Part B | 13.4
402 Part B Tools and Methods in Marine Biotechnology
screening or for genetic screening aimed at identifying mutations that suppress or enhance c-myc induced
carcinogenesis.
TP53 is the most frequently mutated tumor suppressor gene in human cancer, with about 50% of all
tumors exhibiting a loss-of-function mutation. To elucidate the genetic pathways involving p53 and cancer,
Berghmans et al. [13.128] developed transgenic zebrafish lines harboring missense mutation in the tp53
DNA-binding domain. Transgenic zebrafish expressing
the tp53 missense mutation transgene developed malignant nerve sheath tumors. These transgenic lines can
provide a unique platform for modifier screenings to
identify genetic mutations or small molecules that affect tp53-related pathways, including apoptosis, cell
cycle delay, and tumor suppression [13.129]. Very recently, a transgenic zebrafish model was established by
Liu et al. [13.130] to study intrahepatic cholangiocarcinoma (ICC) associated with infection by hepatitis B
(HBV) and C (HCV) viruses. These transgenic lines coexpress HBV X and HCV core protein genes in the liver
and cause eventual ICC formation in the liver. Results
of transcriptome sequencing analysis revealed that gene
expression profiles of ICC formation in the transgenic
fish are similar to those of human ICC formation. Thus,
this in vivo model can serve as an ideal platform for
identifying the molecular events leading to fibrosis and
ICC caused by HBV and HCV infection. It may also
serve as a platform for screening drugs to control hepatic fibrosis and ICC.
13.4.5 Transgenic Fish as Environmental
Biomonitors
Fish have long been used as models in environmental toxicology studies. Prompted by concerns of human
health, tissues of wild-caught fish have been used as indicators for the presence of dangerous pollutants such
as polycyclic hydrocarbons, oxidants, and heavy metals in the waters or fish itself [13.131, 132]. Different
assays have been employed to measure the biological parameters impacted by toxic pollutants, including
DNA damage, defense enzymes (e.g., glutathione peroxidase and superoxide dismutase), genes inducible
by toxic chemicals (e.g., cytochrome P450 1A1 and
1A2), and factors that regulate redox potential (e.g.,
glutathione and ascorbic acid) [13.131]. Although these
assays are very sensitive, they require specialized techniques and equipment, and thus cannot be performed in
the field where the samples are collected. In addition,
the data obtained from wild-caught fish samples give no
indication of the exact time when the fish were exposed
to the toxicants.
Transgenic fish can potentially serve as sensitive
sentinels for aquatic pollution, with the benefit of avoiding the inconveniences mentioned above. The principle
of such fish biomonitoring is to introduce reporter genes
that are easy to observe and are under the control
of promoter/enhancer elements of pollutant responsive
genes. GFP and its variants are thus the best reporters
of choice for easy optical observation, whereas luciferase is the most sensitive. So far, researchers have
made significant progress in establishing transgenic zebrafish carrying luciferase or GFP reporter genes under
the control of aromatic hydrocarbon response elements
(AHRE), electrophile response elements (EPRE), or
metal response elements (MREs) [13.131]. The specificity of each type of response elements is as follows:
AHREs respond to numerous polycyclic hydrocarbons
and halogenated coplanar molecules such as 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD) and polychlorinated biphenyls; EPREs respond to quinones and numerous other potent electrophilic oxidants; and MREs
respond to heavy metal cations such as mercury, copper, nickel, cadmium, and zinc. In the zebrafish cell
line, ZEM2S, these three types of elements can drive the
expression of luciferase reporters in a dose-dependent,
chemical-class-specific manner in response to more
than 20 environmental pollutants [13.131].
Many environmental chemical pollutants possess endocrine disrupting activities; these compounds
present a significant threat to human health. In recent
years, several transgenic zebrafish and medaka lines
have been produced for detecting environmental endocrine disruptors [13.132–138]. Lee et al. [13.136]
developed a transient expression assay for detecting
environmental estrogen via introducing a pair of plasmids (pRER_Gal4ff; pUAS_GFP) by microinjection
into 12-cell stage embryos of zebrafish or medaka.
The resulting transgenic embryos expressed GFP in response to exposure to pollutants containing estrogenic
activity. This assay system will allow easy testing of
environmental oestrogens, as well as detecting the oestrogenic target sites in developing embryos. However,
severe drawbacks of the assay are: i) big fluctuations
among embryos due to variations of the amount of
transgene plasmid in each embryo and ii) cumbersome
of microinjection. To overcome these problems, stable
lines of transgenic zebrafish or medaka carrying reporter systems can be developed. Chen et al. [13.133]
and Brion et al. [13.134] developed transgenic zebrafish
carrying ere-zvtg1-gfp and cyp19a1b-gfp transgenes,
402 Part B Tools and Methods in Marine Biotechnology
screening or for genetic screening aimed at identifying mutations that suppress or enhance c-myc induced
carcinogenesis.
