Journal of Environmental Biotechnology
Vol. 8, No. 2, 105–110, 2008

Original paper (regular paper)

Genetically Modified, Red Fluorescent Zebrafish: Detection, Crossing,Inheritance of Red Fluorescence, and Tolerance to Low Temperatures
KIMIKO AMANUMA*, NOBUYOSHI NAKAJIMA, AKIKO H. HASHIMOTO and YASUNOBU AOKINational Institute for Environmental Studies, 16–2 Onogawa, Tsukuba, Ibaraki 305–8506, Japan* TEL: +81–(0)29–850–2390 FAX: +81–(0)29–850–2588* E-mail: amanuma.kimiko@nies.go.jp(Received; 28 September, 2008/Accepted; 10 October, 2008)
Under Cartagena Protocol domestic law, genetically modified (GM) fish have not yet received permission to be imported
or sold in Japan. However, it is anticipated that ornamental, fluorescent GM fish will be imported and sold prior to any
oﬃcial process for evaluating their eﬀects on biological diversity as GM organisms (GMO). Therefore, we have devised amethod for detecting GM fish that utilizes PCR for the detection of replication origins of plasmids that generally exist as
transgenes integrated in the GMOs’ chromosomes. Using red fluorescent zebrafish (rfZF), sold in Japan, and rpsL GM ZF
established by us, we demonstrated the feasibility of this method for detecting GMO. Next, we examined the biologicalcharacteristics of the GM rfZF. They were able to cross with non-GM ZF; the red fluorescence was inherited by their progeny
according to expected outcomes, based on Mendelian genetics. Examination of the low temperature tolerance of rfZF andnon-GM fish indicated that the lethal, minimum temperature was the same for both fish (5°C). These findings suggest thatthe transgene of the rfZF could spread among wild-type ZF, and that these ZF could overwinter in the southernmost part ofJapan. We conclude that a continuous monitoring is required.
Key words: genetically modified fish, red fluorescent zebrafish, temperature tolerance

1. Introduction
A number of genetically modified zebrafish (Brachydanio
rerio) (GM ZF) and medaka (Oryzias latipes) have beendeveloped by introducing fluorescent protein genes linked todifferent tissue specific promoters into fish. This transgenic
technology has also been applied to develop novel, ornamental GM fish with intense fluorescence. Chou et al. produced GFP medaka with intense fluorescence2), and Gong
et al. introduced GFP, RFP, and YFP into ZF4). The latter
GM fish named GloFish was commercialized in the USA(http://www.glofish.com). The GFP medaka, named TK-1,
and the GFP/RFP ZF, named TK-2, are sold in Taiwan(http://www.azoo.com.tw/). ZF and medaka are indigenous
to South Asia and East Asia, respectively, and are distributed
broadly in these areas. Once these GM ZF or medaka arereleased outside of laboratories, they could potentially influence the biological diversity of native fish by crossing withnative species and competing with them for survival, resulting in an alteration of the ecosystem.
In Japan, under Act on the Conservation and Sustainable
Use of Biological Diversity through Regulations on the Useof Living Modified Organisms (Cartagena Protocol domesticlaw), GM fish have not received permission for importationor sale since the law was enacted in 2004 (http://www.bch.
biodic.go.jp/english/law.html). However, it is anticipatedthat GM fish, most notably ornamental, fluorescent fish, will
be imported and sold without any official process for evaluating their effects on biological diversity as GM organisms(GMO)5). Actually, red fluorescent-colored ZF (rfZF) of
probably GMO origin were imported and sold in a pet shopin early 2007. These rfZF were obtained by the Ministry of
the Environment of Japan and were shown to be GMO, as
they contained the DsRed 2 (Clontech Laboratories, Inc.,
CA, USA) transgene as judged by researchers in the Fisheries
Research Agency of Japan. In light of this, a procedure fordetecting GM fish without any prior information regarding
the integrated transgene is required. In the present study, weestablished a procedure for judging whether or not fish
suspected of being GMO are genetically modified without
possessing any prior information regarding their specifictransgenes. Moreover, using rfZF, we also examined the inheritance of red fluorescence in progeny fish, the ability of
rfZF to crossbreed with native ZF, and the low temperature
tolerance of the rfZF. All of these properties are likely to
influence the effect of any released rfZF on the biologicaldiversity of native species in Japan.

