Identification of Genetically Modified Zebrafish (Danio rerio)

by PCR method.
M. Bielikova (1,2), G. Bukovska (1), S. Vavrova (2), J. Timko (1), J. Turna (1,2)
1 Institute
2

of Molecular Biology, Slovak Academy of Sciences, Dbravsk cesta 21, 845 51 Bratislava, Slovakia
Department of Molecular Biology, Faculty of Natural Sciences, Comenius University, Bratislava, Slovakia

Introduction

(a) Zebrafish wild type

The import, sale and possession of fluorescent transgenic zebrafish

offered under the name GloFish in U.S. aquarium shops are not
permitted in the European Union. Transgenic zebrafish are
harbouring the gfp and/or dsRed genes coding for the green
fluorescent protein GFP (originally isolated from the jellyfish
Aequorea victoria), red fluorescent protein RFP (from marine
sponge Discosoma striata) and also yellow fluorescent protein YFP
which is a genetic mutant of green fluorescent protein.
In cooperation with Slovak inspectorate we have tested several
samples of zebrafish (Danio rerio) which came from Slovak
aquarium shops, where the screening controls have been
performed.
Various methods of biological sampling were applied: invasive (small
tail-clips) and non invasive (wipes from fish mouth on filter-paper
or buccal swab and wipes from fish body on filter-paper) and the
fish genomic DNA was isolated.
Several types of PCR have been performed to determine zebrafish
DNA wild type and transgenic zebrafish:
1. to detect amplifiable genomic zebrafish DNA using primers
specific for the zebrafish parvalbumin gene (IFF232, IFF233A and
IFF233B) and (PARfor1, PARfor2 and PARrev)
2. PCR with primers to specifically amplify the gfp gene (GFP1F,
GFP1R)
3. PCR with primers to specifically amplify the dsRed gene (RFP2206,
RFP2207)
4. PCR with primers to specifically amplify the YFP gene (YFP2005,
YFP2006)
5. PCR with primers to specifically amplify transgenic elements NPTII
(kanamycin/ neomycin antibiotic resistance selectable marker) and
the ancestral plasmid - pUC18 (NPT1in, Puc18b)

(b) Zebrafish transgenic

RESULTS
1. The control for amplifiable DNA using primers specific for the zebrafish parvalbumin gene: IFF232,
IFF233A and IFF233B.
The control for amplifiable isolated DNA was tested by PCR to detect amplifiable genomic DNA using primers
specific for the zebrafish parvalbumin gene (IFF232, IFF233A and IFF233B). The DNA was isolated from wt
Zebrafish. Both methods of biological sampling (invasive and non invasive) have been tested and also the time
after taking the sample (0, 24 and 48 hours).
M

10

11

Marker (100 bp, Fermentas)

1. wt zebrafish DNA, tail-clip, QIAamp DNA Investigator Kit
2. wt zebrafish DNA, tail-clip, ForensicGEM
3. wt zebrafish DNA, swab from fish body, QIAamp DNA Investigator Kit, 0 h
4. wt zebrafish DNA, swab from fish body, Chelex, 0 h
5. wt zebrafish DNA, swab from fish body, ForensicGEM, 0 h
6. wt zebrafish DNA, swab from fish body, QIAamp DNA Investigator Kit, 24h
7. wt zebrafish DNA, swab from fish body, Chelex, 24h
8. wt zebrafish DNA, swab from fish body, ForensicGEM, 24h
9. wt zebrafish DNA, swab from fish body, QIAamp DNA Investigator Kit, 48h
10. wt zebrafish DNA, swab from fish body, Chelex, 48h
11. wt zebrafish DNA, swab from fish body, ForensicGEM, 48h

Methods
Samples
Zebrafish to be presumed to be genetically modified because of the unusual red to
purple coloring (picture b) were collected in aquarium shops in Slovakia by GMO
inspectorate. Totally we had got 10 samples of Danio rerio designated as DR01
DR10. As a negative control and reference for unmodified zebrafish we have
used samples of the wild type zebrafish. Positive controls for the presence of
the GFP, RFP and YFP genes in PCR were: the vector pEGFP-N2 (Clontech) for
GFP and YFP genes and pDsRed2-N1 (Clontech) for RFP gene.

220 bp

2. The control for amplifiable DNA using primers specific for Danio rerio parvalbumin gene PARfor1,
PARfor 2 and PARrev (designed by GMO laboratory, Slovakia) and specific for the fish parvalbumin
gene: IFF232, IFF233A and IFF233B and TuDanioF, TuDanioR.

Sampling of biological material

Various methods of biological sampling were applied and tested: invasive (small
tail-clips) and non invasive (wipes from fish mouth on filter-paper or buccal swab
and wipes from fish body on filter-paper). The tail-clips were preserved in
ethanol; the samples on filter paper or cotton swab were usable for DNA isolation
up to 48 h.
Genomic DNA extraction and PCR
The samples of tail clips were collected by centrifugation, ethanol was remowed
and samples were dried at 37 C. Fish genomic DNA from tail clips and from
cotton swabs or filter paper were isolated by using QIAamp DNA Investigator Kit
(Qiagen) and ForensicGEM (Zygem, New Zealand). The samples from filter
paper or cotton swab were isolated also with Chelex. The quantity and purity of
isolated total DNAs were determined by Nano Drop spectrometer (Lab Tech, UK)
and five various PCRs were performed control for amplifiable DNA, detection of
green, red and yellow fluorescent protein genes and detection of flanking region.

