Winnetal_MarBiotech

Bacteriophage l and Plasmid pUR288 Transgenic FishModels for Detecting In Vivo Mutations

Richard N. Winn,* Michelle Norris, Stacy Muller, Cecilia Torres, and Kathryn Brayer

Aquatic Biotechnology and Environmental Laboratory, Warnell School of Forest Resources, University of Georgia,

Athens, GA 30602, USA

Abstract: We adapted transgenic rodent mutation assays based on fish carrying bacteriophage l and plasmid

pUR288 vectors to address the needs for improved methods to assess health risks from exposure to environ-

mental mutagens and also to establish new animal models to study in vivo mutagenesis. The approach entails

separating the vectors from fish genomic DNA and then shuttling them into specialized strains of E. coli bacteria

to analyze spontaneous and induced mutations in either lacI and cII or lacZ mutational targets. Fish exhibited

low frequencies of spontaneous mutants comparable to the sensitivity of transgenic rodent models. Mutations

detected after treating fish with chemical mutagens showed concentration-dependent, tissue-specific, and

time-dependent relationships. Spontaneous and induced mutational spectra also were consistent with the

specificity of known mutagens, further supporting the utility of transgenic fish for studies of in vivo muta-

genesis.

Key words: transgenic, medaka, Fundulus, mutation, lambda, plasmid.

INTRODUCTION

Detection and analysis of mutations in vivo is the basis for

understanding their ultimate sources and roles after expo-

sure of animals to mutagenic agents in the environment.

Despite the importance of detecting mutations in whole

animals, routine mutation analyses have been hampered by

a lack of methods to efficiently recover and accurately iden-

tify mutant genes. Transgenic rodent models that carry spe-

cific genes for quantitating spontaneous and induced mu-

tations were developed to improve in vivo mutation analy-

ses (Mirsalis et al., 1995). In this approach, a transgenic

animal carries a prokaryotic vector harboring a gene that

serves as a mutational target. After exposure to mutagens,

the vector is separated from the animal’s genomic DNA and

shuttled into indicator bacteria where mutant and nonmu-

tant genes are readily identified (Gossen et al., 1993; Sum-

mer et al., 1989).

Mutation assays in transgenic animals provide numer-

ous benefits for in vivo mutation analyses not available

using other approaches. By combining the simplicity of an

in vitro approach in a whole animal, mutations are quan-

tified directly at the level of single genes, the ultimate end

point of DNA damage and repair. Transgenic mutational

target genes are genetically neutral, affording a distinct ad-

vantage over assays involving endogenous genes that are

limited to very specific tissues or developmental stages. As a

consequence, mutations persist and accumulate without

being subjected to selection in the animal, thereby permit-

ting examination of mutations in virtually any tissue or

developmental stage (Cosentino and Heddle, 1996; Swiger

Received January 31, 2001; accepted March 30, 2001

*Corresponding author: telephone 706-542-6227; fax 706-542-4942; e-mail

[email protected]

Mar. Biotechnol. 3, S185–S195, 2001DOI: 10.1007/s10126-001-0041-2

© 2001 Springer-Verlag New York Inc.

et al., 1999; Tao et al., 1993). Mutations among different cell

types can be compared to determine how such factors as cell

proliferation, metabolism, toxicity, and DNA repair influ-

ence mutagenesis. Large numbers of copies of the locus can

be rapidly screened to provide statistically meaningful re-

sults while also reducing the need for large numbers of

animals. Mutation analyses can be combined also with

analyses of neoplasia or enzyme induction, DNA sequenc-

ing, or other end points within the same animal to disclose

possible mechanisms of mutagen action.

Taking advantage of the adaptability of transgenic mu-

tation assays to different species, transgenic fish were de-

veloped to address the need to improve methods to assess

environmentally induced mutations and also the need for

new and better animal models to study in vivo mutagenesis

(Amanuma et al., 2000; Winn et al., 1995, 2000). Mutation

assays originally developed in rodents were readily adapted

to fish and may increase the efficiency of transgenic assays.