TP53 is the most frequently mutated tumor suppressor gene in human cancer, with about 50% of all
tumors exhibiting a loss-of-function mutation. To elucidate the genetic pathways involving p53 and cancer,
Berghmans et al. [13.128] developed transgenic zebrafish lines harboring missense mutation in the tp53
DNA-binding domain. Transgenic zebrafish expressing
the tp53 missense mutation transgene developed malignant nerve sheath tumors. These transgenic lines can
provide a unique platform for modifier screenings to
identify genetic mutations or small molecules that affect tp53-related pathways, including apoptosis, cell
cycle delay, and tumor suppression [13.129]. Very recently, a transgenic zebrafish model was established by
Liu et al. [13.130] to study intrahepatic cholangiocarcinoma (ICC) associated with infection by hepatitis B
(HBV) and C (HCV) viruses. These transgenic lines coexpress HBV X and HCV core protein genes in the liver
and cause eventual ICC formation in the liver. Results
of transcriptome sequencing analysis revealed that gene
expression profiles of ICC formation in the transgenic
fish are similar to those of human ICC formation. Thus,
this in vivo model can serve as an ideal platform for
identifying the molecular events leading to fibrosis and
ICC caused by HBV and HCV infection. It may also
serve as a platform for screening drugs to control hepatic fibrosis and ICC.
13.4.5 Transgenic Fish as Environmental
Biomonitors
Fish have long been used as models in environmental toxicology studies. Prompted by concerns of human
health, tissues of wild-caught fish have been used as indicators for the presence of dangerous pollutants such
as polycyclic hydrocarbons, oxidants, and heavy metals in the waters or fish itself [13.131, 132]. Different
assays have been employed to measure the biological parameters impacted by toxic pollutants, including
DNA damage, defense enzymes (e.g., glutathione peroxidase and superoxide dismutase), genes inducible
by toxic chemicals (e.g., cytochrome P450 1A1 and
1A2), and factors that regulate redox potential (e.g.,
glutathione and ascorbic acid) [13.131]. Although these
assays are very sensitive, they require specialized techniques and equipment, and thus cannot be performed in
the field where the samples are collected. In addition,
the data obtained from wild-caught fish samples give no
indication of the exact time when the fish were exposed
to the toxicants.
Transgenic fish can potentially serve as sensitive
sentinels for aquatic pollution, with the benefit of avoiding the inconveniences mentioned above. The principle
of such fish biomonitoring is to introduce reporter genes
that are easy to observe and are under the control
of promoter/enhancer elements of pollutant responsive
genes. GFP and its variants are thus the best reporters
of choice for easy optical observation, whereas luciferase is the most sensitive. So far, researchers have
made significant progress in establishing transgenic zebrafish carrying luciferase or GFP reporter genes under
the control of aromatic hydrocarbon response elements
(AHRE), electrophile response elements (EPRE), or
metal response elements (MREs) [13.131]. The specificity of each type of response elements is as follows:
AHREs respond to numerous polycyclic hydrocarbons
and halogenated coplanar molecules such as 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD) and polychlorinated biphenyls; EPREs respond to quinones and numerous other potent electrophilic oxidants; and MREs
respond to heavy metal cations such as mercury, copper, nickel, cadmium, and zinc. In the zebrafish cell
line, ZEM2S, these three types of elements can drive the
expression of luciferase reporters in a dose-dependent,
chemical-class-specific manner in response to more
than 20 environmental pollutants [13.131].
Many environmental chemical pollutants possess endocrine disrupting activities; these compounds
present a significant threat to human health. In recent
years, several transgenic zebrafish and medaka lines
have been produced for detecting environmental endocrine disruptors [13.132–138]. Lee et al. [13.136]
developed a transient expression assay for detecting
environmental estrogen via introducing a pair of plasmids (pRER_Gal4ff; pUAS_GFP) by microinjection
into 12-cell stage embryos of zebrafish or medaka.
The resulting transgenic embryos expressed GFP in response to exposure to pollutants containing estrogenic
activity. This assay system will allow easy testing of
environmental oestrogens, as well as detecting the oestrogenic target sites in developing embryos. However,
severe drawbacks of the assay are: i) big fluctuations
among embryos due to variations of the amount of
transgene plasmid in each embryo and ii) cumbersome
of microinjection. To overcome these problems, stable
lines of transgenic zebrafish or medaka carrying reporter systems can be developed. Chen et al. [13.133]
and Brion et al. [13.134] developed transgenic zebrafish
carrying ere-zvtg1-gfp and cyp19a1b-gfp transgenes,