USED
PRIMERS
IFF232
IFF233A
IFF233B
TUDanioF
TUDanioR
PARfor1*
PARfor2*
PARrev*
GFP1F
GFP1R
RFP2206
RFP2207
YFP2005
YFP2006
NPT1in
Puc18b

(*) Primers were designed by GMO laboratory (Slovakia) specific for Danio rerio parvalbumin gene
Other primers were either taken from literature or from the database of primers
established at CSL for GM detection purposes
( http://www.gm-inspectorate.gov.uk/documents/Detection_and_traceability_report_011205.pdf )

Primers:
Primers: PARfor1, PARfor 2 and PARrev
1. wt zebrafish DNA, swab from fish body, QIAamp DNA Investigator Kit
2. wt zebrafish DNA, swab from fish body, Chelex
3. wt zebrafish DNA, swab from fish body, ForensicGEM
Primers IFF232, IFF233A and IFF233B
Marker (100 bp, Fermentas)
4. wt zebrafish DNA, swab from fish body, QIAamp DNA Investigator Kit
5. wt zebrafish DNA, swab from fish body, Chelex
6. wt zebrafish DNA, swab from fish body, ForensicGEM
Primers TuDanioF,
TuDanioF, TuDanioR
7. wt zebrafish DNA, swab from fish body, QIAamp DNA Investigator Kit
8. wt zebrafish DNA, swab from fish body, Chelex
9. wt zebrafish DNA, swab from fish body, ForensicGEM
Marker (100 bp, Fermentas)

600 bp
387 bp
220 bp

3. The PCR detection of green (GFP1F,GFP1R) and red (RFP2206, RFP2207) fluorescent protein genes and
detection of flanking region (NPT1in, Puc18b) in samples of Danio rerio designated as DR01 DR10
GFP

Sequence 5' - 3'

GACAAGAGCGGCTTCATTGAGG
TCAATACCGATCTTGCCATCACCGT
TCAACTCCGATCATGCCATCACCAT
GCGCGAGTGCATATCCATCCAC
GTGCACTGGTCAGTCAGTTTGCG
CGACTCTTTCAACTACAAGAACT
GAGGATGAGCTGAAACTGTTC
GCTTTTCAAGACATTGAACATGG
TCGAGCTGGACGGCGACGT
GGTGCTCAGGTAGTGGTTGTC
ACAACACCGTGAAGCTGAAGGTGACCAAG
GGTGTAGTCCTCGTTGTGGGAGGTGATGTC
GACGGCGACGTAAACGGCCACAAGTTCA
GCCGATGGGGGTGTTCTGCTGGTAGTGG
ATGCTCTTCGTCCAGATCATCCTGA
GGTCTGACGCTCAGTGGA

RFP
4

10

M 11

12 13

14 15 M

1200
570 bp

505

4. Result of the PCR detection in samples of Danio rerio

DR01DR10 for parvalbumin gene (IFF, PAR), green
(GFP1F,GFP1R) and red (RFP2206, RFP2207) fluorescent
protein genes and detection of flanking region (NPT1in,
Puc18b)

CONCLUSION
In GMO laboratory (Slovakia) we have tested 10 samples of Zebrafish (Danio rerio) designated as DR01 DR10, which were presumed to be
genetically modified.
We verified
various methods of biological sampling - invasive and non invasive,
three methods of fish genomic DNA isolation
we have tested new PCR primers (TUDanioF, TUDanioR)
We can conclude that
non invasive wipe sampling techniques are usable and very convenient to use them in terrain by GMO inspectors and DNA can be isolated up
to 48 hours
all three tested methods: QIAamp DNA Investigator kit (Qiagen), Chelex and ForensicGEM (Zygem, New Zealand) are appropriate for
isolation of amplifiable fish genomic DNA
PCR primers for zebrafish alfa-tubuline gene (TUDanioF, TUDanioR) were more specific as common used primers
We detected two positive fishes DR01 and DR02 out of totally ten zebrafish which were tested. The samples DR01 and DR02 were positive on
both GFP and RFP gene. The presence of YFP gene was not detected.
We have received the same positive results as were detected for transgenic zebrafish in Germany, which contained two events, one containing gfp
and the other containing DsRed. No single vector in the literature is known to contain both these genes.

Marker (100 bp, Fermentas)

Primers:
Primers: GFP1F,GFP1R
1. pEGFP-N2 - positive control plasmid for GFP gene
2. sample DR01*
3. sample DR02*
4. wt zebrafish DNA
5. no template control
Primers:
Primers: RFP2206, RFP2207
Marker (100 bp, Fermentas)
6. pDsRed2-N1 - positive control plasmid for RFP gene
7. sample DR01*
8. sample DR02*
9. wt zebrafish DNA
10. no template control
Primers:
Primers: NPT1in/Puc18
Marker (100 bp, Fermentas)
11. plasmid positive control for flanking region
12. sample DR01*
13. sample DR02*
14. wt zebrafish DNA
15. no template control
Marker (100 bp, Fermentas)
*GMO inspectorate sampled Danio rerio #1 DR01 and #2, DR02

Sample

Test result (+/-)

IFF

PAR

RFP

GFP

Wild type

NPT1inpUC18
-

DR01 tail-clips
(QI*)

DR01 swab
(QI)

DR02 tailclips (QI)

DR02 swab
(QI)

DR03 DR09
swab (QI)

DR03 DR09
swab (chelex)

DR03 DR09
swab (zygem)

N/A

N/A

N/A

N/A

Non template
control

QI * isolated with Qiagen Investigator kit

N/A - Non analysed