrocytes with AmCyan1, AsRed2 and mRFP1 as well as the mouse

proteolipid protein promoter to mark oligodendrocytes with DsRed1.

In brain sections of transgenic mice, RCFP expression was found to be

highly specific using immunohistochemistry and fluorescence micro-

scopy. In contrast to transgenic mice with expression of jellyfish FP

variants, RCFPs formed numerous fluorescent precipitates. These

aggregates were primarily found in cell somata and also in cell

processes. Older mice were more affected than younger ones. Despite

these fluorescent deposits, physiological properties of RCFP expressing

brain cells such as whole-cell membrane currents or glutamate-evoked

calcium signaling seemed to be unaffected.

While brightness and spectral variation of RCFPs are optimal for

expression in transgenic animals used in physiological experiments, the

formation of fluorescent precipitates in various cell types limits their

use for morphological cell analysis in situ.

D 2005 Elsevier Inc. All rights reserved.

Introduction

The jellyfish Aequorea victoria is the source for the most

commonly used reporter gene, the green fluorescent protein

(AvGFP). While the original AvGFP was rather dim and bleached

1044-7431/$ - see front matter D 2005 Elsevier Inc. All rights reserved.

doi:10.1016/j.mcn.2005.08.011

* Corresponding author. Department of Neurogenetics, Max Planck

Institute of Experimental Medicine, Hermann-Rein-Str. 3, 37075 Gottingen,

Germany. Fax: +49 551 3899 758.

E-mail address: [email protected] (F. Kirchhoff).1 Both authors contributed equally.

Available online on ScienceDirect (www.sciencedirect.com).

fast upon excitation, site-directed mutagenesis generated a series of

bright and spectrally distinct variants (Heim and Tsien, 1996; Heim

et al., 1994, 1995; Zhang et al., 1996).

In particular, transgenic mice with FP expression have been

used to study development and cell–cell interactions in the

nervous system. The TgN(hGFAP-S65T-GFP) was the first mouse

line which allowed the selective analysis of a defined population of

brain cells, the astrocytes, which are the major glial cell population

(Zhuo et al., 1997). In this line, the human glial fibrillary acidic

protein (hGFAP) promoter targets FP expression. S65T-GFP was

also used to label oligodendrocytes, the myelin-forming glial cells

(Fuss et al., 2000). Feng et al. (2000) took advantage of the Thy1.2

promoter to drive the expression of several FPs in neurons. Their

study also showed that different neuronal populations could be

studied simultaneously in double-transgenic mice (Feng et al.,

2000). The most common FP, enhanced GFP (EGFP), has also

been used to label astrocytes (Nolte et al., 2001), oligodendrocytes

(Mallon et al., 2002), and microglia (Jung et al., 2000). In the latter,

EGFP is targeted by homologous recombination to the CX3CR1

locus. The prevalent use of EGFP to generate various transgenic

mouse lines, combined with the strong spectral overlap of EGFP

with EYFP, impeded the analysis of structural interactions among

different cell types in transgenic mice.

In addition to these FPs from jellyfish, several new chromo-

proteins (RCFPs) were isolated from various reef coral Anthozoa

species (Fradkov et al., 2000; Gurskaya et al., 2001; Lukyanov et

al., 2000; Matz et al., 1999). RCFPs are characterized by their

bright fluorescence and spectral variation ranging from cyan to far

red. We used AmCyan1 (Anemonia majano, exc. 458 nm, em. 489

nm), DsRed1 (Discosoma sp., exc. 558, em. 583), AsRed2

(Anemonia sulcata, exc. 576 nm, em. 592 nm), and HcRed1

(Heteroactis crispa, exc. 588 nm, em. 618 nm) to generate

transgenic mouse lines with cell-type-specific, cytosolic expression

of RCFPs to study their practical use in a living organism. In

particular, we were interested in the question on how valuable

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P.G. Hirrlinger et al. / Mol. Cell. Neurosci. 30 (2005) 291–303292

RCFPS are for physiological experiments or whether their long-

term expression might impair mouse development and brain

differentiation. In addition, we asked whether or not cell labeling

by RCFPs can be used to visualize and analyze structural

interactions. We used the well-characterized promoters of the

human GFAP, mouse PLP, and mouse Thy1.2 genes to target

expression in astrocytes, oligodendrocytes, and neurons, respec-

tively (Brenner et al., 1994; Caroni, 1997; Wight et al., 1993). In

addition, we used the modified variant of DsRed, monomeric RFP1

(exc. 584 nm and em. 607 nm; kindly provided by Roger Y. Tsien

(Campbell et al., 2002)) for expression in astrocytes to investigate

whether or not the oligomerization of RCFPs interferes with the

labeling of thin processes.

Results

Focusing on the central nervous system, we used the RCFPs

HcRed1, DsRed1, AmCyan1, AsRed2, and mRFP1 to selec-

tively label neurons, oligodendrocytes, and astrocytes. Trans-

Fig. 1. Cell-type-specific expression of fluorescent proteins in the brain of transgen

red (HcRed1, DsRed1, AsRed2, mRFP1) and cyan (AmCyan1) fluorescent protein

glial fibrillary acidic (hGFAP) promoter. (B) The Thy1.2-HcRed1 minigene i

fluorescence in pyramidal cell layers II and V of the cortex is shown. (C) Prot

oligodendrocytes. In the cerebellum, many red fluorescent oligodendrocytes outli

expression of AmCyan1, AsRed2, and mRFP1 in the major astroglial cell popu

expression. While AmCyan1 and mRFP1 were evenly distributed throughout ce

precipitates in the Bergmann glia somata. Panels (B–F) are confocal laser-scan

perfusion-fixed adult transgenic mice. ml, molecular layer; pc, Purkinje cell layer

genic mouse lines with functional fluorescent protein expression

could be obtained from all the constructs tested. In all lines,

RCFPs were expressed in a cell-type-specific manner (Fig. 1).

