generation of two-color transgenic zebrafish using the green and red fluorescent protein reporter...

Generation of Two-color Transgenic Zebrafish Using theGreen and Red Fluorescent Protein Reporter Genes gfpand rfp

Haiyan Wan, Jiangyan He, Bensheng Ju, Tie Yan, Toong Jin Lam, and Zhiyuan Gong*

Department of Biological Sciences, National University of Singapore, Singapore 119260

Abstract: Two tissue-specific promoters were used to express both green fluorescent protein (GFP) and red

fluorescent protein (RFP) in transgenic zebrafish embryos. One promoter (CK), derived from a cytokeratin

gene, is active specifically in skin epithelia in embryos, and the other promoter (MLC) from a muscle-specific

gene encodes a myosin light chain 2 polypeptide. When the 2 promoters drove the 2 reporter genes to express

in the same embryos, both genes were faithfully expressed in the respective tissues, skin or muscle. When the

2 fluorescent proteins were expressed in the same skin or muscle cells under the same promoter, GFP fluo-

rescence appeared earlier than RFP fluorescence in both skin and muscle tissues, probably owing to a higher

detection sensitivity of GFP. However, RFP appeared to be more stable as its fluorescence steadily increased

during development. Finally, F1 transgenic offspring were obtained expressing GFP in skin cells under the CK

promoter and RFP in muscle cells under the MLC promoter. Our study demonstrates the feasibility of

monitoring expression of multiple genes in different tissues in the same transgenic organism.

Key words: dsRed, EGFP, cytokeratin, myosin light chain, skin, muscle.

INTRODUCTION

The green fluorescent protein (gfp) gene, originally isolated

from the jellyfish Aequorea victoria, is widely used as a re-

porter gene for investigation of tissue-specific gene expres-

sion and cellular localization of proteins because the fluo-

rescence of its protein product, GFP, can be conveniently

detected in living cells (Prasher et al., 1992; Chalfie et al.,

1994; Tsien, 1998). Since the introduction of the wild-type

GFP, many mutant forms have been created and screened

for improvement of fluorescence brightness or for altered

spectra of fluorescence shifted toward red or blue (Tsien

and Prasher, 1998). So far, several mutant forms of GFP are

commercially available, and they display different spectra of

fluorescence from that of wild-type GFP. The wild-type

GFP has a maximal spectrum at 508 nm, while several mu-

tant forms have maximal spectra at 440 nm (blue), 477 nm

(cyan), and 527 nm (yellow); these mutant forms are called

blue fluorescent protein (BFP), cyan fluorescent protein

(CFP), and yellow fluorescent protein (YFP), respectively

(Clontech Catalog 2000, pp. 209–222). The availability of

these different fluorescent proteins makes it feasible to carry

out multiple labeling of different organelles or structures

within the same cells or different tissues or cells in the same

organism.

Recently, a new fluorescent protein gene, red fluorescent

protein (rfp), was isolated from a sea anemone relative (Dis-

Received March 15, 2001; accepted September 18, 2001.

*Corresponding author: telephone 65-8742860; fax 65-7792486; e-mail

[email protected]

Mar. Biotechnol. 4, 146–154, 2002DOI: 10.1007/s10126-001-0085-3

© 2002 Springer-Verlag New York Inc.

cosoma sp.) (Matz et al., 1999), and the fluorescent protein

has a red emission spectrum with the maximum at 583 nm.

RFP is distantly related to GFP in primary sequence, and the

fluorescence can also be easily detected without any pre-

treatment. Thus, RFP is also an ideal reporter protein for

living color and can be used for multiple labeling in con-

junction with GFP and its derivatives.

So far, GFP has been widely used for transgenic re-

search in a wide variety of species, including bacteria, yeast,

plants, Caenorhabditis elegans, Drosophila, zebrafish, frogs,

and mice (Chalfie and Kain, 1998). As an important model

organism in genetics and developmental biology, zebrafish

is particularly suitable for GFP expression and detection.

The transparency of zebrafish embryos and external devel-

opment make it feasible to visualize GFP throughout em-

bryo genesis and larval development. Therefore, transgenic

expression of GFP in zebrafish under tissue-specific pro-

moters has become a powerful tool to recapitulate endog-

enous gene expression programs (Higashijimas et al., 1997,

2000; Long et al., 1997, 2000) and to analyze the function of

gene promoters (Meng et al., 1997, 1999; Ju et al., 1999;

Muller et al., 1999; Chen et al., 2001). Recently, gfp under a

heat-shock-inducible promoter has been introduced into

zebrafish, and the inducible expression of GFP provides a

real opportunity to trace cell lineage at any given stage

(Halloran et al., 2000). In addition, gfp transgenic zebrafish

also have the potential use in tracing cell migration, organ

genesis, nuclear and cellular transplantation, purification of

specific cells for a cell-type-specific complementary DNA

library, establishment of in vitro cell lines, etc.