As fish become recognized as valuable animal models, the

generation of transgenic fish will enhance the overall value

of fish in research. In some applications, such as in assessing

health hazards associated with exposure to complex chemi-

cal mixtures or in low-dose chronic exposures, fish offer

distinct advantages over other animal models in providing

insights into the mechanisms of disease processes (Hawkins

et al., 1995).

We review the development and application of trans-

genic fish for mutation analyses to demonstrate that they

meet the fundamental practical requirements for study of

in vivo mutagenesis. Our focus is on the recently intro-

duced transgenic medaka (Oryzias latipes) that carry the

bacteriophage l vector harboring cII and lacI genes as

mutational targets (Winn et al., 2000). Our initial results

also are discussed on a new mutation assay based on the

plasmid pUR288 vector that carries the lacZ gene as a mu-

tational target in transgenic medaka and mummichog

(Fundulus heteroclitus). The findings illustrate that many

features of the fish models are shared among the transgenic

mutation models, including those based on transgenic ro-

dents carrying identical mutational target genes, further

supporting the continued use of fish in studies of in vivo

mutagenesis.

BACTERIOPHAGE l TRANSGENIC MEDAKA

Choice of Species

The medaka was selected for development as a transgenic

mutation model because it is widely used in environmental

toxicology and is the fish species of choice in carcinogenesis

bioassays (Bunton, 1999) and in studies of germ-cell mu-

tagenesis (Shima and Shimada, 1994). In addition, the

medaka’s small size, short generation time, cost-effective

husbandry, well-characterized histopathology, and amena-

bility to transgenic development are ideal for this applica-

tion. The transgenic medaka promises to enhance the ac-

knowledged utility of this species as a comparative animal

model.

l CII AND LACI MUTATION ASSAYS

Bacteriophage l-based mutation systems have numerous

beneficial features for developing a transgenic fish model

with broad applicability. The l-transgenic rodent mutation

assay is used widely, has an extensive database on a variety

of test compounds and test conditions, and has well-

described standardized procedures to enhance its compara-

bility. Bacteriophage lLIZ (∼45 kb) contains two muta-

tional target genes, lacI and cII, flanked at each end by cos

sites that allow excision and packaging of the l phage to

recover the vector from the animal’s genomic DNA. Muta-

tions in the cII and lacI target genes are analyzed by using

different assays described as follows (Figure 1).

lacI Mutation Assay

The most extensively used transgenic rodent in vivo

mutation assay is based on the lacI gene (1089 bp) con-

tained within the lLIZ bacteriophage vector (Kohler et al.,

1991a). Mutations are analyzed by using in vitro packaging

procedures to excise and package the l vector as viable

bacteriophage. The individually packaged phage infect and

lyse the Escherichia coli host whereby mutation-induced in-

activation of the lacI gene is detected by scoring the mutant

plaques by blue-white screening on a Dlac E. coli lawn on

agar plates containing 5-bromo-4-chloro-3-indolyl b-D-

galactoside (X-gal). The assay’s mutation spectrum has been

well characterized (De Boer et al., 1998), as well as its quan-

titative and statistical aspects (Piergorsch et al., 1995). Da-

tabases containing thousands of lacI mutations also have

been established (Cariello et al., 1997; De Boer, 1995). The

system detects most classes of mutations, including base

substitutions, single-base frameshifts, insertions, duplica-

tions, and deletions (Mirsalis, 1993; Provost et al., 1993).

cII Mutation Assay

A positive-selection assay, originally developed for l

transgenic rodents that uses the cII gene as a mutational

S186 Richard N. Winn et al.

