The use of shuttle vectors for mutation analysis in transgenic mice and rats

~ ~|

.'

Fundamental and Molecular Mechanisms of Mutagenesis

ELSEVIER

Mutation Research 307 (1994) 461-478

The use of shuttle vectors for mutation analysis in transgenic mice and rats Mark J. Dycaico, G. Scott Provost, Patricia L. Kretz, Sherrie L. Ransom, Jane C. Moores, Jay M. Short * Stratagene, 11099 N. Torrey Pines Road, La Jolla, CA 92037, USA

(Received 1 June 1993; revision received 29 October 1993; accepted 5 November 1993)

Abstract

The establishment in recent years of transgenic shuttle vector-based mutagenicity assays has provided improved systems for analysis of mutagenic and carcinogenic processes. Results in .the mouse have stimulated the development of an alternate species suitable for mutation analysis and have increased our understanding of the existing models. A previously described shuttle vector (ALIZ), based on a lacI target gene, was constructed in this laboratory for the study of mutagenesis in transgenic mice and in cultured cell lines. The shuttle vector allows for several options in its recovery from the host genome and in mutant identification. Of the 9 transgenic lineages that were generated with the ALIZ vector, one was chosen for use in a standardized mutagenicity assay (Big Blue ®, mouse lineage A1). Characterization of this lineage included copy-number determination, chromosomal localization of transgene integration and analysis of copy-number stability. As part of the validation process, the standardized color-screening assay has been tested in the mouse, both for spontaneous mutant frequencies and with a variety of model mutagenic compounds, and has been shown to identify most major classes of mutations as evidenced by mutant spectra data. A discussion of the relative sensitivity o f the shuttle vector to each of these classes of mutations is included. These studies have now been extended to the generation of transgenic rats containing the same shuttle vector for cross-species analysis. Spontaneous mutant frequencies in two transgenic rat lineages were measured in liver and in germ cells. Preliminary data suggest that spontaneous mutant frequencies in somatic tissue are lower in rats than in mice, a result consistent with historical observations of D N A damage and repair in these two species. Also under evaluation are alternative selectable systems for mutant identification, and hybrid animals obtained from mating ALIZ transgenics with genetically engineered mice possessing an inactivated tumor suppressor gene. It is expected that each of these widely varying endeavors will contribute, not only in furthering our understanding of the role transgenic systems should play in human risk assessment, but in illuminating the mechanisms of mutation in general. Key words: Transgenic mice; Transgenic rats; Shuttle vector; LacI; Species-specific; Mutagenesis; Mutation spectrum

* Corresponding author. 0027-5107/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0027-5107(93)E0230-N

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M.J. Dycaico et al. / Mutation Research 307 (1994) 461-478 fl

1. Introduction

The evolution of transgenic animal technology has made in vivo systems more amenable to experimentation. For example, it is now possible to perform in vivo carcinogenesis studies in transgenic mice containing aberrantly regulated oncogenes and in mice that have had endogenous tumor suppressor genes inactivated (reviews include Adams and Cory, 1991; Fowlis and Balmain, 1993). Similarly, in vivo mutagenesis studies are possible in transgenic mice containing lambda phage shuttle vectors with bacterial lacZ (Short et al., 1988; Gossen et al., 1989; Kohler et al., 1990), lacI (Kohler et al., 1991a,b), or supF target genes (Summers et al., 1989), as well as in mice transgenic for a q~X174 phage shuttle vector containing an am3 amber mutation as a target for reversion mutagenesis (Burkhart et al., 1993). In addition, new species of transgenic animals such as transgenic lambda/lacI rats have been developed and characterized for mutagenesis testing (Dycaico et al., 1993). Combining multiple transgenes in one animal is also possible. For example, investigations in tumorigenesis and mutagenesis can now be carried out concurrently in the FI offspring of "p53 k n o c k o u t " × lambda/lacI transgenic mice. Furthermore, in vitro applications using cell lines derived from transgenic animals, or from the transfection of the transgene constructs, can complement in vivo studies. While the future of environmental mutagenesis, genetic toxicology and oncology will likely include the use of additional transgenic animal models, it is within our capabilities to probe many long unanswered questions with present technology.

1.2. The lambda /lacI shuttle vector Our laboratory has focused on the use of transgenic animals that harbor shuttle vectors for mutagenesis studies in mice and rats. The utility of shuttle vectors lies in the ability to transfer a suitable target D N A sequence from the study organism, such as a mouse or rat, to a convenient organism for analysis, such as a bacterial cell. For example, lambda bacteriophage shuttle vectors employ in vitro lambda packaging extracts as the

colEl

fl

cos

cos

7~. i ~ c : i

ci857

~r

45.5kb

XLIZ 1. 2, 3. 4, 5. 6, 7. 8,

transgenic mice transgenic rats hybrids with p53 knockout transgenics transtected tibroblasts phage color screening lysogenic selectable systems plasmld rescue lytlc selectable systems

Fig. 1. Schematic of the ALIZ shuttle vector and a listing of several systems developed based on the ALIZ vector.