In addition to the characteristic cell morphology highlighted by

fluorescence microscopy, the analysis of the various transgenic

mouse lines by immunohistochemistry using the cell-type

markers glial fibrillary acidic protein (GFAP), myelin-associated

glycoprotein (MAG), and neuronal nuclei antigen (NeuN) for

astrocytes, oligodendrocytes, and neurons, respectively, substan-

tiated the cell-type-specific RCFP expression (Fig. 2). Similar to

transgenic mice with variants of GFP, RCFP transgenic mice

lived more than a year, were fertile, and showed no obvious

signs of developmental or behavioral abnormalities.

Transgenic mice with neuronal HcRed1 expression

(TgN(Thy1.2-HcRed1))

Four different transgenic mouse lines with functional HcRed1

expression were obtained with a distinct set of fluorescently

tagged neuronal populations (Fig. 3). First expression was

ic mice. (A) DNA constructs for oocyte injection were generated by placing

genes into either Thy1.2 and PLP minigenes or downstream from the human

nduced highly selective expression in projection neurons. Here, the red

eolipid protein (PLP)-DsRed1 minigene driven FP expression highlighted

ne the white matter tracts. (D–F) The hGFAP promoter induced high level

lation of the cerebellum, the Bergmann glia. Note the various degree of

ll bodies and processes (D, F), AsRed2 was expressed only as fluorescent

ning recordings obtained from frontal (B) and sagittal (C–F) sections of

; gl, granular layer; wm white matter.

Fig. 2. Immunohistochemical analysis confirmed cell-type-specific RCFP expression in transgenic mice. Cell-type-specific expression of RCFPs was further

substantiated by immunostaining against the cell markers GFAP, myelin-associated glycoprotein, and NeuN (neuronal nuclei antigen) for astrocytes,

oligodendrocytes, and neurons, respectively. (A–D) Expression of AmCyan1 (brain stem) and mRFP (hippocampus) in astrocytes, DsRed1 in

oligodendrocytes of the cerebellar white matter, and HcRed1 in hippocampal CA1 pyramidal neurons. (E–H) Immunostaining against GFAP (E–F),

myelin-associated glycoprotein (G), and NeuN (H). (I–L) Merges of RCFP expression and corresponding immunolabeling. Images were recorded by confocal

laser-scanning microscopy and represented as maximum intensity projections of stacks with a depth of 10 to 50 Am (A, C–E, GI, K and L). Panels (B, F and J)

are single confocal sections. Arrowheads indicate selected cells with co-expression of RCFPs and the respective immunolabel. The arrow in panels (A, E and I)

labels an astrocyte with a low level of AmCyan1 expression.

P.G. Hirrlinger et al. / Mol. Cell. Neurosci. 30 (2005) 291–303 293

detected as early as 6 days of postnatal development. Primarily,

major projection neurons such as pyramidal neurons of layer 2/3

and 6 (Fig. 3B) as well as CA1 neurons of the hippocampus

(Figs. 2D, H, L and 3E) expressed HcRed1. In some of these

lines, efferents of motor neurons of the spinal cord and afferents

of sensory neurons of the dorsal root ganglia were labeled (data

not shown). One line was particularly suited to study retinal

ganglion cells (data not shown). HcRed1 protein was detected

by its fluorescence in all parts of the neuron, such as soma,

axon, and dendrites. The expression of HcRed1 could still be

observed in old mice of more than 300 days of age.

Unexpectedly, numerous neuron populations of different brain

areas expressing HcRed1 showed red fluorescent protein

precipitates of about 1 to 2 Am in size (Figs. 2D and 3). These

aggregates were more pronounced in older mice (postnatal day

200 (P200), Figs. 3D, E, F) than in younger ones (P50, Figs.

3A, B, C). In addition, we saw a significant variability among

neuronal cell types. Aggregation was less common in cortical

pyramidal cells (Fig. 3B), but often seen in cerebellar Purkinje

cells (Figs. 3A, D).

Transgenic mice with oligodendroglial DsRed1 expression

(TgN(PLP-DsRed1))

The PLP-DsRed1 construct gave rise to four different lines with

oligodendroglial FP expression. Two of these showed strong and

highly selective labeling of oligodendrocytes in various brain

regions (Figs. 2C, G, K cerebellum; Figs. 4A cortex; B, C

hippocampus). These two lines were further investigated. Func-

tional transgene expression was detected as early as postnatal day 2

in oligodendrocytes of the brain stem. After 10 days, expression

could also be detected in telencephalic regions such as the corpus

callosum. In young (P21) oligodendrocytes, DsRed1 was also

found in the thin myelin-forming processes (Figs. 4A, B). However,

with cell maturation and myelin compaction, less FP was detected

in processes, and it remained restricted to cell somata and distal

Fig. 3. The fluorescent protein HcRed1 forms clusters in neurons. In various regions of the brain, with increasing age, HcRed1 formed fluorescent protein

deposits mainly in somata as well as in processes of projection neurons. (A and D) HcRed1 formed fluorescent clusters in Purkinje cell somata. Though already

visible at postnatal day 50 (A), HcRed1 deposits accumulated heavily until postnatal day 200 (D). (B and C) Neurons of the cortex or deep cerebellar nuclei of