As both gfp and rfp genes are available, it is interesting

to determine the feasibility of generating 2-color transgenic

animals. In the present study, we used the zebrafish as a

model to transfer gfp and rfp driven by 2 different tissue-

specific promoters. In transgenic zebrafish embryos, both

fluorescent proteins were expressed efficiently and faithfully

according to the specificity of the promoters used. Germ-

line-transmitted F1 generation fish with GFP expression in

skin and RFP expression in muscle were obtained, confirm-

ing the tissue-specific expression of the 2 fluorescent pro-

teins under 2 different tissue-specific promoters.

MATERIALS AND METHODS

Zebrafish Maintenance

Zebrafish were purchased from a local ornamental fish farm

and maintained in our aquarium according to the Zebrafish

Book (Westerfield, 1995). Developmental stages of embryos

are presented as hour postfertilization (hpf) or day postfer-

tilization (dpf) at 28.5°C, according to Kimmel et al. (1995).

DNA Constructs

Gene promoters were isolated by linker-mediated nested

polymerase chain reaction (PCR), as previously described

by Liao et al. (1997). Two promoters were used in the

present study: one from a cytokeratin gene (krt8) for skin

specificity (Ju et al., 1999) and the other from a myosin light

chain 2 gene (mylz2) for muscle specificity (Xu et al., 1999,

2000). The gfp and rfp reporter gene constructs, pEGFP-1

and pDsRed-1, were purchased from Clontech (Palo Alto,

Calif.). The 2 gene promoters, 2.2 kb from the cytokeratin

gene (CK) and 2.0 kb from the myosin light chain 2 gene

(MLC), were inserted into the 2 reporter gene constructs in

front of the coding region at the EcoRI and BamHI sites.

The resulting constructs were named pCK-EGFP, pCK-

RFP, pMLC-EGFP, and pMLC-RFP, respectively.

Microinjection and Fluorescence Detection

Microinjection was carried out as previously described (Ju

et al., 1999). Linearized plasmid DNA was injected into

embryos of 1- to 2-cell stage at a concentration of 150 µg/ml

in 0.1 M Tris-HCl/0.25% Phenol red. When 2 different

DNA constructs were injected, the concentration of each

DNA construct was 75 µg/ml. DNA (300–500 pl) was in-

jected into each embryo. Injected embryos were kept at

28.5°C and monitored and photographed using a Zeiss Ax-

iovert 25 fluorescence microscope. GFP was observed with

the filter BP450-490 (blue light) and RFP was observed with

the filter BP546 (yellow light). Hatched embryos or fry were

anesthetized in 0.1% 2-phenoxyethanol prior to being pho-

tographed.

Screening of Transgenic F1 and Cryosection

Embryos injected with pCK-EGFP/pMLC-RFP were raised

to adults. Each adult founder was crossed with a nontrans-

genic zebrafish, and at least 100 offspring from the cross

were examined for expression of fluorescent proteins.

Among 18 founders examined, 1 produced embryos ex-

pressing both fluorescent proteins. Some of the transgenic

fry were fixed in 4% paraformaldehyde in phosphate-

buffered saline overnight at 4°C and sectioned at 15-µm

thickness by a cryostat prior to the examination of GFP and

RFP expression as described above.

Two-color Transgenic Zebrafish 147

Figure 1. Tissue-specific expression of GFP under the CK and

MLC promoters. A: A 24 hpf embryo developed from a fertilized

egg injected with pCK-EGFP. B: Enlargement of the region boxed

in A. Note the expression of GFP in surface skin cells. C: A 48 hpf

embryo developed from a fertilized egg injected with pMLC-

EGFP. D: Enlargement of the region boxed in C. Note the expres-

sion of GFP in somitic muscle fibers. Yellow color is observed as

a result of saturation of green fluorescence owing to the strong

expression of GFP.

Figure 2. Expression of GFP and RFP in different tissues. A: Em-

bryo injected with pMLC-EGFP/pCK-RFP and viewed under a

blue excitation light for GFP observation. B: The same embryo

viewed under a yellow excitation light for RFP observation.