means of recovery of the transgenic target D N A from the host's genomic DNA. The bacterial lacI gene, which encodes a repressor protein that regulates production of/3-galactosidase in E. coli, has been used as a mutational target in these vectors. The lacI gene has been used extensively as a target for mutagenesis and, using color screening for/3-galactosidase activity, its sensitivity to mutations has been demonstrated (Miller and Schmeissner, 1979; Lebkowski et al., 1985; Schaaper et al., 1986, 1991; DuBridge et al., 1987; Horsfall and Glickman, 1989; Halliday et al., 1990; Yatagai et al., 1991, summarized by Provost et al., 1993). The lambda/lacI shuttle vector can be used in a variety of ways to detect mutations (Fig. 1) (Kohler et al., 1991a,b; Lundberg et al., 1993). Typically transgenic animals or cell lines are exposed to the putative mutagen followed by a suitable expression period. DNA is then prepared from tissues or tissue culture samples and incubated with lambda in vitro packaging extracts (Kretz et al., 1989), which initiate packaging of the shuttle vector into infectious lambda bacteriophage. The recovered shuttle-vector phage are then used to infect restriction deficient (McrA-, McrBC-, M c r F - , M r r - , H s d R - ) E. coli K12 cells (Kohler et al., 1990; Kretz et al., 1991) to be assayed for mutations as lytic lambda phage plaques on a bacterial lawn. The ALIZ (l_acI_, a-lacZ_) vector was designed such that mutations

M.J. Dycaico et al. / Mutation Research 307 (1994) 461-478

that inactivate lacI will allow expression of a /3-galactosidase reporter gene (lacZ) in infected a-complementing E. coli cells. In the presence of 5-bromo-4-chloro-3-indolyl-fl-D-galactopyranoside (X-gal), a substrate for /3-galactosidase, a blue color is visible in lambda phage plaques containing mutant lacI genes while other plaques remain colorless (Kohler et al., 1991a,b; Young et al., 1992, 1993; Ashby et al., 1993; Gorelick et al., 1993; Gunz et al., 1993; Mirsalis et al., 1993; Provost et al., 1993, Shane et al., 1993). The lambda/lacI shuttle vector also lends itself to alternative screening methods. Because the trans-acting repressor protein encoded by lacI regulates gene expression by binding to an operator sequence, options for mutant selection systems exist through the use of various reporter genes. One such approach provides a growth advantage for lysogenic prophage colonies containing mutant lacI genes by making the expression of a-lacZ metabolically required (Lundberg et al., 1993; Kretz et al., 1992). Approaches that place other reporter genes under the regulation of the lac repressor are also possible (Kretz et al., 1993a,b; see later section). In addition, the ALIZ shuttle vector includes plasmid maintenance sequences, the colE1 origin of replication and the ampicillin resistance gene, to permit both target recovery and mutant screening as a plasmid rather than a lambda phage. This has been accomplished by excising the plasmid directly from the chromosomally integrated ,tLIZ vector using the restriction endonucleases BgllI and BarnHI. A variety of mutation detection methods, such as those mentioned above, can be applied following Table 1 Transgenic C57BL/6 mouse lineages (ALIZ) Founder lineage

Sex

Phage rescue

Copy number

Germ-line integration

A1 AB CR DA-S DA-W HE JA OM PA

male female female male male male female male male

yes yes yes yes yes no yes yes yes

~ 40 1- 3 1- 3 6-10 est. 3 - 5 est. 1- 3 1- 2 30-40 1- 3

yes yes yes yes yes sterile yes yes yes

463

subsequent purification, ligation and transformation of suitable E. coli strains. This plasmid approach has been described previously by Gossen et al. (1993). That the ALIZ vector can provide a diversity of recovery and screening techniques exemplifies the versatility of transgenic shuttlevector systems in general and their capacity to serve as models for in vivo mutagenesis.

2. The mice

lambda/lacl

shuttle vector in transgenic

2.1. Transgene localization Nine C57BL/6 transgenic mouse lineages have been generated (Table 1), each containing from 1 to approximately 40 chromosomally integrated copies of the ,~LIZ shuttle vector. The chromosomal integration site of the ALIZ transgene in the Big Blue ® mouse (designated the A1 mouse lineage), has been characterized using fluorescence in situ hybridization (FISH). Established as a valuable technique for localizing and mapping specific endogenous genes on mammalian chromosomes, FISH is also useful as a method for determining the integration site of transgenes in transgenic animal lineages (Lichter et al., 1991; Trask, 1991; McNeil et al., 1991). While there are several variations to the in situ hybridization technique, the approach used here involves preparing probes for the gene of interest by directly labelling the DNA with fluoresceinated nucleotides. These probes are then hybridized to metaphase chromosomes that have been isolated from the animal and fixed upon microscope slides. Under the appropriate experimental conditions, the probe anneals specifically to the complementary gene sequence on the chromosome. The fluorescent signal generated can be observed directly with a fluorescence microscope, thereby allowing the chromosomal position of the gene to be established. To determine the location of the transgenes in the A1 mouse lineage, fluorescently labelled probe DNA was prepared from the )tLIZ shuttle vector. After hybridization of the probe with chromosome spreads prepared from mouse spleen

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cells, the transgenes were d e t e r m i n e d to be in a discrete location on a single c h r o m o s o m e . This is as expected for a lineage that p r o d u c e s transgenic offspring at the predicted Mendelian frequency and also confirms previous Southern blot data indicative of a single insertion site. In addition, the reproducibly strong signal obtained from these hybridizations is consistent with a high copy number. This confirms data previously obtained by both d e n s i t o m e t r y analysis and scintillation counting of tail D N A dot blots that estimated the c o p y - n u m b e r of transgenes in the A1 lineage to be about 40, and that showed the transgene to be stably integrated over 5 generations (Short et al., 1992b).