P50 mice displayed no sign of protein aggregation, in contrast to neurones from older mice (P200; E, F). All images were recorded by laser-scanning

microscopy of brain sections obtained from perfusion-fixed transgenic mice.


processes. Similarly to the TgN(Thy1.2-HcRed1) mice, the DsRed1

protein also formed fluorescent aggregates which accumulated in

the cell somata (Fig. 4C), as well as the processes (Figs. 4A, B). In

addition, we observed regional variation and increased FP

aggregation in older mice (more than a few weeks old) compared

to early postnatal mice.

Fig. 4. DsRed1 was abundantly expressed in somata and processes of oligodendr

highlighted the three-dimensional structure of oligodendrocytes in cortex (A) a

transgenic brain sections. (C) After reducing the detector gain of the CLSM, DsRe

in panel [B]). These precipitates were already detectable in the first postnatal we

Transgenic mice with astroglial AsRed2 or AmCyan1 expression

As expected, the other RCFPs AsRed2 and AmCyan1 gave

rise to transgenic mice with cell-type-specific fluorescent labeling

as well. In these mice, the human GFAP promoter was used to

target RCFPs into another neural cell population, the astrocytes

ocytes, but also forms fluorescent deposits. (A and B) DsRed1 expression

nd hippocampus (B) recorded by confocal laser-scanning microscopy of

d1 clusters became evident within the oligodendroglial soma (same view as

eks (P21; A–C) and accumulate with age.


(Figs. 1D, E, 2A and 5). In the case of the GFAP-AsRed2 construct,

however, we obtained one putative female founder, which had only

non-transgenic offspring. After sacrificing this TgN(GFAP-

AsRed2) mouse, we found that AsRed2 was expressed in the brain,

but only in solid fluorescent aggregates. The cell-type-specific

expression could be determined by their selective presence in

Bergmann glia somata, which can unequivocally be identified by

their location within the cerebellar Purkinje cells (Fig. 1E). In

contrast, the GFAP-AmCyan1 transgene generated a mouse line

with bright fluorescent astrocytes in several regions of the central

nervous system (Figs. 1D, 2A, 5) such as cortex, brain stem, and

striatum. However, AmCyan1 expression was highest within the

cerebellar Bergman glial cells. As it has been described previously,

reporter gene expression driven by the human GFAP promoter can

label at least two distinct astroglial cell populations which are

characterized by different expression levels of the transgene (Grass

et al., 2004; Matthias et al., 2003). Also, in our transgenic mice

expressing AmCyan1 under the control of the human GFAP

promoter, a second distinct astroglial cell population with a low

transgene level and lack of GFAP immunoreactivity could be

discerned (Fig. 2A).

Although the AmCyan1 protein formed numerous and very

bright clusters, it did not impair fertility nor aging of transgenic

mice. As observed in neurons and oligodendrocytes, RCFP

aggregates were abundant in astroglial somata and processes

(Fig. 5). The clustering of AmCyan1 protein increases with the age

of the animal. At postnatal day 10, only a few protein aggregates

could be detected in Bergmann glia (Fig. 5A), while at postnatal

Fig. 5. AmCyan1 formed fluorescent protein deposits along astroglial processes. T

cell populations, cerebellar Bergmann glia (A, C) and protoplasmic astrocytes of c

be detected at postnatal day 10 (A, B). They steadily increased with age (P21; C

day 21, the number of fluorescent particles was substantially

increased (Fig. 5C).

Transgenic mice with astroglial mRFP1 expression

We then asked whether or not transgenic RCFP precipitation is

a unique property of FPs known as oligomers. AmCyan1, DsRed1,

and AsRed2 form tetramers (Lukyanov et al., 2000; Matz et al.,

1999), while HcRed1 forms dimers (Gurskaya et al., 2001). We

reasoned that proteins with an intrinsic propensity for oligomeri-

zation might be particularly prone to unspecific aggregation when

expressed in an ectopic environment such as the brain of

transgenic mice. Therefore, we used the monomeric DsRed variant

mRFP1 (Campbell et al., 2002) in combination with the human

GFAP promoter to generate transgenic mice with labeled

astrocytes.

We successfully generated five different mouse lines with

functional mRFP1 expression in astrocytes of various CNS regions

(Figs. 2B, F, J and 6). From these, two lines were selected for more

intense investigation, due to their high level of expression in many

astrocytes. In these lines, the mRFP1 distribution within the CNS

was comparable to other transgenic lines using the GFAP promoter

(see above and (Grass et al., 2004; Matthias et al., 2003; Nolte et

al., 2001)). To our surprise, we detected numerous mRFP1

precipitates in astroglial somata as well as in the processes. These

aggregates were more abundant in older mice (3 weeks and older)

than in younger ones (P14), although they were already detectable

within the first postnatal week.

he hGFAP promoter targeted expression of AmCyan1 to different astroglial

ortex (B) and striatum (D). Fluorescent clusters of AmCyan1 could already

, D). Note the numerous FP deposits along the cell processes.