2. Materials and Methods
2.1. Zebrafish
rfZF suspected to be GMO were obtained from a pet shop

106

AMANUMA et al.

by the Ministry of the Environment of Japan. They wereoriginally examined in the Institute of Fisheries Research
Agency of Japan where researchers demonstrated the presence of the DsRed 2 transgene in the rfZF. rpsL GM ZF,
which had been established by us using AB line of ZF andmaintained for over 10 generations, carries approximately
350 copies of the pML4 plasmid in their chromosomal
DNA1). As non-GM fish, ZF AB line was used. All kinds offish were maintained in rearing water (0.1% Instant Ocean
salts) at 24–25°C with a 14 h light/10 h dark cycle.
2.2. Polymerase chain reaction (PCR)
Primer sets used for amplification of ori and ZF betaglobin chain were [forward 5’-ACGAGGGAGCTTCCAGGGGGA-3’ and reverse 5’-TCGTTCGGCTGCGGCGAGCGGT-3’] and [forward 5’-AGGTGTCTCATCGTGT
ACCCCT-3’ and reverse 5’-CTTACCCTGAAGTTGTCGGGA-3’], respectively. The primer set for detecting both
DsRed1 and DsRed2 was [forward 5’-TGTCCCCCAGTTCCAGTACG-3’ and reverse 5’-CGATGGTGTAGTCCTCGTTGTG-3’].
We performed PCR reactions in a final volume of
20 microL of rTaq polymerase reaction mixture (attachedrTaq polymerase) containing additionally 0.2 mM dNTPs,
2.5 mM MgCl2, 0.1 microgram DNA, 20 pmole of eachprimer and 1 unit of rTaq polymerase (Toyobo Co. Ltd.,
Osaka, Japan). The reaction conditions were 35 cycles at
94°C for 1 min, 55°C for 1 min and 72°C for 2 min. PCR
products were separated by agarose gel electophoresis andvisualized with ethidium bromide. As a positive control,
PCR against the beta-globin chain was performed simultaneously to confirm that the PCR worked properly.
2.3. Temperature Tolerance Experiment
Four month old F1 fish and AB line were used. F1 fishwith red fluorescence (distinct red and light red (orange)),and without fluorescence (yellow fish) had been obtained bymating males and females rfZF. Seven fish of each typewere put into 2-L of rearing water in plastic tanks. Thesetanks were placed in temperature-controlled incubators(Sanyo Electric Co., Ltd., Osaka, Japan), and the temperature
was decreased 1°C per day. Fish were fed Tetramin tropicalflake food once a day. Aeration was performed once a day,and half of the rearing water was changed twice a week.
Ten hatched fry (post-fertilization day, pfd, 6), whichwere hemizygous rfZF obtained by mating rfZF ID No. 3
and fish from the AB line, were put into 0.3-L of rearing
water in a plastic tank and placed in the same incubator as
described above, in which the temperature was decreased
1°C per day. Fry were fed once a day a kind of zooplankton,
termed Rotifera, prepared in our institute. Aeration was
performed once a day, and the rearing water was exchanged
once or twice a week. After 2 weeks, when the temperatureof the incubator had decreased to 10°C, 5 of the 10 fry
were returned to the tank at 24°C, and the other 5 fry were
kept in the low temperature tank. The former 5 fry were fed
both Rotifera and brine shrimp (Artemia franciscana). The
phenotypes of all these fry were red and black striped.
We checked all the fish in the incubator at least once aday. With the decrease in temperature, fish gradually reduced
their movements and finally came to rest at the bottom of
the incubator. The fish were considered to be dead when
their hearts stopped beating and/or when they were unable
to recover after return to a higher temperature.