To ascertain m o r e precisely the site of integration of the transgenes, co-localization experiments were p e r f o r m e d using the F I S H technique. For these experiments, probes m a d e from end o g e n o u s mouse genes of known c h r o m o s o m a l origin and probes for the transgenes were hybridized simultaneously to c h r o m o s o m e spreads. Fig. 2 shows the results of a co-hybridization experiment using the A L I Z probe and a gene probe specific for c h r o m o s o m e 4. A single bright signal (T) was obtained from the A L I Z probe annealing with the integrated shuttle-vector scquences. Hybridization of the c h r o m o s o m e 4specific probe resulted in two less intense signals (P), one emanating from the same c h r o m o s o m e

Fig. 2. Fluorescence in situ hybridizations of mouse chromosomes probed with transgene and chromosome-specific DNA probes. Metaphase chromosome spreads were prepared from mouse spleens using established procedures (Verma and Babu, 1989). Fluorescent probe DNA was generated from ALIZ (transgene) and H30 (chromosome 4 specific gene) DNA, using the Prime-lt ~" Fluor fuorescent labeling kit (Stratagene, La Jolla). In situ hybridizations were performed using the In Situ Hybridization kit (Stratagene, La Jolla). T, Transgene; P, Chromosome-specific probe. The chromosomes were overlaid with antifade containing propidium iodide and observed using an Olympus inverted epifluorescence microscope with 100 x objective and a 450-490-nm excitation wavelength, 520-nm emission wavelength filter.

M.Z Dycaico et al. / Mutation Research 307 (1994) 461-478

as the concatemeric transgene and one emanating from its homologous allele. The co-localization of the probes for the two genes demonstrates that, in the A1 mouse lineage, the ALIZ shuttlevector sequences are integrated in chromosome 4. Although exact mapping of the transgenic integration site has not been attempted, indirect evidence suggests that the shuttle-vector sequences may be integrated in the general region of the brown locus. This was inferred from comparison of the position of the transgenic signal with maps of the positions of known genes for mouse chromosome 4 (Lyon and Searle, 1989). In co-localization experiments with the gene for the brown locus the signal obtained from probe hybridization to the wild-type version of chromosome 4 was often clearly visible, yet a signal was never observed on the homologous chromosome which containined the integrated transgenes. This suggests that the brighter signal from the transgene may have overlapped and obscured the weaker signal from the chromosome 4 specific single copy gene.

2.2. Assay standardization With any given mutation assay the observed results are influenced by the experimental conditions used. Experimental conditions and procedures that affect mutant frequencies in the color-screening transgenic mutagenesis assay include: bacterial plating cell titers, plaque density per plate, X-gal concentration, agar medium components, incubation time and mutant screening methodologies. Therefore, for the purpose of mutagenesis testing, the development of standard protocols and internal assay sensitivity controls are important for achieving reproducible results. Using a standardized approach, which includes mutant plaque color controls, data gathered on mean spontaneous mutant frequencies demonstrate excellent intra-and inter-laboratory reproducibility within a given transgenic lineage (Rogers et al., 1993; Young et al., 1993). For instance, liver spontaneous mutant frequencies measured in 10 Big Blue ® B6C3F l hybrid mice (C57BL/6 A1 lineage transgenic × C3H) averaged 4.1 × 10 -5 (_+0.8 x 10 -5 S.D.) in one labo-