target, was introduced as a logistically simple, cost-effective

alternative to the lacI mutation assay (Jakubczak et al.,

1996). The cII mutation assay was the focus of l transgenic

medaka studies because it has numerous characteristics that

increase the efficiency of developing and testing the fish

mutation model (Winn et al., 2000). The assay is based on

the role the cII protein plays in the commitment of bacte-

riophage l to the lysogenic cycle in E. coli host cells. A

specialized E. coli strain (hfl−) extends the longevity of the

cII product, facilitating selection of mutant cII l. The l

phage with wild-type cII produce lysogens that are indis-

tinguishable in the E. coli lawn, whereas l phage that carry

a mutation in cII are selected as they form plaques on the

bacterial lawn when incubated at 24°C. The smaller size of

cII target (296 basepairs vs. 1080 bp for lacI) also facilitates

efficient characterization of specific mutations because the

entire gene can be directly sequenced. Despite the more

limited analysis of the cII locus compared to lacI, results

from rodent and fish studies support its continued use as an

acceptable alternative target to lacI (Harbach et al., 1998;

Watson et al., 1998; Winn et al., 2000; Zimmer et al., 1998).

Production of l Transgenic Medaka

Transgenic medaka were produced using lLIZ vector DNA

ligated at cohesive termini (cos ligation) to form linear con-

catamers before microinjection into one-cell-stage medaka

zygotes (Winn et al., 2000). The cos ligation of the l vector

apparently improves the number of rescued phage per ge-

nome by protecting the integrity of most intervening cohe-

sive termini that are important in phage assembly (Dycaico

et al., 1994). Nine germ-line transmitting medaka founders

were identified. Mosaic integration of the transgene in the

germ-line of the founders was indicated by the variable

frequencies of transmission observed among offspring.

Mendelian inheritance of the transgene in the offspring ob-

tained from sibling crosses (>5 generations) supported the

conclusion that the l vector is integrated in a single chro-

mosomal site in each transgenic lineage.

Recovery of l Vector from Fish Genomic DNA

The l-based mutation assays rely on in vitro packaging

extracts to excise the intact l vector from the transgenic

Figure 1. Bacteriophage l-based transgenic medaka mutation as-

says using lacI (1089 bp) or cII (297 bp) mutational targets. After

isolating genomic DNA, the l vector (∼45 kb) is excised and

packaged similarly for each using in vitro packaging procedures.

Individually packaged phage infect and lyse the E. coli host

whereby mutation-induced inactivation of the lacI gene is detected

by scoring mutant plaques by blue-white screening on a Dlac E.

coli lawn on agar plates containing X-gal. In the cII assay, packaged

phage infect a hfl− E. coli host which extends the longevity of the

cII product to facilitate selection of mutant cII. Phage containing

wild-type cII produce lysogens and are indistinguishable in the E.

coli lawn, whereas l phage with a mutation in cII are selected by

forming plaques when incubated at 24°C.

Transgenic Fish Models for Detecting Mutations S187

animal’s genomic DNA and package it into viable phage

particles for subsequent infection of E. coli host cells. The

efficiency with which the vector can be recovered was

shown to be a practical requirement for transgenic rodent

mutation assays (Dycaico et al., 1994). Using standard in

vitro packaging methods and cII mutation assay procedures,

efficiencies in recovering l vector from fish genomic DNA

were variable, with the numbers of plaque forming units

(PFU) recovered associated with the number of l copies

carried among the medaka lineages (Winn et al., 2000).

Lineage l310, shown to carry ∼74 l copies/haploid genome,

exhibited unprecedented vector recovery with an average of

∼60,000–70,000 PFU/µg DNA from various tissues. The

highly efficient recovery of the vector from this lineage in-

dicated that it would satisfy practical assay requirements

and would permit analysis of mutations in individual fish,

precluding the pooling of multiple samples. Previous stud-

ies of transgenic rodents (Dycaico et al., 1994) and fish

(Winn et al., 1995) demonstrated a similarly enhanced re-

covery of the vector from animals carrying multiple copies

of bacteriophage vectors.