C,E: Embryos injected with pCK-EGFP/pMLC-RFP under blue

light to observe GFP. D,F: The same embryos under yellow light to

observe RFP. The stages of embryos are indicated on the right.

148 Haiyan Wan et al.

RESULTS

Tissue Specificity of CK and MLC Promoters

The 2 promoters used in the present study have been pre-

viously characterized in transient transgenic zebrafish em-

bryos (Ju et al., 1999) or by direct injection of naked DNA

into adult muscles (Xu et al., 1999). When pCK-EGFP was

injected into zebrafish embryos, GFP was specifically ex-

pressed in the surface skin cells in early embryos (Figure 1,

A and B), consistent with our earlier observation (Ju et al.,

1999). In embryos injected with pMLC-EGFP, GFP expres-

sion was specifically detected in trunk skeletal muscles (Fig-

ure 1, C and D). These expression patterns have been con-

firmed in stable transgenic zebrafish lines by expression of

GFP under the 2 promoters (our unpublished observa-

tions). Therefore, the 2 promoters used in the present study

were highly tissue-specific.

Two Fluorescent Proteins Expressed in TwoDifferent Tissues

To determine whether the 2 fluorescent proteins driven by

different tissue-specific promoters can be expressed cor-

rectly in targeted tissues, pMLC-EGFP/pCK-RFP and pCK-

EGFP/pMLC-RFP were respectively coinjected into ze-

brafish embryos. As shown in Figure 2, both fluorescent

proteins were presented faithfully in the expressing tissues

according to the respective promoters.

In embryos injected with pMLC-EGFP/pCK-RFP, GFP

and RFP were correctly expressed in muscle and skin cells,

respectively (Figure 2, A and B). RFP was first detected in

skin cells at 16 hpf, while GFP was first expressed in muscle

fibers of somites at 22 hpf. It is interesting to note that

strong expression of RFP can be detected as orange color

under the excitation light for GFP (Figure 2, A); however,

no GFP fluorescence can be observed under the excitation

light for RFP (B).

In embryos injected with pCK-EGFP/pMLC-RFP

(Figure 2, C–F), GFP was first detected at the superficial

layer of injected embryos at 6 hpf (shield stage), and the

expression continued in the outmost layer of skin cells dur-

ing embryogenesis. This is in contrast to the earliest detec-

tion of RFP expression at 16 hpf in embryos injected with

pMLC-EGFP/pCK-RFP. In muscle cells, RFP was first ob-

served at 30 hpf, also later than the earliest detection of GFP

(22 hpf) under the same MLC promoter. Thus, GFP is

detected earlier than RFP in the same tissue under the same

promoter.

Unexpectedly in the embryos injected with pCK-EGFP/

pMLC-RFP, GFP was also observed in muscle fibers at the

same stage as RFP detection. It is apparent that all GFP-

expressing muscle fibers also express RFP (Figure 2, C).

This phenomenon is likely due to the formation of a het-

eroconcatemer of the 2 injected plasmid DNAs, and thus

the muscle-specific elements from pMLC-RFP affected the

expression of gfp, as previously reported by Muller et al.

(1997). During embryogenesis, RFP accumulation increased

steadily in muscle fibers. In comparison, GFP fluorescence

in muscle fibers became relatively weak and seemed to dis-

appear after 72 hpf (Figure 2, E).

Despite the observation of “ectopic” expression of GFP

in muscles of the embryos injected with pCK-EGFP/pMLC-

RFP, we never observed the expression of RFP in the

muscles of the embryos injected with the reciprocal com-

bination, pCK-RFP/pMLC-EGFP. Neither was “ectopic”

expression of GFP or RFP observed in skin cells from em-

bryos injected with either combination, pCK-EGFP/pMLC-

RFP or pCK-RFP/pMLC-EGFP.

Two Fluorescent Proteins Expressed in IdenticalCells or Tissues

To investigate whether there is any interference between the

2 fluorescent proteins if they are expressed in identical cells,

coinjection of pCK-EGFP/pCK-RFP or pMLC-EGFP/

pMLC-RFP was carried out. In both cases, GFP and RFP

were correctly expressed in the same tissues and presented

in identical sets of cells (Figure 3). However, as observed

from injection experiments with 2 heterogenous promoter

constructs, GFP generally appears about 10 hours earlier

than RFP. The first detection of GFP in skin and muscle was

around 5 hpf and 22 hpf, respectively; in comparison, the

appearance of RFP in the 2 tissues was 16 hpf and 30 hpf,

respectively. The timing of GFP and RFP appearance in

embryos injected with different combinations of DNA con-

structs is summarized in Figure 4.