Fig. 6. The monomeric red fluorescent protein mRFP1 formed fluorescent protein deposits in transgenic mice. Similar to the TgN(hGFAP-AmCyan1) mice, the

monomeric red fluorescent protein mRFP1 was selectively targeted to astroglial cells of the cerebellum (A), hippocampus (B), cortex (C), and brain stem (D).

Surprisingly, numerous fluorescent precipitates were also found in this transgenic mouse line, even during the early postnatal weeks (P14; A–D).


Fluorescent protein precipitates are aggregates of the respective

RCFPs

To further characterize the identity of the fluorescent ag-

gregates, we performed spectral analysis using multi-channel

laser-scanning microscopy (Figs. 7A, B). We selected areas of

cells with normal soluble RCFP expression as well as those with

fluorescent clusters. Both types of regions were scanned in the

wavelength (lambda) scanning mode using the multi-channel

detector of the laser-scanning microscope. In particular, we

investigated the precipitates of AmCyan1 and DsRed1 (Figs.

7A, B). No significant differences of the various emission spectra

could be detected between regions of obvious soluble or insoluble

RCFPs. Therefore, we conclude that the observed fluorescent

protein aggregates consist mainly of the respective, unmodified

RCFP.

Transient transfection of cell lines is not sufficient for RCFP

aggregation

To study the RCFP deposition in culture, we transfected

different cell lines such as human embryonic kidney (HEK293)

and Chinese hamster ovary (CHO) cells with vectors in which the

RCFPs AsRed2, AmCyan1, and mRFP1 (Figs. 7C–E) are driven

by the cytomegalovirus (CMV) promoter. Cells were investigated

24 and 48 h after transfection by confocal laser-scanning micro-

scopy. All the tested RCFPs displayed a similar, uniform cytosolic

expression within the cells (Figs. 7C–E). Fluorescent deposits

were not detected. From these data, we conclude that short-term

(48 h and less) expression is not sufficient to induce RCFP

deposition.

The jellyfish variants of GFP do not form aggregates

In general, the formation of fluorescent deposits is not a

phenomenon of ectopic overexpression of fluorescent proteins. We

also studied the distribution of FP variants derived from the

jellyfish GFP in transgenic mice. For this purpose, we investigated

several transgenic mouse lines expressing EGFP (2 lines, Fig. 7F)

or ECFP (1 line, Fig. 7H) under the control of the hGFAP promoter

in astrocytes and expressing EYFP driven by the Thy1.2 promoter

in neurons (7 lines, Fig. 7G) (unpublished and Nolte et al., 2001).

In contrast to mice expressing RCFPs, we did not detect clusters of

fluorescent proteins in mice expressing the jellyfish FP variants

(Figs. 7F, G, H), even at older ages.

RCFP deposits do not change the properties of resting membrane

currents and glutamate evoked Ca2+ signaling

To evaluate whether transgenic RCFP expression interferes

with the cellular physiology, we applied whole-cell patch-clamp

recordings and Ca2+ imaging to acutely isolated brain slices (Fig.

8). In current clamp mode, positive current injections into

identified cortical pyramidal cells (resting membrane potential

Vmem = �62 mV T 6 mV, n = 15, P4–P20) evoked a series of

action potentials which resemble wild-type recordings (Figs. 8A–

C) (Larkum et al., 2001). Simultaneous filling of the patch-

clamped neuron with Lucifer Yellow revealed its detailed structure

Fig. 7. Comparison of reef coral fluorescent proteins with GFP variants from jellyfish. (A, B) The emission spectra of fluorescent deposits (squares) and soluble

RCFPs (circles) (A, AmCyan1, exc. 458 nm; B, DsRed1, exc. 543 nm) did not reveal significant differences. Using the multi-channel detector of the laser-

scanning microscope, emission spectra were recorded in somatic cell regions with either fluorescent aggregates (small yellow circles) or uniform fluorescence

(large yellow circles) of transgenic brain sections. Emission spectra are displayed as mean values T SD of n = 21 (AmCyan1, aggregated), n = 21 (AmCyan1,

soluble), n = 36 (DsRed1, aggregated), and n = 14 (DsRed1, soluble) recordings. (C–E) Transient transfection of various cell lines with RCFP vectors under

the cytomegalovirus promoter was not sufficient to induce FP aggregation. (C) Human embryonic kidney (HEK) cells transfected with AsRed2; (D) HEK cells

transfected with AmCyan1; (E) Chinese hamster ovary (CHO) cells transfected with mRFP1. (F–H) Transgenic mice generated with similar constructs but

using the jellyfish FP variants EGFP, EYFP, and ECFP did never show fluorescent protein precipitates. CLSM recordings from TgN(hGFAP-EGFP),

TgN(Thy1.2-EYFP), and TgN(hGFAP-ECFP) revealed uniform fluorescence in hippocampal protoplasmic astrocytes (F) and pyramidal neurons (G) as well as

cortical astrocytes contacting brain capillaries (H), respectively.


(Fig. 8B). Analysis of oligodendrocytes in the white matter of the

cerebellum and midbrain (Vmem = �64 mV T 10 mV, n = 20, P6–

P25; Figs. 8D–F) as well as astrocytes in the cortex (Vmem = �77

mV T 12 mV, n = 4, P6; Figs. 8G–I) in voltage-clamp mode

showed unequivocally the dominance of symmetrical almost non-

rectifying, Ohmic potassium currents (Berger et al., 1991; Fuss et

al., 2000; Grass et al., 2004; Matthias et al., 2003). Both cell types

showed a slightly higher expression of inward currents when



hyperpolarizing voltage steps were applied (Figs. 8E and H). This

is often observed in mature glial cells. At large depolarizing steps,

oligodendrocytes showed some apparent inactivation of outward

currents and larger tail currents (Fig. 8H). This behavior is very

typical for oligodendrocytes and represents a shift in the K+

equilibrium potential due to the restricted extracellular space in

myelinated tissue (Berger et al., 1991).