465

ratory study and 4.0× 10 -5 ( + 1 . 0 × 10 -5 S.D.) in a separate laboratory using similar procedures and conditions. Similar spontaneous mutant frequencies were observed in the C57BL/6 Big Blue ® strain; mean spontaneous liver mutant frequencies from 10 C57BL/6 mice were 4.1 x 10 -5 (_+0.69 S.D.) and 4.4 x 10 -5 (___0.91 S.D.) respectively in the different laboratories. In these initial studies, greater than 500 000 plaques were recovered from each animal. However, there was no statistically significant difference in the mutant frequencies after 200000-300000 plaques were recovered, based on a two tailed t-test of log transformed data, assuming a power of 80 and unequal variance. In another study, statistical analysis of inter-laboratory comparisons showed little excess variability in the transgenic assay among several levels of hierarchical protocol, with the possible exception of cases where the isolated DNA was stored for longer than 90 days prior to packaging (Piegorsch et al., 1994). Furthermore, using the standardized protocols and a minimum of 5 benzene-treated animals per test group, statistically significant (p < 0.05) inductions of less than 50% above background mutant frequencies were also detected by screening as few as 200000-300000 pfu per animal (Callahan and Short, in preparation). To validate the transgenic color-screening system, numerous mutagenic and non-mutagenic compounds have been tested using standardized protocols. Compounds tested in both the lambda/lacI mice and in other in vivo assays, have yielded results consistent with previous studies. Compounds tested include 2-acetylaminofluorene, di(2-ethylhexyl)phthalate, heptachlor, phenobarbital (Gunz et al., 1993), benzo[a]pyrene (Kohler et al., 1991; Young et al., 1992; Shane et al., 1993), cyclophosphamide (Kohler et al.,1991a), dimethyl benzanthracene (Thompson et al., 1992), dimethyl nitrosamine, methyl methanesulfonate (Mirsalis et al., 1993), ethyl nitrosourea (Kohler et al., 1991a; Provost et al., 1992), and methyl nitrosourea (Kohler et al., 1991b; Provost et al., 1993). Additional compounds evaluated (unpublished data) include benzene, the well characterized clastogen; 4-acetylaminofluorene, a noncarcinogenic analog of 2-acetylaminofluorene; o-

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M.J. Dycaico et al. / Mutation Research 307 (1994) 461-478

anisidine, a putative non-genotoxic bladder carcinogen; and tris (2,3-dibromopropyl) phosphate. 2.3. Target gene sensitivity

Previous analysis of mutations recovered from transgenic animals and analyzed by sequencing indicate that most classes of mutations are detected with the l a m b d a / l a c I transgenic assay, including base substitutions, single-base frameshifts, insertions, duplications and deletions (Kohler et al., 1991a; Mirsalis et al., 1993; Provost et al., 1993). In order to be detected, these types of mutations are restricted to the lacI target gene or the operator sequences of the ot-lacZ reporter gene (Fig. 1 and Fig. 3). Deletion mutations of up to approximately 7.5 kb are possible within the ALIZ vector, assuming a minimum lambda pack-

~

x

aging size of 38 kb. We have named this group of mutations intragenic to describe deletions occurring within the lambda vector (Short, 1992a). In addition to these intragenic deletions, there is another catagory of deletion mutations that we have termed intergenic, which describes a mutation where one deletion endpoint is positioned within the target gene of one integrated transgene and the other endpoint is positioned within a flanking transgene (Fig. 3). In the case of a lambda shuttle vector, such deletions can only be recovered by lambda packaging if the mutagenic event deletes and splices the two halves of the lambda phage in a manner that generates a packageable phage (38-51 kb). However, since mutant detection requires both the presence of at least one functional a - l a c Z gene and the absence of all functional lacl genes within the bacteriophage

45.5kb f

lacl

Chromosomal DNA

u p t o 7.5kb

Inh'ag~Ic

44.3 - ~ l ( b

89.8 - 98.5kb Inter~mlc 135.3- 143.8kb . . . 1.8Mb

Fig. 3. Intragenic and intergenic deletion possibilities in concatemeric ALIZ. Thick black bars indicate regions of detectable deletion endpoints. Thin black bars indicate minimum lengths that endpoints must span for detection of intergenic deletions.

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M.J. Dycaico et al. / Mutation Research307 (1994) 461-478

particle, the size range of the packaged and scorable ALIZ vector is 38-46.6 kb. The probability of a detectable intergenic deletion event appears relatively low. Yet, because transgenes typically integrate into the chromosome in multiple copies arranged in head-to-tail concatemers (Brinster et al., 1981; Constantini and Lacy, 1981), a large number of potential deletion endpoints are available. For the approximately 40 copy Big Blue ® mouse (A1 lineage) the largest deletion theoretically possible is approximately 1.8 megabases (Fig. 3). Without consideration of the possible mechanisms leading to deletions, the number of detectable deletion mutations expands quasi-geometrically with increasing copy number. The total number of intragenic and intergenic mutations detected using the colorscreening assay can be expressed as outlined in the empirically derived equation, Eq. 1"

intragenic component + [ n ( n 2 1) ] [ x 2 + ( f - 1 ) x + l ]

(1)

intergenic component

where S is the theoretical number of all possible deletions that can be detected, n is the shuttlevector copy number, x is the number of base pairs within the target gene, and f is the number of base pairs of non-lacZ sequence flanking the target region to one side that permits both a packageable phage and an observable mutation (Fig. 3). Each deletion mutation differing by one base is counted as a separate mutation in this formula. For comparison purposes, if we assume that every base pair of the target gene is 100% sensitive to deletion mutations, then a 40 copy lambda/lacI transgenic animal is capable of detecting approximately 7.4 x 109 deletion mutations (x = 1200 bp, f = 6300 bp); this consists of 3.3 x 108 intragenic deletions and 7.0 x 109 intergenic deletions. If deletion sizes are equally represented, these numbers indicate that most of the deletion sensitivity of multiple copy transgenes stems from the intergenic, rather than the intragenic, mutations. For comparison, this sensitivity

is essentially equal to that of a hypothetical assay using an endogenous hemizygous gene comprising 10 kb of target sequence flanked on each side by 75 kb of sequence that can be deleted without killing the cell. Eq. 2 represents the total number of detectable deletions in such an assay, where r and t are the lengths (in base pairs) of deletable sequence flanking the endogenous gene on either side of the target, respectively:

