I – Introduction
A GMO, or genetically modified organism, is a plant, animal, microorganism or other organism
whose genetic makeup has been modified in a laboratory using genetic engineering or transgenic
technology. This creates combinations of plant, animal, bacterial and virus genes that do not
occur in nature or through traditional crossbreeding methods.
A transgenic fish is one that contains genes from another species
Genetically modified fish (GM fish) are organisms from the taxonomic clade which includes the
classes Agnatha (jawless fish), Chondrichthyes (cartilaginous fish) and Osteichthyes (bony fish)
whose genetic material (DNA) has been changed using genetic engineering techniques.
A transgenic fish is an improved variety of fish provided with one or more desirable foreign gene
for the purpose of enhancing fish quality, growth, resistance and productivity.
Typically, genes of one or more donor-species are isolated, and spliced into artificially
constructed infectious agents, which act as vectors to carry the genes into the cells of recipient
species. Once inside a cell, the vector carrying the genes will insert into the cell’s genome. A
transgenic organism is regenerated from each transformed cell (or egg, in the case of animals),
which has taken up the foreign genes. And from that organism, a transgenic variety can be bred.
In this way, genes can be transferred between distant species, which would never interbreed in
nature.
GM fish are used in scientific research and kept as pets. They are being developed as
environmental pollutant sentinels and for use in aquaculture food production. In 2015, the
AquAdvantage salmon was approved by the US Food and Drug Administration (FDA) for
commercial production, sale and consumption, making it the first genetically modified animal to
be approved for human consumption. Some GM fish that have been created have promoters
driving an over-production of "all fish" growth hormone. This results in dramatic growth
enhancement in several species, including salmonids, carps and tilapias.
Picture 1: AquAdvantage salmon
II – Principle of gene transfer in aquatic species:
1. Choice of target genes
The most popular gene used in aquatic species is growth hormone (GH) for reasons that
are obvious. GH has been widely used in terrestrial species and as the gene sequence is highly
conserved; the product is readily utilized across species boundaries. It may also be noted that, at
least in some cases, enhanced growth is associated with more effective utilization of food.
Picture 2: Results in induction of GMOs in aquatic species
Cold water temperatures are often a major problem in aquaculture in temperate climates
when an unusually cold winter can severely damage both production and brood fish stocks of
fish. Some marine teleosts have high levels of serum anti-freeze proteins (AFP) or glycoproteins
(AFGP) which reduce the freezing temperature by preventing ice-crystal growth. Fletcher, Hew
and Davies (2001) have shown that there is one class of AFGP and four classes of AFP. Most are
expressed primarily in the liver and some show clear seasonal changes (Melamed et al., 2002).
Work has particularly focussed on the production of AFP from the winter flounder (Pleuronectes
americanus), and the gene has been successfully introduced into the genome of Atlantic salmon,
integrated into the germ line and passed on to F3 offspring where it was expressed in the liver.
However, a number of Ala, Pro-specific endopeptidases are required for production of mature
proteins and these are not present in Atlantic salmon. Furthermore, the AFP gene in winter
flounder, and possibly other Arctic species, exists in many copies. Thus, much further work is
required in order to develop effective antifreeze activity in Atlantic salmon (Hew et al., 1999).
Work on AFP has also been conducted in goldfish (Wang et al., 1995) and milkfish (Wu et al.,
1998).
2. Isolation of the gene of interest
Usually the gene of interest will already be available as an element of a “library” of short
sections of the total genome of the donor strain or species. If this is the case the procedure
followed is to multiply the gene using the PCR reaction. If, however, the gene is to be taken from
a genome not previously investigated, a more complex procedure will need to be followed. The
use of the technique of the Polymerase Chain Reaction (PCR) enables the gene in both the cases
noted above to be multiplied to the level of several million copies needed for the generation of
the construct.
3. Cloning the gene of interest
When many copies of the target gene have been generated, the gene is placed in a
“construct”. Once the gene of interest has been ligated enzymatically into the construct, this
whole complex is ligated into bacterial plasmids, which act as “production vectors” and enable
the gene to be replicated many times within the bacterial cells. The bacteria are then plated out. It
is possible to tell from reporter genes whether the vector has been taken up by the bacterial cells.