Spontaneous cII Mutant Frequencies

Determining the frequency of spontaneous mutations at the

cII locus was essential in establishing the utility of the fish

model for detecting induced mutations. The sensitivity of

mutation assays is defined by the magnitude of induced

mutational response compared to the frequency of sponta-

neous mutation (Heddle et al., 2000). Mutation analyses

were performed using offspring from transgenic parents

that were homozygous for the l transgene. Ranges of spon-

taneous mutant frequencies observed in individual trans-

genic fish were slightly lower than, but comparable to,

ranges of cII mutant frequencies in l transgenic rodents,

indicating that fish have at least equivalent, if not a some-

what greater sensitivity of the two in detecting induced

mutations. Frequencies of cII mutants in medaka lineage

l310 varied significantly among tissues with the lowest fre-

quency in testes, followed by whole fish and liver (Winn et

al., 2000) (Table 1). Subsequently, spontaneous mutant fre-

quencies were obtained for eyes, whole blood, and skin

(Table 1). A benefit afforded by the efficient recovery of the

vector and low variability in spontaneous mutant frequen-

cies in fish is that few animals (6–7 fish/treatment) are

required to detect a significant induction of mutations over

background. Analysis of spontaneous mutant frequencies in

l310 fish indicate that no de novo mutations have accu-

mulated in the germ-line of this lineage in over five gen-

erations.

Response of cII to Chemical Mutagens

Responsiveness of the cII target gene to mutagen treatment

was examined after exposing fish to ethyl-N-nitrosourea

(ENU) and dimethylnitrosoamine (DMN). ENU is a well-

characterized mutagen that directly ethylates oxygen and

nitrogen in the bases of DNA (Singer, 1976; Singer et al.,

1978). ENU is widely used as a mutagen in saturation mu-

tagenesis screens (Mullins et al., 1994; Solnica-Kretzel et al.,

1994) and as a standard control mutagen in transgenic ro-

dent assays (Mirsalis et al., 1994). The cII target was highly

responsive to ENU treatment, reflecting the induction of

mutations consistent with the mutagen’s concentration, tis-

sue-specificity, manifestation time, and known modes of

ENU action (Winn et al., 2000). cII mutant frequencies in

whole fish were induced 2.7-fold and 4-fold over untreated

fish sampled 15 days after 1 hour of treatment at 60 and 120

mg/liter ENU, respectively. Frequencies of cII mutants in

fish sampled at 5, 15, 20, and 30 days after ENU treatment

illustrated the tissue-specific influence of sampling time on

mutagenesis. Mutant frequencies in livers of fish treated

with ENU did not increase significantly above those of un-

treated fish sampled five days after treatment but increased

significantly by 3.5-fold at 15 days and continued to in-

crease to nearly 7-fold above background at 30 days. In

contrast, mutant frequencies in the testes were induced 5.2-

fold over the mean background mutant frequency at 5 days

and reached a peak of 10-fold induction at 15 days, followed

by a nonsignificant decline to 8.8-fold above background at

20 and 30 days after ENU treatment.

Table 1. Recovery and Spontaneous cII Mutant Frequencies in

Tissue from Transgenic Medaka Lineage l310

Tissue

Total

fish

Total

mutants

Mean

PFU

Mean mutants

×10−5 PFU

(± SEM)

Whole fish 12 599 1,468,917 3.9 (0.4)

Testes 26 537 1,123,455 1.9 (0.2)

Liver 29 1352 1,342,701 3.1 (0.2)

Eyes 10 207 426,500 2.8 (0.1)

Skin 4 95 1,666,250 1.5 (0.3)

Blood 3 70 1,018,333 2.4 (0.4)


We examined induction of mutations in cII further by

using dimethylnitrosoamine (DMN), a potent liver carcino-

gen used extensively as a model mutagen in assays with

transgenic rodents. DMN is among a class of nitrosamines

that induce hepatocarcinogenesis in fish with progressive

stages similar to those characterized in rodent hepatic neo-

plasia (Hawkins et al., 1995). We observed significant in-

ductions of cII mutants in liver 15 days after treatment with

DMN at 0, 300, and 600 mg/liter administered over 96

hours in a static renewal exposure regimen (Figure 2). Mu-

tants were induced 7.1-fold (16.7 ± 2.8 × 10−5), and 16.4-

fold (38.4 ± 6.6 × 10−5) above controls (2.1 ± 0.25 × 10−5)

at 300 and 600 mg/liter, respectively.