S=

-

(a)

+ [x(r+t)]

(b)

+ rt

(2)

(c)

In this equation, component (a) represents deletions contained within the target, component (b) represents deletions extending from the target out to either of the flanking regions, and component (c) represents deletions spanning from one flanking region, across the target, to the other. While sensitivity to deletion mutations is increased by the concatemeric integration of transgenes, this sensitivity is ultimately affected by the assay's ability to detect other classes of mutations. When testing mutagens that stimulate predominately deletions, assays with a high sensitivity to point mutations (relative to deletions) will show mutant inductions lower than assays that detect only deletions. Assays with a low sensitivity to point mutations have a lower background mutant frequency and thus will likely exhibit mutant induction levels higher than those obtained in the lambda transgenic assay when testing deletiongenerating compounds. In other words, an assay's greater relative sensitivity to point mutations can lead to attenuation of its observed deletion sensitivity. It is possible to design an assay using a gene that is positioned in the chromosome in a manner that permits large flanking regions to be deleted without inactivating the cell (e.g., at the end of a chromosome). In this instance the ability to detect mutagen-induced deletions would increase, but at the expense of reduced detection of point mutations, since the spontaneous (background) deletion frequency would be higher. This underscores some of the compromises confronted in selecting a mutagen testing-system.

M.J. Dycaico et aL / Mutation Research 307 (1994) 461-478

468

2.4. Spectral analysis of mutations

mutation is believed to be deamination of 5methylcytosine moieties resulting in C to T transversions (Cooper and Krawczak, 1989). Since the CpG dinucleotides are common sites of eukaryotic methylation (Bird, 1986), this result suggests that spontaneous deamination of 5-methylcytosine at this dinucleotide is a significant contributor to eukaryotic spontaneous mutation. This is consistent with the observed paucity of CpG dinucleotides in eukaryotic DNA (Jones et al., 1992), which presumably evolved as a result of the gradual conversion of these sites through mutation. The mutant spectra in transgenic animals treated with mutagenic compounds is in accordance with previously observed spectra for the respective compound. For example, of the 99 mutants sequenced from mice exposed to the direct-acting alkylating agent, methyl nitrosourea (Provost et al., 1993), 71% occurred at the 3' G of G p G dinucleotides as observed in previous studies (Zarbl et al., 1985; DuBridge et al., 1987;

Sequence analysis of mutations detected using the transgenic mutagenesis assay provides a source of information about mutations at a molecular level. In addition, it permits the accumulation of valuable spontaneous mutation spectra for comparative purposes. Spontaneous mutants recovered from various tissues in the C 5 7 B L / 6 and B6C3F 1 strains derived from the A1 mouse lineage have provided the basis for sequence analysis to determine the spectra of spontaneous mutation in these transgenic mice (Table 2). Of the 89 spontaneous mutants that have been characterized in this laboratory, 85% are base substitutions while insertions, deletions and single-base frameshifts comprise the remainder. In these transgenic mice, roughly 30% of all spontaneous mutations in somatic tissues and 50% of spontaneous mutations in germ cells are G : C to A : T transitions occurring at CpG dinucleotides. The mechanism of CpG dinucleotide

Table 2 S p e c t r u m of s p o n t a n e o u s m u t a t i o n in lambda/lacl mice (A1 l i n e a g e ) Spleen

Transitions A : T to G : C

Liver

Germ

Lung

Brain

Marrow

Total

C57BL/6 B6C3F1

5 1

1 0

1 0

0 0

1 0

0 0

8 l

G : C to A : T (at C p G sites)

C57BL/6 B6C3Ft

4 0

5 2

7 9

2 2

1 2

0 0

19 15

G : C to A : T ( n o n - C p G sites)

C57BL/6 B6C3F~

1 0

0 1

1 2

0 1

0 0

0 1

2 5

C57BL/6 B6C3FI

0 0

2 0

6 1

0 2

0 0

0 0

G : C to C : G

C57BL/6 B~,C3F t

4 0

1 1

0 0

0 1

0 0

0 0

A : T to C : G

C57BL/6 B~,C3F I

4 1

0 1

1 0

0 0

0 0

0 0

A : T to T : A

C57BL/6 B~,CsF l

0 0

0 1

0 0

0 0

0 0

0 0

Other ~

C57BL/6 B6C3F 1

3 0

2 2

3 1

0 1

0 0

0 1

Totals

C57BL/6 B6C3F I

21 2

11 8

19 13

2 7

2 2

0 2

Tra n t ~ersions G : C to T : A

~' O t h e r , insertions, d e l e t i o n s a n d single b a s e frameshifts.