Astrocytes display various responses to the excitatory trans-

mitter glutamate (Verkhratsky and Kettenmann, 1996). In the

hippocampus of early postnatal mice (P2 to P4), we investigated

glutamate-evoked Ca2+ transients by confocal laser-scanning

microscopy (Figs. 8J–L). Frontal cortical slices of TgN(hGFAP-

mRFP1) mice were loaded with the Ca2+ indicator dye Fluo4-AM.

Signals from astrocytes could unequivocally be identified by the

astroglial mRFP1 expression. Short bath application of glutamate

(1 mM in aCSF for 1 min) evoked reliable Ca2+ signals with various

kinetics. In some of the cells, spontaneous Ca2+ oscillations prior to

glutamate application could be observed (Figs. 8J–L, cell #3).

Discussion

A series of transgenic mice were generated, which functionally

express various reef coral fluorescent proteins (RCFPs) driven by

various promoters. The mouse Thy1.2, the mouse proteolipid

protein (PLP), and the human glial fibrillary acidic protein

(hGFAP) promoters are well-characterized DNA elements targeting

specific expression in the major neural cell types: neurons,

oligodendrocytes, and astrocytes, respectively. Indeed, RCFP

expression was highly cell-type specific in the nervous system of

our transgenic mice. As expected from the above promoters which

are rather sensitive to their site of chromosomal transgene

insertion, each of the RCFP transgenes showed a variable

percentage of labeled cells not only in different brain regions but

also among different mouse lines.

Transgenic expression of various reef coral fluorescent proteins is

possible in several cell types of the brain

In contrast to the abundant and versatile use of jellyfish GFP

variants as reporters in transgenic mice (for reviews, see

Hadjantonakis et al., 2003; Hadjantonakis and Nagy, 2001), there

are only a few reports describing transgenic expression of RCFP

variants. So far, RCFPs have mainly been used to generate

transgenic plants (Jach et al., 2001; Wenck et al., 2003),

invertebrates (Verkhusha et al., 2001), or lower vertebrates (Finley

Fig. 8. RCFP expression did not change physiological properties such as whole-c

clamp analysis of cortical pyramidal cell (P16) expressing HcRed1. (A) Confoc

Dialysis with Lucifer Yellow-containing pipette solution highlighted the investig

injections induced a series of action potentials. (D–F) Patch-clamp analysis of

Epifluorescence image recorded by a CCD camera. The arrowhead points to the p

symmetrical, almost non-rectifying in- and outward currents. (F) The I/V relat

equilibrium potential indicating that the resting membrane conductance was prima

expressing astrocyte in voltage-clamped mode. (G) Epifluorescence image recorde

of hyper- and depolarizing voltage steps induced symmetrical, almost not-rectifyin

showed current reversal close to the K+ equilibrium potential indicating that th

Glutamate-evoked Ca2+ signaling in acutely isolated brain slices of hGFAP-mRFP

from a 4-day-old TgN(hGFAP-mRFP1) mouse. Numerous astrocytes were detecta

widespread loading of the slice with the green fluorescent Ca2+ indicator dye F

changes (in arbitrary units) reflect the variety of Ca2+ signals in astrocytes. The ci

panel (L). Note Ca2+ oscillations in cell 3 prior to glutamate application.

et al., 2001; Zhu and Zon, 2004). However, there are also few

reports describing the successful generation of transgenic rats with

DsRed2 expression in liver (Sato et al., 2003), as well as transgenic

mice with DsRed1 expression in different tissues composed of

smooth muscle (Magness et al., 2004). Very recently, transgenic

mice with ubiquitous expression of mRFP1 have been described

(Long et al., 2005; Zhu et al., 2005). Unfortunately, the authors did

not show high-resolution images allowing a detailed evaluation at

the cellular and subcellular level. Transgenic expression of RCFPs

in the nervous system has so far only been described in two

studies: in different types of neurons, DsRed1 was targeted by the

Thy1.2 promoter (Feng et al., 2000), and in catecholamine neurons

of the retina and the substantia nigra, DsRed2 was driven by the

tyrosine hydroxylase promoter (Zhang et al., 2004). Others,

however, reported severe problems in establishing DsRed1 trans-

genic mouse lines via embryonic stem cell-mediated transgenesis

(Hadjantonakis et al., 2002). Using the cre/loxP system, problem-

atic expression during embryo development can be circumvented

(Vintersten et al., 2004). Since the human GFAP promoter is active

as early as day 11 of gestation in radial glia which represent a vital

neural progenitor cell population during embryonic development

(Malatesta et al., 2003), our data suggest that RCFP expression

does not impair brain differentiation or mouse development.

Our study shows that various RCFPs can be used in several cell

types as a conventional transgenic label. Since RCFPs display awide

spectral range, these mice are extremely helpful in cell-specific

analysis when combined with a wide variety of dyes for functional

imaging such as fluorescent calcium or voltage indicator dyes. Our

physiological analysis of various cell types with transgenic RCFP

expression suggests that important cellular properties are not

affected and well comparable with those of wild-type cells.