This usually involves some colour change in the colonies containing inserted DNA. The many
times amplified DNA construct is then enzymatically cut out of the plasmids (after these have
been removed from the bacterial cells) and it is ready to be used for insertion into eggs of the
host species.
Picture 3: Step in DNA Cloning
4. The construct
A construct is a piece of DNA which functions as the vehicle or vector carrying the target gene
into the recipient organism. It has several different regions as shown in Picture 4. There is a
promoter region which controls the activity of the target gene, a region where the target DNA is
inserted, usually some type of reporter gene to enable one to ascertain whether the target has
combined successfully with the construct and a termination sequence.
Picture 3: Diagram of DNA sequence of a basic plasmid and incorporated construct.
The sources of these several DNA sequences may be different species although promoter
and target genes would ideally be derived from the same species
As shown in Picture 5, constructs have been reported from 92 studies. The number of
different constructs is greater than the number of target genes used in aquaculture and a
substantial research effort has been made in this area. From the early 1990s research focussed on
developing “all fish” constructs in preference to using mammalian promoters.
The use of all-fish constructs has dramatic effects on expression of transgenes, e.g.
Devlin et al. (1994), developed an all salmon gene construct which accelerates the growth of
transgenic salmonids by over 11 fold. In tilapia, Maclean (1994) found that using carp beta actin
instead of rat beta actin promoter led to a ten fold increase in production of hormone in
transgenic animals.
Picture 5: Summary of major research effort in inducing GMOs in aquatic species.
Other important work suggested that the optimal stage at which the transgene is
introduced might vary between cells and species eg. Garcia del Barco et al. (1994) using
Zebrafish showed that there were differences in the regulatory requirements for cells and
embryos, and suggested therefore that constructs should be assayed in both cells and embryos.
Other work shows how critical the nature of the gene construct is. Devlin et al. (1995a)
showed that using an opAFPGHc gene construct in coho salmon eggs gave rise to some alevins
which had the typical brown colouration, while the remainder displayed a distinct green
colouration. The results suggest that the green phenotype arose from the presence of the
opAFPGHc construct and therefore could be indicative of transgene uptake/transmission. All the
offspring were tested by PCR for presence of the transgene and 182 of 184 alevins were correctly
assigned on this basis. However, it was found that later in development all fish turned green (the
normal colour later in development) and so the transgenic fish were showing accelerated growth.
Later in development it was found that most of the transgenic fish showed signs of cranial
abnormality probably due to accelerated growth. While the construct was useful in that transgene
uptake could be monitored, further work was needed to ensure that healthy fish could be
produced.
5. Techniques for inducing transgenics
Transgenic fish have largely been produced through microinjection into fertilised eggs or
early embryos. Electroporation of sperm has been shown to be successful in some species eg.
Zebrafish (Khoo et al., 1992) Chinook salmon (Sin et al., 1994) and Loach (Tsai, Tseng and
Liao, 1995). Liposomes have also been utilized as vectors (Khoo 1995). Ballistic methods using
microprojectiles have been investigated in Artemia with a view to their use in generating
transgenic crustacea (Gendreau et al., 1995) and also in seaweed species (Qin et al.,1994).
“Baekonisation”, an electric, flat field type of electroporation was utilized to transfer DNA into
Zebrafish embryos (Zhao, Zhang and Wong, 1993), this method appeared to be successful but
has not been taken up in the same way as other forms of electroporation and microinjection
methods.
More recently the use of embryonic stem cells (ESC) as a method for inducing
transgenesis has been advocated. These cells are undifferentiated and remain totipotent, so they
can be manipulated in vitro and subsequently reintroduced into early embryos where they can
contribute to the germ line of the host. In this way genes could be stably introduced or deleted
(Melamed et al., 2002). Despite the early success of ESC technology in mice, the uptake of the
technology for fish has been slow, although early precursor cells (Mes 1) have been cultivated
from Medaka and show many of the same features as mouse ESC. Studies by Hong, Winkler and
Schartl (1996, 1998) and Hong, Chen and Schartl (2000) showed that 90 percent of host cell
blastulae transplanted with Mes 1 cells developed into mosaic fry, and these cells became
integrated into organs derived from all three germ layers, and differentiated into various types of
functional cells.