= =

55 34

M.J. Dycaico et al. / Mutation Research 307 (1994) 461-478

Richardson et al., 1987). Furthermore, 91% were G : C to A : T transitions suggesting that O6-meth ylguanine, one of the primary methylnitrosureainduced mutagenic lesions, contributed significantly to the induced mutation spectrum. In another example, transgenic mice treated with dimethyl nitrosamine (Mirsalis et al., 1993), a metabolically activated alkylating agent, produced primarily (80%) G : C to A : T transitions as observed in earlier studies (Horsfall and Glickman, 1989; Devereux et al., 1991). Similar correlations have also been observed with benzo[a]pyrene (Kohler et al., 1991a), and ethyl nitrosurea (Kohler et al., 1991a; Provost et al., 1992; Provost and Short, in preparation). Therefore, compound-specific mutagenic effects can be supported by spectral analysis.

3. The rats

lambda/lacl

shuttle vector in transgenic

3.1. Rationale

The transgenic mutagenesis assay described above has been studied extensively in the A1 lineage in two strains of mice, C57BL/6 and B6C3F1. The B6C3F 1 transgenic is produced by crossing the C57BL/6 founder lineage with the C3H inbred mouse strain, thus the chromosomal integration site of the transgene is identical in both. This makes it possible to study the influence of genetic background on mutant frequencies within Mus musculus. Introducing the same shuttle vector into a different species, however, provides a means to examine the effects of species-specific differences on mutant frequencies and mutational spectra measured in the l a d target. The ultimate benefit of doing so may be the elucidation of mechanisms that underlie mutational phenomena and lead to cellular transformation in different species. The rat (Rattus norvegicus) was chosen as the second species for several reasons. First, differences in tumor induction are known to exist between mice and rats (Tennant et al., 1986; Haseman and Huff, 1987). Whether or not the information obtained in the mouse and rat transgenic

469

assays parallels these differences will be useful in building models that link induced carcinogenesis to specific mutational events. Second, the laboratory rat has historically been popular among toxicologists. It is a well-studied animal, for which many experimental techniques have been established (Waynforth and Flecknell, 1992). Third, the rat is roughly 8-10 times the size of the mouse and increased body size is advantageous for isolating sufficient quantities of genomic DNA. 3.2. Generating transgenic rats

The transgenic rat lineages were generated using pronuclear injection techniques (Hogan et al., 1986). A detailed discussion of the obstacles in producing transgenic rats and the modifications used to deal with them can be found elsewhere (Heideman, 1991). To date, we have produced 17 transgenic founders, obtained by microinjecting the ALIZ clone into the embryos of Fischer 344 inbred rats. Of the 12083 Fischer embryos injected thus far, 6942 (57%) appeared morphologically intact and were returned to recipient females. Of these, 257 pups (3.7%) survived to be screened for the presence of the shuttle vector. This resulted in the founders (6.6% of screened pups) shown in Table 3. A fundamental goal, when generating founders for the lambda/lacI assay, is establishing lineages that are high in copy number. In this and other transgenic mutagenesis assays, both deletional sensitivity and efficient vector recovery dictates that the transgene should be present in several copies per cell such that the ratio of recovered target vector per genome is high. Since the mechanism of DNA integration during transgenic development is poorly understood, we have experimented with microinjecting various forms of ALIZ. In transgenic mice, it is common for microinjected DNA fragments to be present in long head-to-tail concatemers, which suggests mechanisms such as pre-integration intracellular concatenation (Hamada et al., 1993) or post-integration homologous recombination (Brinster et al., 1985). We therefore performed some microinjections using the monomeric form of ALIZ, which

M.J. Dycaico et a l . / Mutation Research 307 (1994) 461-478

470

Table 3 Transgenic Fischer 344 rat lineages ( a L I Z ) Founder Sex lineage

DNA Phage Copy form a rescue number

Germ-line integration

SP VA DI

male male male

C C M

yes yes no

2- 4 2- 4 l- 2

yes yes yes

NO TA WA

female M female M female M

no yes no

1- 2 5- 6 1- 2

sterile yes yes

GA TR HO

female M female O female C

ND no yes

1- 2 1 2 ~ 200

no yes yes

EM FD JD b

female C male C male C

yes yes yes

5- 6 1- 2 25-35

yes yes yes

RI DU EV

female C female C male C

ND yes

5- 6 ND < 1 est. ND 20-30 est. ND

UR QX

female C male C

yes yes

5 - 6 est. ND 10-15 est. ND

yes

a Form of the microinjected ALIZ shuttle vector: monomers; C, concatemers; 0 , overlapping monomers. h Suspected to be a double integrated transgenic. ND, not yet determined.

M,

resulted in the lineages DI, NO, TA, WA and GA. In other experiments, pronuclear injection was accomplished using ALIZ concatemers formed by ligating the monomers at their cohesive termini (cos-ligation) in vitro prior to pronuclear injection. Lineages SP, VA, HO, EM, FD, JD, RI, DU, EV, U R and QX were all produced in this way. Lastly, others have reported transgenic work that employs in vivo homologous recombination to reconstruct large D N A segments by coinjecting smaller, overlapping D N A fragments (Shimoda et al., 1991; Pieper et al., 1992). Thus, some experiments involved coinjecting standard cos termini monomers along with monomers formed by first ligating the cos ends, then digesting the concatemers with a single hitting restriction enzyme that cuts near the middle of the shuttle vector (XbaI). This provides a mixture of overlapping fragments. The T R lineage is the sole result of those experiments. The data presented here suggest that the cosligation method is the most reliable. With the