Transgenic expression of RCFPs is accompanied by fluorescent

protein deposition

We were successful in transgenic labeling of neurons, astro-

cytes, and oligodendrocytes with spectrally different RCFPs.

Different cell types of the brain could be highlighted and easily

identified; for example, for use in physiological studies such as

patch-clamp recordings or Ca2+ imaging. The property of RCFPs

to form fluorescent aggregates, however, deteriorates a morpho-

logical analysis. Fluorescent deposits of all RCFPs (AmCyan1,

DsRed1, AsRed2, HcRed1, and mRFP1) were found in the somata,

as well as processes of cells. Not all cell types of the same class

(like different neuronal populations or oligodendrocytes in differ-

ent brain regions) were similarly affected. In some cells, we

ell membrane currents or glutamate-evoked Ca2+ signals. (A–C) Current

al laser-scan recording showing several HcRed1-expressing neurons. (B

ated neuron (arrowhead). (C) Neuronal depolarization by positive curren

a DsRed1-expressing oligodendrocyte (P6) in voltage-clamped mode. (D

atch pipette. (E) A series of hyper- and depolarizing voltage steps induced

ionship of the evoked currents showed current reversal close to the K+

rily carried by K+ ions. (G– I) Patch-clamp analysis of a cortical, mRFP1

d by a CCD camera. The arrowhead points to the patch pipette. (H) A series

g in- and outward currents. (I) The I/V relationship of the evoked currents

e resting membrane conductance is primarily carried by K+ ions. (J–L

1 transgenic mice. (J) Single optical section through a brain slice obtained

ble due to their intense red fluorescence. (K) CLSM recording revealed the

luo4-AM. (L) Glutamate-evoked (1 mM for 1 min) fluorescence intensity

rcles in panels (J and K) outline the cells which responses are displayed in

-

)

t

)

-

)


detected fluorescent clusters, while they were missing in others. In

addition, we observed an increase in cluster formation during aging

of mice. We conclude that the continuous accumulation of proteins

in a given cell leads to concentration and time-dependent

precipitation of RCFPs. In transient short-term transfection experi-

ments, we did not observe any fluorescent deposits. It is most

likely due to the cell-type-specific catabolism of ectopic proteins,

which determines the aggregation threshold. In addition, this

threshold by itself is an intrinsic function of RCFPs, since the

spectral comparison of aggregated versus soluble RCFP did not

show significant differences. Therefore, we exclude gross mod-

ifications of RCFPs by oxidation or other mechanisms as a cause

for aggregation. In contrast, the aggregation of RCFPs seems to be

an intrinsic property of this protein family, since transgenic mice

with jellyfish FP variants are devoid of fluorescent aggregates (our

own study and Belachew et al., 2003; Feng et al., 2000; Mallon et

al., 2002; Nolte et al., 2001). The aggregation is independent of the

oligomerization of RCFPs, which exists normally as tetramers or

dimers (Matz et al., 1999), since exchange of amino acids which

are responsible for tetramerization of DsRed (Campbell et al.,

2002) does not prevent deposition of insoluble protein as seen with

the monomeric RFP1. RCFP aggregation is a time-dependent

process. While cells in culture expressing the proteins for a short

time (<48 h) show no RCFP aggregates, long-term expression in

transgenic mice is accompanied by a widespread RCFP deposition.

This is underlined by the observation that young animals show less

aggregates than older ones.

Protein aggregates have been considered a major pathogenic

principle in a number of neurodegenerative disorders such as

Alzheimer disease or Parkinson disease (Ross and Poirier, 2004;

Shastry, 2003; Taylor et al., 2002). RCFP aggregates, however, do

not obviously interfere with the normal life of the transgenic mice.

These mice have a normal life expectancy and their breeding

behavior is comparable to other mice, except of the TgN(GFAP-

AsRed2), which is probably due to an unfavorable genomic

integration. When investigated in the cage, obvious behavioral

abnormalities were not detected. Therefore, protein aggregates as

such seem not to be toxic. Disease-related protein aggregates are

specific properties of these proteins or their intermediates formed

during precipitation (Agorogiannis et al., 2004).

Conclusion

Reef coral fluorescent proteins represent a versatile class of

proteins which possess a broad spectral variation. As such, they are

perfectly suited to label various cell populations in transgenic mice

and can be used in combination with various fluorescent indicator

dyes in physiological studies. However, their tendency to form

fluorescent protein deposits in cell somata as well as processes

limits their use for morphological analysis as well as their

application as fusion proteins, at least in transgenic mice.