3. Results and Discussion
3.1. Visual observation of rfZF
Ten rfZF brought into the National Institute for Environmental Studies had no black stripes and differed slightly incolor; some looked distinctly red, while others were light redor orange (Fig. 1). A large number of progeny, most ofwhich were red-colored, were obtained by mating, indicatingthat the rfZF fish could reproduce. A few yellow fish without
red fluorescence were found in the progeny, indicating that
rfZF had been produced from mutant yellow (sometimes
called golden) ZF, and that there were hemizygous andhomozygous fish in the rfZF population.
3.2. Detection of GM fish by PCR for the replication
origin of the plasmid
A transgene is generally prepared as a plasmid, a ‘transgene construct’, which carries the actual transgene sequence(protein coding sequence linked to a promoter), a selectable
marker such as an antibiotic resistant gene and an origin of
replication (ori). To prepare a large amount of the ‘trans-

Fig. 1. Red fluorescent ZF used in this study.
Numbers 2 and 3 appeared distinctly red, and numbers 1, 4 and 5 looked light red or orange. The sex of each fish was established aftermating with non-GM fish.

Characterization of red fluorescent GM zebrafish

gene construct’ for establishing the GMOs, plasmids withefficient ori are generally used. To our knowledge, almost
all of the plasmids currently used for large scale preparations contain the ori derived from colicin E1 (ColE1); pBR
and pUC plasmids bear either the ColE1 ori or the ColE1
ori with a single base substitution, respectively. GM fish are
generally established by microinjection of the ‘transgene
constructs’ into fertilized eggs, followed by selection ofgerm-line transformants. Therefore, most established GM
fish lines possess plasmid-derived ori sequences integrated
into their chromosomal DNA.
We designed PCR primers for detecting both types of
ColE1 ori. As shown in Fig. 2, amplified products were observed in DNA prepared from two kinds of GM fish, rfZF
and rpsL GM ZF, and from E. coli bearing the plasmid
pUC19, whereas no products were amplified using the DNAof the negative control, i.e. non-GM fish. We confirmedthat the amplified product in the rfZF DNA sample wasobtained from the ColE1 ori by DNA sequencing.
To confirm that PCR had proceeded correctly, PCR

107against the beta-globin chain was performed at the sametime. An amplified band of the beta-globin chain was observed in the negative control samples (non-GM fish),suggesting that PCR had not been inhibited in these samples. Thus, GM fish containing the ColE1 ori can be distinguished from non-GM fish by PCR against the ColE1 ori.
Theoretically, this ori-directed PCR is also applicable fordetecting other GMOs carrying a plasmid vector, but not
for GMOs with a phage vector or a retrovirus vector.
3.3. Crossing rfZF with native ZF and the inheritanceof red fluorescenceFig. 2. PCR amplification of the plasmid replication of origin andbeta-globin gene.
Genomic DNA prepared from the following samples were subjected to PCR.
Lane 1, rfZF; lane 2, non-GM fish (AB line); lane 3, rpsL GM
fish; lane 4, DNA from E. coli bearing pUC19.

To examine the crossbreeding of rfZF with native ZF andthe inheritance of red fluorescence, each of 5 rfZF wascrossed with ZF from the AB line. We succeeded in obtaining offspring from all the breeding combinations. All theoffspring possessed black stripes, which is consistent withthe observation that pigment mutants are generally recessive.
Approximately half of the offspring of fish ID1, 4 and 5
were red and almost all of the offspring of fish ID2 and 3
were red (Table 1), indicating that their parents were hemizygous and homozygous with regard to the red fluorescencephenotype, respectively. When a hemizygous male and ahemizygous female were crossed, approximately 75% of the
fish had a red body color (Table 2). Thus, the red fluorescence phenotype was inherited, as expected, according to
Mendelian genetics.
Several offspring obtained from each combination were
examined to determine whether they carried the DsRed
transgene by PCR. As shown in Fig. 3, the red fish bodycolor was consistent with the presence of the DsRed transgene in these fish. All red-colored fish contained the DsRed
sequence, whereas the non-colored fish did not. These findings indicate that the DsRed gene was integrated stably into
the chromosome of vfZF.
3.4. Temperature Tolerance Experiment
Four types of fish (7 fish for each type) were utilized inexperiments to compare the low temperature tolerances ofTable 1. Inheritance of red fluorescence after crossbreeding red fluorescent ZF with the AB line.
F0