The different frequencies of induced mutations ob-

served in various fish tissues at equivalent mutagen treat-

ments illustrate a valuable attribute of the transgenic fish

mutation assay. The interval between mutagen treatment

and analyses, termed mutation manifestation time, is af-

fected by several variables, including tissue/cell type, muta-

gen, and mutagen-treatment regimen, that must be consid-

ered in designing and interpreting mutation studies (Hara

et al., 1999; Sun et al., 1999; Walker et al., 1999). For ex-

ample, the relatively higher magnitude of mutation induc-

tion and their shorter manifestation time in testes com-

pared to liver suggests that these differences reflect differ-

ences in cell proliferation rates and ENU action among

these tissues. These results also indicated that a 15-day sam-

pling time may be sufficient to detect a significant 2-fold

induction in most fish tissues, although an expression time

>30 days may be required for weak mutagens; a 5-day ex-

pression time is probably suitable only for the most potent

mutagens. Because cell proliferation is a prerequisite for

DMN-produced methyl DNA adducts to become fixed as

mutations (Mirsalis, 1993), results from the DMN exposure

study illustrate further the importance of considering cell

proliferation in designing exposure regimens. A distinct

value of using transgenes that are genetically neutral is that

mutations persist and accumulate without being subjected

to selection in the animal. Repeated exposures may, there-

fore, more closely mimic realistic environmental exposures

if chemicals are administered during periods of cell prolif-

eration induced by a previous exposure. As a consequence,

accumulation of mutations in transgene targets indicates

that repeated or chronic mutagen treatments should in-

crease the sensitivity of the assay (Heddle et al., 2000). The

amenability of fish to a wide range of exposure regimens

suggests that transgenic fish are ideally suited for such

evaluations.

Spontaneous and Chemically InducedMutational Spectra

An important benefit of transgenic mutation systems is the

ability to analyze mutations at the level of the DNA se-

quence. Sequencing the target gene recovered from the ani-

mal can provide important information on the spectra of

mutations induced by specific compounds and indicate

possible mechanisms of mutagen action. Sequencing the

target gene also may prove to be particularly useful in ex-

amining whether small increases in mutant frequencies after

exposure to chemicals at low environmental concentrations

are accompanied by shifts in mutational spectra. After veri-

fying the phenotype of cII mutant phage using selective

plating conditions, a 446-bp product that included the en-

tire 294 bp cII gene was sequenced. Spontaneous and

chemically induced cII mutational spectra in fish appear to

be similar to spectra in transgenic rodents. Single-base sub-

stitutions comprised the majority of spontaneous and ENU-

(Winn et al., 2000) and DMN-induced mutations in fish,

with a large percentage of the G:C → A:T mutations at CpG

sites (Table 2). The different modes of action of ENU and

DMN are reflected in the mutational spectra. The propor-

tions of mutations at A:T basepairs were higher in ENU-

treated livers compared to untreated livers, with the bulk of

the increase being A:T → T:A transversions, consistent with

the greater mutagenic effect of ENU at A:T basepairs (Shane

et al., 1997; Walter et al., 1998). ENU produces O6-

ethylguanine, O4-ethylthymidine, and O2-ethylthymidine in

DNA, promoting G:C → A:T and A:T → G:C transitions

and A:T → T:A transversions (Shelby and Tindall, 1997;

Shibuya and Morimoto, 1993), respectively. The majority of

Figure 2. cII mutant frequencies (± SEM) in livers from medaka

15 days following exposure to 0, 300, or 600 mg/liter DMN in a

static renewal regimen. Mutant frequencies were significantly el-

evated over the mean background (2.1 ± 0.25 × 10−5) at 300 (16.7

± 2.8 × 10−5) and 600 (38.4 ± 6.6 × 10−5) mg/liter DMN.


frameshifts observed in the spontaneous mutation spectrum

in the fish were either insertions or deletions within a

known cII hotspot (Watson et al., 1998). Their percentage

decreased in the ENU-exposed fish consistent with studies

showing ENU does not induce high numbers of frameshift

mutations (Shelby and Tindall, 1997). Single-base substitu-

tions were the most frequent mutations in the liver of

DMN-treated fish, with G:C → A:T transitions being more

numerous than transversions which is characteristic of

DMN exposure and attributable to the mispairing of O6-

methylguanine with thymine, eventually leading to G:C →A:T transitions during replication (Wang et al., 1998).