Experimental methods

Cloning of vectors used to generate transgenic mice

Vectors encoding AmCyan1, DsRed1, AsRed2, and HcRed1

were purchased from BD Biosciences/Clontech (Heidelberg). The

mRFP1 vector was kindly provided by Roger Y. Tsien (University

of California, San Diego). For cell-type-specific expression in

astrocytes, the human GFAP promoter was PCR amplified from a

pGFAP-EGFP vector (Nolte et al., 2001) using sense primer 5V-ACC TCC ATTAAT CCC ACC TCC CTC TCT GTG CTG-3V andanti-sense primer 5V-GCT CCT CGC CCT TGC TCA CCA-3V andthe AseI/AgeI fragment of the product was cloned into

pAmCyan1-C1 and pAsRed2-N1. The mRFP1 was amplified

from pRSETB-mRFP1 using sense primer 5V-ACC GGT CGC

CAC CAT GGC CTC CTC CG-3V and anti-sense primer 5V-TCAAGC TTC GAA TTC TTA GGC GCC-3V. The BamHI/EcoRI

fragment of the product was cloned into the corresponding sites of

pGFAP-AmCyan1-C1. To label oligodendrocytes, the cDNA

sequence coding for DsRed1 was isolated from pDsRed1-1

(Clontech) as a HindIII-NotI fragment and inserted into the PLP

promoter construct as described previously (Fuss et al., 2000). To

label neurons, a construct was generated by cloning the open

reading frame of HcRed1 into the XhoI site of the Thy1.2-

minigene as described (Caroni, 1997; Feng et al., 2000). The

Thy1.2 minigene consists of 6.5 kb of the murine Thy1.2 gene

from the promoter to the intron following exon 4, but lacking exon

3 and its flanking introns (see Fig. 1A).

Cell culture and transfection

To prove functional cytosolic expression of the various FPs,

human embryonic kidney cells (HEK293) and Chinese hamster

ovary (CHO) cells were transiently transfected. HEK293 cells were

cultured in Dulbecco’s modified Eagle Medium (Gibco) supple-

mented with 5% fetal bovine serum. CHO cells were cultured in

F12 HAM medium supplemented with 10% fetal bovine serum.

For transfection, cells were plated onto coverslips (12 mm in

diameter) in 24-well plates and transfected at approximately 75%

density. Expression of all RCFPs was driven by the cytomegalo-

virus (CMV) promoter. Transfection was performed with Lip-

ofectamini 2000 (Invitrogen) according to the manufacturer’s

protocol. Cells were washed once with phosphate-buffered saline

(PBS) 24 or 48 h after transfection and fixed with 4%

paraformaldehyde in 100 mM sodium phosphate buffer (PFA).

Generation of transgenic mice

Transgenic mice were generated by injection of their respective

linear transgene-containing construct into the pronucleus of

fertilized oocytes obtained from FVB/N or C57BL/6J mouse

strains. Mice were kept in the mouse facility of the institute

according to national and European guidelines for the welfare of

experimental animals. Putative transgenic founders were identified

by PCR genotyping of tail-tip DNA and subsequently crossbred to

wild-type mice. Positively genotyped F1 litters of transgenic

founders were screened for functional FP expression using

epifluorescence and laser-scanning microscopy. All transgene

constructs gave rise to functional expression in adult mice. Lines

with functional FP expression were bred further and investigated.

Cell-type-specific expression of FPs was readily identified, since

RCFP-mediated fluorescence highlights the characteristic mor-

phology of astrocytes, oligodendrocytes, and neurons.

Tissue fixation and immunohistochemistry

Transgenic mice were anesthetized with 2.5% 2,2,2-tribromoe-

thanol in H2O (100 Al/10 g body weight) and perfused trans-


cardially with Hank’s Balanced Salt Solution (HBSS, Gibco)

followed by perfusion with PFA for 15 min. The brain was

removed and incubated in PFA over night at 4-C. For analysis oftransgenically expressed fluorescent proteins, 100 Am sagittal or

horizontal vibratome sections (Leica VT 1000S, Leica Instruments,

Nussloch, Germany) were investigated by epifluorescence or laser-

scanning microscopy. Immunohistochemical labeling was per-

formed on 50 Am free-floating vibratome slices at room temper-

ature. Cells were permeabilized with 0.4% Triton-X100 in PBS for

30 min and then blocked for 30 min in 4% fetal calf serum (FCS)

with 0.2% Triton-X100 in PBS. The slices were incubated over

night at 4-C in 1% FCS and 0.05% Triton-X100 in PBS with the

following first antibodies: monoclonal anti-GFAP (mouse, 1:200,

Novocastra), monoclonal anti-neuronal nuclei antigen (NeuN,

mouse, 1:100, Chemicon), or monoclonal anti-myelin associated

glycoprotein (MAG, 1:200, Chemicon). Slices were washed twice

in PBS for 5 min and incubated with Cy2- or Alexa 488-

conjugated secondary antibodies (1:1000, Dianova; 1:2000 Mobi-

tec) for 2 h in 1.5% FCS in PBS. After washing twice in PBS,

slices were mounted with Immu-Mount (Shandon).

Confocal laser-scanning microscopy

Fixed brain sections were investigated by confocal laser-

scanning microscopy (Zeiss LSM 510NLO, Axiovert 200M)

equipped with laser lines at 458/488/514, 543 and 633 nm. The

following laser lines and emission filters were used for excitation

and emission: 458 nm/BP 473–526 or BP 480–520 IR

(AmCyan1), 458 nm/BP 480–520 IR or BP 462–548 (ECFP),

488 nm/BP 500–530 IR (EGFP), 488 nm/BP500–550 IR (Cy2

and Alexa 488), 514 nm/BP 526–580 (EYFP), 543 nm/LP 560

(HcRed1/mRFP1), 543 nm/BP 565–615 IR (DsRed1), 543 nm/BP

558–623 (AsRed2). Three-dimensional image stacks were

recorded with frame sizes of 512 � 512 pixels or 1024 � 1024

pixels. Shown are maximum intensity projections from stacks with

a depth of 10 to 50 Am, except of the immunohistochemistry of

TgN(hGFAP-mRFP1) which is a single optical plane. All confocal

images were stored and processed with the Zeiss LSM Software,

ImageJ or Adobe Photoshop.