Although RFP was detected later than GFP, the inten-

sity of its fluorescence increased steadily during develop-

ment. By 72 hpf, only RFP fluorescence was detected in skin

cells in embryos injected with pCK-EGFP/pCKRFP (Figure

3, C and D). Similarly, under the MLC promoter, RFP

fluorescence became predominant after 5 dpf and GFP fluo-

rescence was greatly overshadowed, even under the optimal

excitation light for GFP (Figure 3, G and H).


Two-color Transgenic F1

To examine the expression of GFP and RFP in stable trans-

genic zebrafish, embryos injected with pCK-EGFP/pMLC-

RFP were raised to adulthood and screened for transgenic

offspring. One founder was identified to produce embryos

expressing both GFP and RFP. The transmission of trans-

gene to F1 is 10.6% (n = 85), indicating transgene mosa-

icism in the founder. In F2 offspring, standard Mendelian

ratio was observed, indicating stable integration of trans-

gene in germline. The transgenic offspring displayed green

fluorescence in skin and red fluorescence in muscle (Figure

5, A and B). Cross section of transgenic offspring confirmed

that GFP expression was restricted to the superficial layer of

skin cells and RFP was expressed specifically in skeletal

muscle (Figure 5, C and D). Similar to the observation in

the transient expression assay, RFP in cross sections under

a blue filter can also be viewed as orange (Figure 5, C).

Since the 2 fluorescent proteins were always expressed

in the same individuals, the 2 gene constructs were likely

Figure 3. Expression of GFP and RFP in the same tissue. A,C:

Embryos injected with pCK-EGFP/pCK-RFP and viewed under a

blue excitation light for GFP observation. B,D: The same embryos

viewed under a yellow excitation light for RFP observation. E,G:

Embryos injected with pMLC-EGFP/pMLC-RFP under blue light

to observe GFP. F,H: The same embryos under yellow light to

observe RFP. The stages of embryos are indicated on the right. The

yellow color of skin cells in C is due to the saturation effect of

fluorescence and correlates to cells with the highest intensity of red

fluorescence.


integrated into the same chromosome locus. More likely,

the 2 constructs had been concatenated and integrated. Un-

like the expression in transient transgenic embryos, where

weak GFP expression was also detected in muscle (Figure 2,

C), no GFP expression was detected in muscles from stable

transgenic embryos and fry.

DISCUSSION

Using the gfp reporter gene under zebrafish fish gene pro-

moters, faithful expression of the transgene has been re-

peatedly demonstrated (Higashijimas et al., 1997, 2000;

Long et al., 1997, 2000; Meng et al., 1997, 1999; Ju et al.,

1999; Muller et al., 1999). In the present study, using 2

different fluorescent reporter genes, we have further dem-

onstrated the faithful expression of the 2 transgenes simul-

taneously in the same fish and thus proved the feasibility of

generating 2-color transgenic animals. Multicolor trans-

genic animals should be useful in examination and tracing

of the development of 2 or more tissues and organs simul-

taneously in the same animal, thus providing a valuable tool

for a better and closer comparison. In particular, the com-

Figure 5. Expression of GFP and RFP in a transgenic F1 fry (5 dpf)

from a founder injected with pCK-EGFP/pMLC-RFP. The same

embryo is show for GFP expression (A) and for RFP expression

(B). A transgenic F1 fry of the same stage was cross-sectioned

through the trunk for observation of GFP expression (C) and RFP

expression (D). The same section was used in C and D under

different filters.

Figure 4. Summary of the timing

of appearance of GFP and RFP in

embryos injected with different

combinations of DNA constructs.

Green bars indicate GFP expression

and red bars indicate RFP

expression. The injected DNA

constructs and expressing tissues

are indicated on the left and right,

respectively. The hours after

fertilization are indicated at both

the top and bottom.


parison is valuable for 2 adjacent tissues or cell types. For

example, expression of the 2 fluorescent proteins can be

targeted to endocrine and exocrine cells of pancreas under

2 suitable promoters, and thus a useful transgenic model

could be created for detailed study of pancreas development

and the interaction of different types of cells in the pan-

creas.