Spontaneous and ENU-Induced Mutations in lacI

A significant advantage of the l-based transgenic medaka is

afforded by the ability to analyze mutations in the lacI target

gene also contained in the lLIZ vector. Despite limited

studies of the lacI target in the l transgenic medaka, recent

work demonstrated the feasibility of analyzing spontaneous

and ENU-induced mutations in the lacI locus recovered

from these fish (Shih et al., 2001). Comparable numbers of

l-lacI phage were recovered from untreated and ENU-

treated fish, with PFU numbers approaching those typically

obtained from transgenic rodents. Frequencies of sponta-

neous lacI mutants in whole fish also were comparable to

ranges in most rodent tissues (Dycaico et al., 1994; Kohler

et al., 1991b) and somewhat lower than cII mutant frequen-

cies in fish (Winn et al., 2000). The frequencies of lacI

mutants were increased 7-fold after a 1-hour exposure to

120 ppm ENU over untreated fish, compared with a 4-fold

induction in cII with identical ENU treatment. Sequencing

of the lacI mutants disclosed spontaneous and ENU-

induced mutational spectra similar to those in rodent mod-

els.

PLASMID PUR288 TRANSGENIC MEDAKAAND MUMMICHOG

Choice of Species

The mummichog also was selected for this application be-

cause this species has many attributes that are well-suited

for transgenic development and environmental toxicology.

The mummichog has well-described embryology, a small

adult size, controllable year-round spawning, a transparent

chorion, short embryogenesis and generation time, and a

proven amenability to transgenic production (Winn et al.,

1995). In addition, as a common inhabitant of coastal and

estuarine environments, the mummichog is one of the most

extensively used organisms in studies of the risks of envi-

ronmental contaminants (Atz, 1986; Eisler, 1986) and in

ecotoxicological studies (Lotrich, 1975; Vogelbein, 1990).

lacZ Mutation Assay

A transgenic mouse model based on a plasmid pUR288

vector containing the lacZ gene as the mutation target was

introduced as an alternative to the bacteriophage l-based

mutation assays (Boerrigter et al., 1995) and was adapted to

the two species of fish discussed here (Figure 3). In this

approach, the pUR288 vector, flanked by HindIII restriction

sites and containing the entire lacZ sequence as the muta-

tional target (3096 bp), is recovered from the transgenic

animal’s genomic DNA by binding the lac repressor protein

to the operator sequence located in front of the lacZ gene.

After exposure to a mutagen, the transgenic animal’s geno-

mic DNA is digested with HindIII to release monomeric

plasmid sequences. The plasmid then is separated from the

genomic DNA by affinity capture using magnetic beads to

recover the lacZ plasmid sequences. After being circularized

Table 2. Mutational Spectra for Spontaneous cII Mutants (Liver,

Testes, and Whole Fish Combined) and DMN-Exposed Livers

Spontaneous

300

mg/liter

600

mg/liter

Total mutations 89 42 44

Mutations outside cII 5 0 6

Independent mutations 74 37 34

Transitions % (N) % (N) % (N)

G:C → A:T 20 (15) 67 (25) 73 (25)

A:T → G:C 12 (9) 5 (2) 3 (1)

Transversions

G:C → T:A 20 (15) 11 (4) 0

G:C → C:G 11 (8) 0 6 (2)

A:T → T:A 4 (3) 3 (1) 3 (1)

A:T → C:G 7 (5) 3 (1) 0

CpG 47 (7) 20 (5) 12 (3)

Frameshift

(+) 13 (10) 0 3 (1)

(−) 11 (8) 11 (4) 9 (3)

Other 0 0 3 (1)


by ligation, concentrated plasmids are transferred into

lacZ−, galE− E. coli by electroporation in the presence of

phenyl-b-D-galactoside (P-gal) (Gossen et al., 1992). Mu-

tant frequencies are determined as the ratio between the

numbers of colonies on selective (P-gal) LB agar plates ver-

sus the number of colonies on nonselective plates.