F1a
Phenotype

ID

1
2
3
4
5

Sex

F
F
M
M
M

No. of F1 fishobtained
39
44
111
108
158

Black stripes

Genotype F0 parentb

Red fluorescence

No. of fish%

No. of fish

%

39
44
111
108
158

100
100
100
100
100

22
44
110c
48
72

56
100
99
44
46

hemi
homo
homo
hemi
hemi

F1 were obtained by crossing each F0 with several non-GM ZF of the AB line.
The genotype of the F0 parent was determined from the percentage of rfZF in the F1 generation.
c
One fish without red color was seen. We concluded that it was probably a contaminant from the non-GM F1 generation of other crossings,
because in the same morning we also collected non-GM fertilized eggs from crossings between non-GM fish.
ab

AMANUMA et al.

108

Table 2. Inheritance of red fluorescence after crossbreeding hemizygous red fluorescent ZF.
Offspring

rfZF parents

PhenotypeFemaleMale

No. ofoffspring obtained

F0 No. 1
F1 Orange

F0 No. 4
F0 No. 5

1064Black stripes

Red fluorescence

No. of fish%

No. of fish

%

0
0

0
0

7
52

70
81

Fig. 3. PCR amplification of the DsRed gene.
Genomic DNA samples from F1 fish, whose body colors were yellow, orange (light red), and distinctly red, and non-GM fish (AB line)
were subjected to PCR.

the GM and non-GM fish. Three types consisted of the F1
fish of rfZF: these were distinct red fish (rfZF), light red(orange) fish (rfZF) and yellow fish (non-GM), obtained bymating rfZF. The remaining type was the AB line. The
temperature was decreased 1°C per day, mimicking thetemperature change in autumn and winter in Japan. Whenthe temperature reached 10°C, all 4 types of fish stopped
swimming actively and started to eat poorly. At 7°C, alltypes of the fish did not move at the bottom of the tankunless startled. At 5.5°C, 5 fish out of 7 of the AB line weredead, while the other three types of fish were alive. The
differences in sensitivity to low temperature may be explained by the fact that the AB line is an inbred strain andthe others are probably not. At 4.5°C, all the fish were dead(Fig. 4). No differences in low temperature tolerance wereobserved between distinct red fish (rfZF), light rfZF andyellow non-GM fish. These findings are consistent with areport by Cortemeglia and Beitinger3), which found relatively
small absolute differences in mean temperature tolerances
between wild-type and transgenic ZF (Glofish) (5.3°C and
5.6°C, respectively), following an initial acclimation to
20°C.
The F1 hemizygous hatched fry (post-fertilization day,pfd, 6) obtained by mating rfZF ID No. 3 with non-GM ZF(AB line) were challenged with cold by decreasing the temperature of the incubator by 1°C per day. The temperature

control fry, obtained at the same time, were kept at 25°C.
Although both fry were fed similarly, their sizes were significantly different after 2 weeks; the median length of thecontrol fry was approximately 10 mm or above, while themedian length of the fry in the cold incubator was approximately 7 mm. At 11°C, the fry started to swim unusually,
displaying oscillatory movements. On the following day, at
10°C, 5 fry out of 10 were returned to the tank at 25°C.
The remaining 5 fry were exposed to further decrements intemperature. At 8°C, one of the 5 fry died, and the remaining 4 fry died by the time the temperature was reduced to
6.5°C (Fig. 5). When compared to adult fish, both the rfZF
and AB line fry were more sensitive to cold temperature(Fig. 5).
To examine the reproductive ability of the cold-exposed
fry, the fry that had been exposed to cold temperature andthen returned to the normal rearing temperature (25°C)
were reared to adulthood. Several cold-exposed females
were mated with native males (AB line), and several coldexposed males were mated with native females. Both matinggroups successfully produced fertilized eggs, and the eggs
developed normally, suggesting that exposure to cold, produced by gradually lowering the temperature to 10°C, is notnecessarily detrimental to the reproductive capacity ordevelopment of ZF.