Another example of new and possibly more efficient ways for gene transfer is the use of
pantropic retroviral vectors. These are able to infect a wide range of host cells and have been
used to infect newly fertilized Medaka eggs with a reporter gene, which appeared to become
integrated into the entire germ line of some of the P1 females (Lu, Burns and Chen, 1997). In
Zebrafish when retroviral infection and microinjection were compared, the two methods were
equally efficient in passing the transgene into eggs, but there was wider variability in the extent
of reporter gene expression among those founders that were microinjected (Linney et al.,1999).
However, the use of retroviruses is not without problems.
The microinjection method is suitable for relatively small numbers of organisms being
manipulated whereas electroporation, sperm/liposome mediation and bombardment methods are
more suitable for mass treatments. The most popular method of insertion of transgenes in
aquaculture is microinjection; in 92 studies reviewed from 1985 to the present, 68 used
microinjection, eleven used sperm mediated methods, six used electroporation and five used both
sperm mediation and electroporation. However, the problem of mosaic expression of the
transgenes is common, and this gives rise to varying proportions of transgenic genotypes in the
progeny.
Picture 6: Microinjection Process
6. Expression of gene
The uptake and integration of a transgene does not guarantee that the gene will express
itself in the new genetic environment. Tests must be carried out to determine whether there is
expression and if there is expression, at what level this takes place. Clearly, in commercial
aquaculture only those transgencs expressing the target gene at a sufficiently high level will be of
interest.
7. Inheritance of gene
A fish which expresses the target gene at an acceptable level may not be able to transmit
the gene to progeny. This is because many transgenics are mosaic individuals and unless the
gonads are included in the tissues possessing the transgene the transgenic animals will not breed
true. Appropriate breeding tests must, therefore, be carried out.
The high proportion of mosaic individuals is one reason why the proportions of progenies
of different genotypes resulting from parents that are putatively hemizygous for a transgene do
not necessarily conform to mendelian expectations. Another reason is the integration of two or
more copies of the transgene at different sites in the recipient genome. Further breeding tests will
be required in order to establish a pure breeding line of transgenic fish.
References:
Journal:
Alam, M.S., Lavender, F.L., Iyengar, A., Rahman, M.A., Ayad, H.H., Lathe, R., Morley, S.D. &
Maclean, N. 1996. Comparison of the activity of carp and rat -actin gene regulatory sequences in tilapia
and rainbow trout embryos. Mol. Reprod. Dev. 45: 117-122.
Devlin, R.H., Yesaki, T.Y., Donaldson, E.M. & Hew, C.L. 1995a. Transmission and phenotypic
effects of an antifreeze/GH gene construct in coho salmon (Oncorhynchus kisutch). Aquaculture, 137:
161-170.
Devlin, R.H., Biagi, C.A., Yesaki, T.Y. Smailus, D.E. & Byatt, J.C. 2001. Growth of domesticated
transgenic fish. Nature 409: 781-782.
Garcia del Barco, D., Martinez, R., Hernandez, O., Lleonart, R. & de la Fuente, J. 1994.
Differences in transient expression directed by heterologous promoter and enhancer sequences in fish
cells and embryos. Journal of Marine Biotechnology, 1: 203-205.
Gendreau, S., Lardans, V., Cadoret, J.P. & Mialhe, E. 1995. Transient expression of a luciferase
reporter gene after ballistic introduction into Artemia franciscana (Crustacea) embryos. Aquaculture, 133:
199-205.
Hew, C.L. & Fletcher, G.L. 2001. The role of aquatic biotechnology in aquaculture. Aquaculture,
1: 191-204.
Hew, C.L., Jun, D.S., Gong, Z., Garth, F.L., Margaret, S., Peter, D.I. & Robert, D. 1996.
Biotechnology for aquaculture: Transgenic salmon with enhanced growth and freeze-
resistance. Biotechnol. Apl., 13: 49 pp..
Hew, C.L., Poon, R., Xiong, F., Gauthier, S., Shears, M., King, M., Davies, P. & Fletcher, G.
1999. Liver-specific and seasonal expression of transgenic Atlantic salmon harboring the winter flounder
antifreeze protein gene. Transgenic Research, 8: 405.
