Transgenic Technology in Marine Organisms 13.2 Production of Transgenic Marine Organisms 391
Part B | 13.2
fusion proteins that are engineered by assembling the
DNA-binding motif of the transcription activator-like
effector (TALE) protein from Xanthomonas with Fok1
nuclease. These molecules function similarly to ZFNs.
Since TALEN-binding sites are expected to occur about
once in every 35 base pairs on random DNA sequences,
they will provide a greater flexibility in the selection of the target sites [13.61]. Very recently, another
new method of gene inactivation has emerged from
studies conducted on bacteria and archaea. In bacteria
and archaea, there exists arrays of clustered, regularly
interspersed short palindromic repeat (CRISPR) and
CRISPR-associated (CAS) genes that can provide acquired resistance against phages by degrading the phage
DNA or RNA [13.28, 62, 63]. From further studies on
the structural features of the CRISPR and CAS systems,
rational designing of CRISPR RNAs, and the CAS protein have been worked out for direct in vivo cleavage of
RNAs or DNAs in zebrafish or other organisms [13.64].
13.2.2 Selection of Marine Organism Species
Gene transfer studies have been conducted in several
different aquatic organisms including fish, shrimp, bivalves and algae. Since 1985, transgenic fish have been
produced for many fish species such as salmon, rainbow
trout, common carp, goldfish, loach, channel catfish,
northern pike, seabream, tilapia, walleye, zebrafish, and
Japanese medaka [13.1–3]. Depending on the purpose
of the transgenic studies, the embryos of some fish
species prove to be more suitable than others. In selecting a fish species for gene transfer studies, for instance,
a series of parameters should be considered. These parameters are:
i) Life cycle length
ii) Year round supply of eggs and sperm
iii) Culture conditions
iv) Size of the adult at maturity, and
v) Availability of background information on genetics, physiology, and endocrinology of the fish
species [13.5].
Japanese medaka (Oryzias latipes) and zebrafish
(Danio rerio) are regarded as ideal fish species for conducting gene transfer studies. Both fish species have
short life cycles (3 months from eggs to mature adults),
produce hundreds of eggs on a regular basis without
exhibiting a seasonal breeding cycle, and can be maintained in the laboratory for 23 years. Their eggs are
relatively large (1:01:5 mm in diameter) and possess
very thin and semi-transparent chorions. These features
allow easy injection of DNA into the newly fertilized
eggs if appropriate glass needles are used. Furthermore,
inbred lines and various morphological mutants of both
fish species are available. These fish species are ideal
candidates for producing transgenic fish for:
i) Studying developmental regulation of gene expression and action
ii) Identifying cis-acting elements that regulate the expression of a gene
iii) Measuring the activity of promoters
iv) Producing transgenic models for human diseases
and environmental toxicology. While the advantage
of using zebrafish or medaka as transgenic model
animals is due to the wealth of genetic and genomic databases, a major drawback of these two fish
species is their small body size, which makes them
less suitable for endocrinological and biochemical
studies.
The handicap of using medaka or zebrafish as experimental animals in gene transfer studies can be
overcome by the use of fish species such as loach, killifish (Fundulus), goldfish, and tilapia. The body sizes
of these fish species are big enough to collect sufficient
amounts of blood or tissue samples from individual fish
for biochemical and endocrinological studies. Another
important attribute of these fish species is their shorter
maturation time compared to rainbow trout or salmon.
In particular, tilapia is a very appropriate medium-sized
fish species for gene transfer studies, since it only requires about 4 months to reach reproductive maturation.
It is possible to produce almost three generations of
transgenic tilapia in 1 year. Unfortunately, the lack
of a well-defined genetic background and the asynchronous reproductive behavior of these fish species
present some problems for the collection of a sufficient
number of newly fertilized eggs to conduct gene transfer, unless a large number of reproductively mature fish
is available.
Rainbow trout, salmon, channel catfish, and common carp are commonly used as large body size model
fish species for transgenic fish studies. Since an extensive knowledge of the endocrinology, reproductive
biology, and physiology of these fish species is available, they are well suited for studies on comparative endocrinology, as well as aquaculture application.