Characterization of red fluorescent GM zebrafish

109

Fig. 4. Low temperature tolerance of red fluorescent ZF.
Survival curves of three groups of fish with different body colors (7 fish for each group, including distinctly red, yellow and black stripedfish) are shown. The curve of the light red (orange) fish is not shown since it was the same as that of the distinctly red fish. The temperature of the incubator was decreased 1°C per day.

Fig. 5. Low temperature tolerance of F1 hemizygous fry.
F1 hemizygous fry, identified by the red fluorescent color and black stripes, were obtained by mating rfZF and non-GM fish (AB line). Ten
F1 fish were divided into 2 groups of 5 fish each; one was challenged with continual drops in temperature until death (fry A), while theother group was returned to normal temperature after the temperature in its incubator reached 10°C (fry B). The survival curve of theadult F1 rfZF from Fig. 3 is shown for reference.

4. Conclusion
4.1. Detection of GM fish without information regarding the GMO
Based on the results obtained in this study, we proposethat the described method is useful for judging whether or
not fish suspected of being GMO are, in fact, geneticallymodified, because almost all ornament GM fish may havebeen established utilizing plasmid vector.
DNA, generally most-readily extracted from the fin, isutilized as a template in PCR, using primers for the ori andbeta-globin chain. The amplification of ori confirms thatthe fish is a GMO. Furthermore, identification of the integrated transgene is desirable to confirm this conclusion.
When no amplification of ori is observed, but amplificationof globin is observed, the fish is likely not a GMO. Whenamplification of neither the globin gene nor the ori is detected, there is a problem either with the extracted DNA
sample and/or the PCR reaction. In these cases, new DNA
extracts should be prepared again, and/or the PCR mixtures
should be prepared again more carefully before performing
the reactions again.

4.2. Potential risk for biological diversity
We demonstrated here that rfZF are able to cross withnative ZF. Furthermore, we showed they could not survive
below a temperature of 4.5°C, but that they could live at
6.5°C for at least one day. Moreover, fry exposed to cold
temperature (10°C) were able to grow to adulthood andnormally reproduce. These findings indicate that the transgene in the rfZF could spread among wild-type ZF, and that
rfZF, as well as wild-type ZF, could overwinter in the natural
water environments of Japan, for example, in the southernmost part of Japan or near hot springs. Continuous surveillance is required to avoid spreading either the transgenes
among natural, non-GM ZF or the GM fish themselves.

Acknowledgements
We thank Ichiro Oohara (the Fisheries Research Agencyof Japan) and Hiroshi Horiuchi (the Ministry of the Environment of Japan) for helpful comments and discussion.
We also thank Tamaki Takahashi, Yukari Sakashita, andMami Kobayashi for maintaining the GM fish, and Satomi

AMANUMA et al.

110

Karube for maintaining the AB line.

References
1) Amanuma, K., H. Takeda, H. Amanuma, and Y. Aoki. 2000.
Transgenic zebrafish for detecting mutations caused by compounds in aquatic environments. Nat. Biotechnol. 18: 62–65.
2) Chou, C.Y., L.S. Horng, and H.J. Tsai. 2001. Uniform GFPexpression in transgenic medaka (Oryzias latipes) at the F0
generation. Transgenic Res. 10: 303–315.

3) Cortemeglia, C., and T.L. Beitinger. 2006. Projected US distributions of transgenic and wildtype zebra danios, Danio rerio,
based on temperature tolerance data. J. Therm. Biol. 31: 422–
428.
4) Gong, Z., H. Wan, T.L. Tay, H. Wang, M. Chen, and T. Yan.
2003. Development of transgenic fish for ornamental and bioreactor by strong expression of fluorescent proteins in the skeletal muscle. Biochem. Biophys. Res. Commun. 308: 58–63.
5) Kanou, K., Y. Furuhata, T. Yoshida, and H. Horiuchi, 2007.
Japanese J. Ichthyology (in Japanese) 54(2): 248–250.