The plasmid pUR288 vector and associated lacZ rodent

mutation assay has several advantages over bacteriophage-

based rodent assays that may be useful in a transgenic fish

model. The pUR288 plasmid-based system is more efficient

than the bacteriophage system at rescuing lac genes from

transgenic mice (Gossen et al., 1993) due, in part, to the

high capacity of the lacI repressor magnetic bead to purify

the plasmid from the restriction-enzyme-digested genomic

DNA in a single step. In addition, in contrast to the llacI

mutation assay, mutations are detected by a selective pro-

cedure rather than by scoring colors, thereby reducing am-

biguity in analyses. Possibly more importantly, the plasmid-

based mutation assay appears to have a superior ability to

detect point mutations, small deletions and insertions, as

well as large-scale deletions and rearrangements induced by

clastogenic agents, such as radiation (Tao et al., 1993). Res-

cue of plasmids is not as size-dependent as are bacterio-

phage l vectors that require two intact cos sites for vector

packaging whereby deletions extending into regions adja-

cent to the l transgene may prevent recovery of the vector.

Production of Plasmid pUR288 Transgenic Fish

Plasmid pUR288 DNA (from Dr. Jan Vijg, University of

Texas Health Science Center, San Antonio, TX, USA) was

prepared as linear concatamers and microinjected into the

one-cell zygotes of medaka and mummichog using similar

procedures to those previously described (Winn et al., 1995,

2000). Fifteen medaka shown to be positive for the plasmid

sequence by PCR analyses (15/165 fish) were mated with

nontransgenic fish, and germ-line transmission was con-

firmed in six founders (6/15 fish). Mosaic integration of the

transgene into the germline was indicated by the variable

transmission frequency among the founders (4%–44%). In-

tegration of the plasmid in a single chromosomal site in the

transgenic lineages was confirmed from subsequent Men-

delian inheritance of the transgene beyond three genera-

tions. Nineteen mummichog were confirmed positive for

the plasmid sequence by PCR (19/304 fish), and Mendelian

inheritance of the plasmid was confirmed in two founders

(2/19 fish).

Figure 3. The plasmid pUR288 (∼5 kb)

is flanked by HindIII restriction sites and

contains the entire lacZ gene (3089 bp) as

the mutational target that is recovered

from the transgenic animal’s genomic

DNA by binding the lac repressor protein

to the operator sequence in front of lacZ.

Genomic DNA is digested with the

restriction enzyme to release monomeric

plasmids, which are recovered using

affinity capture with magnetic beads.

Plasmids are circularized by ligation and

transferred into lacZ-, galE- E. coli by

electroporation in the presence of p-Gal.

Mutant frequencies are determined as the

ratio between the number of colonies on

selective (P-gal) plates versus the number

of colonies on non-selective plates.


pUR288 Recovery and lacZSpontaneous-Mutant Frequencies

By using the positive selection mutation assay (Boerrigter et

al., 1995; Boerrigter, 1998; Dolle et al., 1996), pUR288 plas-

mids were recovered from genomic DNA isolated from one

medaka and mummichog lineage (Table 3). Rescue of plas-

mids ranged from 910,000 to 3,400,000 CFU/sample in

medaka lineage pUR127, to 52,000 CFU/sample in the

mummichog lineage, indicating that the recovery of the

vector is sufficient to perform the mutation assay. Medaka

lineage 127 and mummichog lineage 99 showed spontane-

ous lacZ mutant frequencies within ranges comparable to

those of transgenic mice carrying the identical target (Boer-

rigter et al., 1995). Studies are underway to characterize the

remaining lineages and to test the relative responsiveness of

the lacZ target after exposing fish to chemical mutagens.