Emission spectra of AmCyan1 and DsRed1 were recorded at

10.7 nm resolution using the Zeiss LSM510 Meta system. FPs

were excited at 458 nm and 543 nm, respectively. For analysis of

emission spectra, regions of interest were examined in areas

consisting of obvious aggregates or where a diffuse fluorescence

indicated soluble protein. Spectra were normalized to the max-

imum (set as 100) and averaged.

Recording of calcium signals by laser-scanning microscopy

Astroglial calcium responses to glutamate (1 mM in artificial

cerebrospinal fluid, aCSF; in mM: 125 NaCl, 2.5 KCl, 2 CaCl2, 1

MgCl2, 1.25 NaH2PO4, 25 NaHCO3, and 25 d-glucose; 330

mosm/l, pH7.4) were observed in acutely isolated brain slices

obtained from TgN(hGFAP-mRFP1) transgenic mice. Frontal

slices (200 Am) were prepared from the brain of postnatal 4-day-

old mice. The slices were placed in carbogenated (95% oxygen/5%

carbon dioxide) aCSF at 35-C for 10 min, subsequently kept at

room temperature for 45 min, stained with 1 AM Fluo-4-AM

(Molecular Probes) for 45 min, and washed for at least 30 min in

aCSF. Slices were transferred to the recording chamber and

continuously perfused with aCSF (room temperature; 20–23-C).

Fluo-4 fluorescence was excited at 488 nm and recorded using a BP

500–530 emission filter. Before starting Ca2+ imaging, a mRFP1

image was acquired (Ex 543nm, Em LP 560) to unequivocally

identify astrocytes. 1 mM glutamate in aCSF was applied to the bath

for 1min, followed bywashingwith aCSF until the end of recording.

Fluo-4 fluorescence was recorded at about 1-Hz repetition rate with

a spatial resolution of 0.5 Am (512 � 512 pixels).

Electrophysiology

Acute 200 Am frontal sections were obtained from mice

(TgN(hGFAP-mRFP1), TgN(mPLP-DsRed1) and TgN(mThy1.2-

HcRed1)) of postnatal days 4–20. Mice were decapitated, the brain

was dissected and placed in ice-cooled, carbogen-saturated aCSF.

Slices were stored in aCSF at 35-C for 10 min and then placed at

room temperature (20–23-C) for at least 20 min before experi-

ments were started. Subsequently, slices were transferred to the

recording chamber and kept submerged by a platinum grid with

nylon threads (Edwards et al., 1989) for mechanical stabilization.

The chamber was mounted on an upright microscope (Axioscope 2

FSmot, Zeiss, Germany) and continuously perfused with aCSF

(room temperature; 20–23-C) at a flow rate of 2–5 ml/min. Cells

(neurons, oligodendrocytes, and astrocytes) were identified by their

red fluorescence in the epifluorescence illumination (excitation 560

nm; Polychrome II, TILL Photonics, Grafelfing Germany). Images

of astrocytes and oligodendrocytes were taken with a CCD camera

(Princeton Instruments, Trenton, USA) and the MetaMorph

imaging software (Visitron Systems, Puchheim, Germany).

Whole-cell voltage-clamp recordings were obtained with an

EPC9/2 double patch-clamp amplifier (HEKA, Lambrecht/Pfalz,

Germany). Patch electrodes were pulled from borosilicate glass

capillaries (Hilgenberg, Malsfeld, Germany) on a horizontal pipette

puller (P-2000, Sutter Instruments, Germany) and filled with (in

mM) 125 K-Gluconate, 1 CaCl2, 2 MgCl2, 4 Na2ATP, 10 EGTA,

10 HEPES (pH adjusted to 7.2 with KOH). The resistance of the

pipettes was between 2 and 6 MV.

Currents were low-pass filtered at 3 kHz, recorded at 20 kHz

using Patchmaster 1.0 software (HEKA, Lambrecht/Pfalz, Ger-

many), and stored for off-line analysis. The analysis was performed

with IGOR Pro Version 4.0 (Wavemetrics, Inc., USA). Glial cells

were voltage-clamped at �70 mV (Vhold). Whole-cell membrane

currents were evoked by a series of hyper- and depolarizing

voltage steps ranging from �130 to 50 mV with an increment of 10

mV. Under voltage-clamp neurons were clamped at �60 mV. After

switching into the current-clamp mode, various hyper- and

depolarizing currents were injected (�30 pA to +250 pA) into

the soma.

During electrophysiological experiments, selected neurons were

dialyzed with Lucifer Yellow (3 mM in pipette solution, Molecular

Probes) for 5–10 min. After filling, slices were fixed in PFA for 2

h and visualized by confocal microscopy as described above.

Acknowledgments

This work was supported by the Deutsche Forschungsgemein-

schaft through the DFG Research Center for Molecular Physiology

of the Brain (CMPB grant to F.K.) and a priority program grant

(SPP 1172 to F.K.). In addition, the authors wish to thank Adriane

Mosley for the valuable comments on the manuscript, Simone

Schmidt for the DNA injections, Daniel Flemming and Rainer


Libal for the animal husbandry and mouse database management.

The generous support of Klaus-Armin Nave to this project is

particularly acknowledged.

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Expression of reef coral fluorescent proteins in the central nervous system of transgenic mice