exception of the T A lineage, all founders with copy number greater than 4 have resulted from this technique. Cos-ligation also appears to aid in improving the number of rescued phage per genome, perhaps by ensuring protection of most of the intervening cohesive termini, the integrity of which is important in phage assembly. This is supported by the fact that the T A lineage, which was produced by injecting monomeric ALIZ and possesses 5 - 6 copies, yields DNA that allows phage rescue at efficiencies resembling 1-2 copy transgenics. Furthermore, this may also explain why phage cannot be rescued from the rest of the monomer-derived lineages, which all have copy numbers in the 1-2 range. One notable outcome of this research is the H O lineage, which possesses over 200 copies of the ALIZ vector. At 45 kb per monomer, this amounts to 9 megabases of DNA, about 6% of the average length of a rat chromosome. Bacteriophage can be rescued from the DNA of this lineage at an equally impressive efficiency. Single-locus integration of the transgene has been verified both by monitoring transgene inheritance from several breedings of the founder and by visual observation of chromosomes using the FISH technique describe earlier. That the HO lineage provides unprecedented phage rescue efficiencies would have made it useful for the purposes of the established mutagenesis assay if it were not for the presence of a damaged lacl sequence among its 200 copies. This heritable mutation leads to dark blue plaques on the color-screening assay at a frequency of approximately 10 -2. Because in vivo spontaneous mutant frequencies in the mouse and rat are on the order of 10 -5, mutation analysis of the lacI gene in the H O lineage is not possible using the methods described here. However, it may be possible to exploit other genes within the phage for developing alternative screening/selection strategies in order to take advantage of this high copy-number lineage. Two other high copy-number lineages were obtained, JD and EV. While the EV founder is too young to breed at the time of this writing, germ-line integration has recently been established for JD. Breeding performance and trans-

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gene characterization are currently under investigation in both lineages. Preliminary tests on the DNAs from both founders suggest that all transgenic lacI segments exhibit a wild-type phenotype in bacteria.

3. 3. Spontaneous mutant frequencies To observe how spontaneous mutant frequencies in the rat compare to those previously measured in the lambda/lacI mouse assay, two of the older, established lineages were chosen for preliminary study (Fig. 4 and Fig. 5). The low copy number and, therefore, low rescue efficiencies shared by these two lines lead to a more time

471

consuming experiment compared to the A1 mouse studies, hence the small sample number per group. Fig. 4 shows a summary of the study, which measured spontaneous mutant frequencies in a somatic tissue (liver) and a mixed population of germ cells isolated from the testes. Previous data for both strains of lacI mice are also included for comparison. The germ cell mutant frequencies in both rat lineages were similar to those detected in the mouse. Although the numbers per test group were small, there was a statistically significant difference observed in liver between mice and rats (p < 0.006). Fig. 5 shows the animal-to-animal variations in liver for both the SP and TA rat lineages along with the B6C3F 1

liver

germ cell 4

0 X

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E

A1

A1

C57BL/6 B6C3F1

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A1

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TA

C57BL/6 B6C3F1

Fig. 4. Spontaneous mutant frequencies for liver and a mixed population of germ cells in lambda/lacI transgenic rodents. Both strains of the A1 mouse lineage are compared to two separate F344 rat lineages, SP and TA. Each bar corresponding to the A1 lineage represents the average frequency for 10 individual male mice at 6 wks of age. The SP and TA bars represent the average frequencies for groups of 3 individual male rats at 14 and 10 wks of age, respectively. Standard deviations are included. All frequencies were measured using the standardized Big Blue ® plaque-color-screening assay (Stratagenc, La Jolla). Liver frequencies were determined from examining a minimum of 300000 pfu per animal; those for germ cell were measured using a minimum of 500000 pfu per animal. Data points are as follows ( × 10-5): A1(C57BL/6) germ cell, 0.6:1: 0.3; AI(B6C3F 1) germ cell, 1.1 5: 0.3; SP germ cell, 0.7 + 0.5; TA germ cell, 0.6 5: 0.3; A1(C57BL/6) liver, 4.1 5: 0.7; AI(B6C3FI) liver, 4.1 _+ 0.8; SP liver, 2.2 5: 0.7; TA liver, 1.4 5: 0.3. In each comparison between mouse liver vs. rat-liver frequencies, p < 0.006 using a 2-tailed t-test.