UTILITY OF TRANSGENIC FISHMUTATION MODELS

Transgenic rodent models developed for assessing sponta-

neous and induced mutations have demonstrated numer-

ous benefits for studies of in vivo mutagenesis over other

approaches (Mirsalis et al., 1995). Among their more dis-

tinctive advantages is their amenability to comparisons of

mutagenesis among different cells, tissues, and species. Al-

though transgenic fish mutation models were introduced

only recently, the results demonstrate that fish share many

features of mutation analyses in rodent models and support

their continued use in studies of in vivo mutations. Studies

on bacteriophage l fish mutation assays based on lacI, and

to a greater extent, on cII, establish a good basis for evalu-

ating the relative utility and efficacy of using transgenic fish

models for mutation analyses. The efficient vector recovery

and low variability among fish has facilitated analyses of

mutations in a variety of tissues using a relatively small

number of animals. Spontaneous mutant frequencies in the

two target genes are low, comparable to that in transgenic

rodents, indicating sufficient sensitivity for detecting in-

duced mutations. Treating fish with chemical mutagens re-

sulted in concentration-dependent inductions in each target

gene and in tissue-specific and time-dependent inductions

in the cII locus. Sequencing of the cII and lacI mutants

recovered from fish illustrate that the mutational spectral

shifts are consistent with known mechanisms of mutagen

action. Initial results on developing the pUR288 plasmid

medaka and mummichog demonstrate that these models

also have promise as alternative mutation models that may

be useful for detecting a broad spectrum of mutations com-

parable to the existing pUR288 plasmid mouse model.

Two additional transgenic fish mutation assays adapted

from rodent assays have been introduced. Transgenic mum-

michog were produced based on the bacteriophage

fX174am3cs70, in which mutations are detected by rever-

sion of am3 to wild-type phage by one transition and two

transversions of a single A:T basepair (Winn et al., 1995).

The spontaneous mutation frequency in these fish was com-

parable to that of transgenic mice (Burkhart et al., 1993). A

transgenic zebrafish (Danio rerio) was derived from a mouse

mutation assay (Gondo et al., 1996), based on the pML4

plasmid containing mutations in the rpsl gene as the mu-

tational target (Amanuma et al., 2000). Spontaneous and

chemically induced mutant frequencies were consistent

with studies in the transgenic mouse. Further testing of

these systems may provide valuable comparisons of muta-

tions detected in different transgene targets and species.

By extending transgenic mutation systems to another

species such as fish, various factors influencing mutagenesis

can be identified in identical DNA sequences in different

cells, tissues, and species in applications that could not be

attempted or performed otherwise. Transgenic fish muta-

tion assays also offer excellent opportunities to improve the

assessment of genetic risks associated with exposure to

chemicals in aquatic systems. By taking advantage of the

amendability of fish at all life stages to a variety of exposure

regimens ranging from microinjection of embryos (Walker

et al., 1996) to static renewal and flow-through chronic

exposures (Kane et al., 1996), transgenic fish can be used in

a wide range of applications to enhance the assessment of

risks from exposure to waterborne and sediment-associated

Table 3. Transmission Frequency, Recovery, and Spontaneous

Mutant Frequencies in pUR288 Transgenic Medaka and Mum-

michog Lineages

Lineage

Transmission

frequency

(%)

Total

(CFU)

Mutant

frequency

×10−5

CFU

Medaka

127 44 910,000– 3,400,000 6.6–9.4

Mummichog

99 37 52,000 5.6–12.0


chemical contaminants. When used in combination with

measures of other toxicological end points in fish, mutation

analyses may prove useful to more fully characterize bio-

logical impacts of environmental hazards. Finally, the trans-

genic fish may make important contributions to emerging

issues related to environmentally induced developmental

and heritable diseases.

ACKNOWLEDGMENTS

This work was supported in part by grant R24RR11733

from the National Institutes of Health National Center for

Research Resources, grant RR251139 from the Georgia Ad-

vanced Technology Development Center, and grant

RR389930 from the Georgia Research Alliance.

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