Hong, Y., Chen, S. & Schartl, M. 2000. Embryonic stem cells in fish: current status and
perspectives. Fish Physiology and Biochemistry, 22: 165-170.
Hong, Y., Winkler C. & Schartl, M. 1996. Pluripotency and differentiation of embryonic stem cell
lines from the medaka fish (Oryzias latipes). Mech. Dev. 60: 33-34.
Hong, Y., Winkler, C. & Schartl, M. 1998. Production of medakafish chimeras from a stable
embryonic stem cell line. Proc. Nat. Acad. Sci. 95: 3679-3684.
Khoo, H.W., Ang, L.H., Lim, H.B. & Wong, K.Y. 1992. Sperm cells as vectors for introducing
foreign DNA into zebrafish. Aquaculture, 107: 1-19.
Linney, E., Hardison, N.L., Lonze, B.E., Lyons, S. & Di Napolis, L. 1999. Transgene expression in
zebrafish: a comparison of retroviral and DNA microinjection approaches. Developmental Biology, 213:
207-216.
Lu, J.K., Burns, J.C. & Chen,T.T. 1997. Pantropic retroviral vector integration, expression, and
germline transmission in medaka (Oryzias latipes). Molecular Marine Biology and Biotechnology, 6: 289-
295.
Maclean, N. 1994. Animals with novel genes C.U.P.
Martinez, R., Arenal, A., Estrada, M.P., Herrera, F., Huerta, V., Vazquez, J., Sanchez, T. & de la
Fuente, J. 1999. Mendelian transmission, transgene dosage and growth phenotype in transgenic tilapia
(Oreochromis hornorum) showing ectopic expression of homologous growth hormone. Aquaculture, 173:
271-283.
Melamed, P., Gong, Z.Y., Fletcher, G.L. & Hew, C.L. 2002. The potential impact of modern
biotechnology on fish aquaculture. Aquaculture, 204: 255-269.
Sin, F.Y.T., Walker, S.P., Symonds, J.E. & Sin, I.L. 1994. Sperm-mediated gene transfer in
chinook salmon. High Performance Fish: Proceedings of an International Fish Physiology Symposium At
the University of British Columbia in Vancouver, Canada, July 16 21 1994. Ed. D. D. MacKinlay. pp.360-
365.
Qin, S., Zhang, J., Li, W., Wang, X., Tong, S., Sun, Y. & Zheng, C. 1994. Transient expression of
GUS gene in phaeophytes using biolistic particle delivery system. Oceanol. Limnol. Sin. Haiyang Yu
Huzhao, 25: 353-356.
Tsai, H.J., Tseng, T.S. & Liao, I.C. 1995. Electroporation of sperm to introduce foreign DNA into
the genome of loach (Misgurnus anguillicaudatus). Canadian Journal of Fisheries and Aquatic Science,
52: 776-787.
Wang, R., Zhang, P., Gong, Z. & Hew, C.L. 1995. Expression of the antifreeze protein gene in
transgenic goldfish (Carassius auratus) and its implication in cold adaptation. Molecular Marine Biology
and Biotechnology, 4: 20-26.
Wu, S.M., Hwang, P.P., Hew, C.L. & Wu, J.L. 1998. Effects of antifreeze protein on cold tolerance
in juvenile tilapia (Oreochromis mossambicus Peters) and milkfish (Channos
channos Forskaal). Zoological Science, 37: 39-44.
Zhao, X., Zhang, P.J. & Wong, T.K. 1993. Application of Baekonization: A new approach to
produce transgenic fish. Molecular Marine Biology and Biotechnology, 2: 63-69.
Website:
https://www.semanticscholar.org/paper/Circular-No-.-989-FIRI-%2F-C-989-(-E-)-
GENETICALLY-Beardmore-Porter/ff4dff7bc2f38aaf8c833b6518a276559c06dd89
http://www.biologydiscussion.com/gene/gene-transfer/physical-methods-of-gene-transfer-
genetics/72013
http://www.fao.org/3/y4955e/y4955e06.htm
https://biocyclopedia.com/index/biotechnology/genes_genetic_engineering/techniques_of_geneti
c_engineering/biotech_microinjection.php