When the 2 fluorescent proteins were expressed in the

same tissues, GFP generally appeared several hours earlier

than RFP. This was not likely to be due to the timing of

transcription since the same promoter was used. It seems

that GFP is more sensitive for detection than RFP and re-

quires a lower concentration for visualization. The gfp DNA

used in the present study was a mutant form, encoding a

GFP variant containing a critical amino acid substitution in

the chromophore region, and the codons have also been

modified based on preferred human codon usage (Cormack

et al., 1996). The resultant GFP is called enhanced GFP, or

EGFP, which is 35-fold stronger in fluorescence intensity

than the wild-type GFP in human cells and presumably in

other vertebrate cells as well (Yang et al., 1996).

At later stages, however, it seemed that the fluorescence

intensity of RFP in both muscle and skin cells was stronger

than that of GFP. It is thus likely that RFP is more stable

than GFP and that a higher steady level of RFP can be

reached within the cells. Under our conditions, GFP could

only be detected under its excitation light (blue) but not

under the excitation light for RFP (yellow); while RFP,

when it was expressed at a high level, could be detected as

orange under the blue light and frequently interfered with

the detection of GFP. However, strong expression of GFP

seemed to have no effect on detection of RFP. Despite the

potential interference of strong RFP with GFP detection, the

simultaneous visualization of both GFP and RFP under the

same excitation light could be beneficial under certain cir-

cumstances as no switch of light or double exposure is

required for real-time observation.

The reporter genes are commonly used to determine

temporal and spatial patterns of gene expression. For tem-

poral expression, based on our unpublished data in stable

gfp transgenic zebrafish lines, detection of GFP fluorescence

is generally 2 to 3 hours later than detection of gfp messen-

ger RNA in developing embryos because of the delay of the

translation event and accumulation of sufficient GFP for

detection. According to the current study, the appearance of

RFP fluorescence is delayed by about 10 hours compared

with that of GFP fluorescence, and thus it is likely that the

detection of RFP is 12 to 13 hours later than the detection

of its mRNA. Therefore, gfp reporter gene should be a better

choice than rfp if the timing of gene activation is critical in

the analysis.

Although stable transgenic animals have been made for

many species, the fate of exogenously introduced DNA and

how it is integrated into the host chromosomes remain

unclear. It has been reported that the foreign DNA, once

injected into fertilized eggs, forms DNA concatemers and

undergoes a rapid amplification and subsequent degrada-

tion (Flytzanis et al., 1985; Stuart et al., 1988; Chong and

Vielkind, 1989). Because of this, as previously demon-

strated, analysis of DNA cis-elements can be achieved by

coinjection of a promoter-reporter gene construct and a

testing DNA cis-element. Conceivably, the cis-element will

be ligated to the promoter in vivo, thus eliminating the

laborious process of constructing a proper test DNA plas-

mid in vitro. This approach has proved useful in zebrafish

(Muller et al., 1997, 1999). In our present study, as shown

in Figure 2, C, indeed muscle expression of GFP was ob-

served due to the presumable concatemerization of a

muscle element from a heterologous DNA construct. How-

ever, the expression of GFP in this way was relatively weak,

and probably only effective in early embryos in which a

large amount of amplified exogenous DNA remains avail-

able. Consistent with this, the muscle expression of GFP in

embryos injected with pCK-EGFP/pMLC-RFP became

weak and even undetectable at late embryonic stages when

most of the exogenously DNA was presumably degraded.

Furthermore, we never observed muscle expression of RFP

in embryos injected with pCK-RFP/pMLC-EGFP because

more accumulated RFP is required for fluorescence visual-

ization. The “ectopic” expression of GFP and RFP was never

observed in skin cells, probably because the skin-specific CK

promoter is not as strong as the muscle-specific MLC pro-

moter. The lack of muscle expression was further confirmed

in stable transgenic F1 embryos in which only one or a few

copies of transgenes are integrated. Thus, our observation

indicated that a reliable analysis of DNA cis-element re-

mains the approach to introduce in vitro ligated DNA con-

structs.

In summary, in the present study, we have generated

2-color transgenic zebrafish using gfp and rfp reporter

genes, and both genes were correctly expressed in the tar-

geted tissues according to the specificity of the promoters

used. The 2-color transgenic model should facilitate com-

parative studies of development of multiple tissues or or-

gans and differentiation of different cell types in the same

individual.


ACKNOWLEDGMENTS

This work was supported by an academic research grant

from National University of Singapore (NUS). H.W. was

supported by a postgraduate scholarship from NUS.

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