However, the maturation time for each of these fish
species is relatively long. For example, rainbow trout
and salmon require 2–3 years to reach reproductive
maturity, and common carp or channel catfish require
Part B | 13.2
fusion proteins that are engineered by assembling the
DNA-binding motif of the transcription activator-like
effector (TALE) protein from Xanthomonas with Fok1
nuclease. These molecules function similarly to ZFNs.
Since TALEN-binding sites are expected to occur about
once in every 35 base pairs on random DNA sequences,
they will provide a greater flexibility in the selection of the target sites [13.61]. Very recently, another
new method of gene inactivation has emerged from
studies conducted on bacteria and archaea. In bacteria
and archaea, there exists arrays of clustered, regularly
interspersed short palindromic repeat (CRISPR) and
CRISPR-associated (CAS) genes that can provide acquired resistance against phages by degrading the phage
DNA or RNA [13.28, 62, 63]. From further studies on
the structural features of the CRISPR and CAS systems,
rational designing of CRISPR RNAs, and the CAS protein have been worked out for direct in vivo cleavage of
RNAs or DNAs in zebrafish or other organisms [13.64].
13.2.2 Selection of Marine Organism Species
Gene transfer studies have been conducted in several
different aquatic organisms including fish, shrimp, bivalves and algae. Since 1985, transgenic fish have been
produced for many fish species such as salmon, rainbow
trout, common carp, goldfish, loach, channel catfish,
northern pike, seabream, tilapia, walleye, zebrafish, and
Japanese medaka [13.1–3]. Depending on the purpose
of the transgenic studies, the embryos of some fish
species prove to be more suitable than others. In selecting a fish species for gene transfer studies, for instance,
a series of parameters should be considered. These parameters are:
i) Life cycle length
ii) Year round supply of eggs and sperm
iii) Culture conditions
iv) Size of the adult at maturity, and
v) Availability of background information on genetics, physiology, and endocrinology of the fish
species [13.5].
Japanese medaka (Oryzias latipes) and zebrafish
(Danio rerio) are regarded as ideal fish species for conducting gene transfer studies. Both fish species have
short life cycles (3 months from eggs to mature adults),
produce hundreds of eggs on a regular basis without
exhibiting a seasonal breeding cycle, and can be maintained in the laboratory for 23 years. Their eggs are
relatively large (1:01:5 mm in diameter) and possess
very thin and semi-transparent chorions. These features
allow easy injection of DNA into the newly fertilized
eggs if appropriate glass needles are used. Furthermore,
inbred lines and various morphological mutants of both
fish species are available. These fish species are ideal
candidates for producing transgenic fish for:
i) Studying developmental regulation of gene expression and action
ii) Identifying cis-acting elements that regulate the expression of a gene
iii) Measuring the activity of promoters
iv) Producing transgenic models for human diseases
and environmental toxicology. While the advantage
of using zebrafish or medaka as transgenic model
animals is due to the wealth of genetic and genomic databases, a major drawback of these two fish
species is their small body size, which makes them
less suitable for endocrinological and biochemical
studies.
The handicap of using medaka or zebrafish as experimental animals in gene transfer studies can be
overcome by the use of fish species such as loach, killifish (Fundulus), goldfish, and tilapia. The body sizes
of these fish species are big enough to collect sufficient
amounts of blood or tissue samples from individual fish
for biochemical and endocrinological studies. Another
important attribute of these fish species is their shorter
maturation time compared to rainbow trout or salmon.
In particular, tilapia is a very appropriate medium-sized
fish species for gene transfer studies, since it only requires about 4 months to reach reproductive maturation.
It is possible to produce almost three generations of
transgenic tilapia in 1 year. Unfortunately, the lack
of a well-defined genetic background and the asynchronous reproductive behavior of these fish species
present some problems for the collection of a sufficient
number of newly fertilized eggs to conduct gene transfer, unless a large number of reproductively mature fish
is available.
Rainbow trout, salmon, channel catfish, and common carp are commonly used as large body size model
fish species for transgenic fish studies. Since an extensive knowledge of the endocrinology, reproductive
biology, and physiology of these fish species is available, they are well suited for studies on comparative endocrinology, as well as aquaculture application.
However, the maturation time for each of these fish
species is relatively long. For example, rainbow trout
and salmon require 2–3 years to reach reproductive
maturity, and common carp or channel catfish require