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strain A1 mouse lineage. The liver mutant frequencies for both rat lineages are statistically similar with respect to each other but significantly lower than those of the mouse. Future studies will be necessary to confirm whether rat-liver frequencies are indeed two fold lower than those observed in the mouse, but the similarity between the SP and T A rat data in this study supports this conclusion. These preliminary results indicating lower mutant frequencies in rat somatic tissue may be a manifestation of the physiological environment of a larger animal. Evidence from measuring urinary 8-hydroxy-2'-deoxyguanosine levels in rodents suggests that the rate of oxidative damage from endogenous metabolic processes is lower in the rat than in the mouse (Shigenaga et al., 1989). Moreover, excision repair has been shown to be more active in rats than in mice (Hart and Setlow, 1974; Calabrese, 1991), thus, assuming similar repair fidelity, lower spontaneous mutant frequencies would be expected in the rat. Martin

A1 (B6C3F1)

and Palumbi (1993) have identified an inverse relationship between silent rates of nucleotide substitution and body size. This hypothesis ties lower body size with a shorter "nucleotide generation time" which is defined as the time of stasis for a given nucleotide position before it is duplicated via either DNA repair or replication. This increment of time, in turn, is governed by factors that include cellular generation time and metabolic rate. Another factor affecting liver frequencies could be the influence of the chromosomal integration site among the 3 transgenic lineages. The integration site of transgenes within the host chromosome is believed to be fairly random (Palmiter and Brinster, 1986) and is possibly facilitated by short patches of homology between the sequence at the site of chromosomal integration and the terminal sequence of the injected D N A (Hamada et al., 1993). In one instance where integration occurred in an unstable region of chromosome X, the position of the transgene affected the sponta-

TA

SP

0 v

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0 1

2

3

4

5

6

7

8

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Fig. 5. Animal-to-animal variation observed in experiments measuring spontaneous m u t a n t frequencies in transgenic rat and mouse liver (see Fig. 4 legend). Shown are the values obtained for individual animals (black bars) along with the m e a n and standard deviation for each group (gray bars), m, m e a n frequency of the group.

M.J. Dycaico et al. / Mutation Research 307 (1994) 461-478

neous mutant frequency of a lambda/lacZ shuttle vector in mice (Gossen et al., 1992). However, previous studies in our laboratory revealed that chromosomal position had little effect on the spontaneous mutant frequencies among 3 separate lambda/lacI mouse lineages (Moores et al., 1992). The reported spontaneous mutant frequencies of two other lambda/lacZ transgenic mouse lineages were likewise similar to those determined for these 3 lambda/lacI lineages and thus also appear free of integration effects (Gossen et al., 1989; Kohler et al., 1990). Moreover, the measured frequencies of the two rat lineages in the present study are also similar with respect to each other. These observations support two assumptions: that the observed difference in spontaneous mutant frequencies between mouse and rat liver is valid, and that among the lineages studied in this laboratory, the effect of the integration site is less than 2-fold on mutant frequency within a given species. Nevertheless, given the complexity of mammalian genome organization and its variability between cell types (Pienta et al., 1991), it is likely that chromatin structure influences the degree of D N A damage in localized areas and that this could be a potential caveat for any in vivo assay that relies on mutation targets that are located at a specific locus within the genome. Further comparative work on additional lineages is needed to address this issue.

4. Selectable systems for screening mutants in lambda//ac/rodents All of the mutant frequency data discussed above have been obtained by using the colorscreening assay. Because both mutant and nonmutant targets are scored in this assay, it is considered a non-selectable system. Although this system generates easily identifiable mutants, standardized plating conditions are optimal at ~ 15 000 plaques per 25 × 25 cm plate. Based on these numbers, and the statistical requirements, 15-20 plates per rodent are required for each tissue analyzed. This can contribute significantly to the cost of the assay in terms of plates, medium,

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X-gal substrate, and labor. Selectable versions of this assay in which only vectors that harbor mutant lacI target genes are scored may be preferred since they have the potential of increasing both the speed and ease of performing the assay while also reducing the cost. In addition, systems that select for mutated target genes may alter the sensitivity of the assay and mutant spectrum from that detected with the color-screening system. Using transgenics that carry the ALIZ shuttle vector, it was possible to develop selectable systems that depend on lacI inactivation. Because of the transacting nature of the LacI protein, it can be used to regulate the expression of a wide variety of reporter genes. This is done by cloning the reporter gene downstream of a lac operator sequence and including this recombinant region in the E. coli host ceils destined for infection by phage rescued from the transgenic animal. The expression of a chosen reporter gene could provide a selective growth advantage to the host cell a n d / o r to the incoming ALIZ phage particle. Alternatively, it is possible to choose a reporter gene that, when expressed, can allow the cell a n d / o r bacteriophage to survive otherwise toxic conditions. In either situation, the expression of the reporter gene is determined by the status (mutant or wild-type) of the incoming lacI target gene and provides the switch between a selective growth advantage and a growth disadvantage. Depending on the host cell and environmental conditions chosen for the assay, the bacteriophage may replicate lyrically, resulting in plaques as the detectable endpoint, or it may replicate lysogenically, resulting in bacterial colonies as the detectable endpoint. Additionally, because the lacI target gene can be excised from the ALIZ vector as an independent plasmid, it is possible to perform excision and selection for mutants simultaneously within a single host cell. The lacI target gene can be recovered in the form of either lysogenic colonies, lambda plaques, or colonies containing the excised plasmid and this allows further flexibility in the design of systems that select for mutants. One selectable system that uses lacI as the target gene and lacZ as the reporter gene is the minimal medium lactose system (Kretz et al.,

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1992; Lundberg et al., 1993). The identification of mutants using this system depends on lysogenic replication of the incoming bacteriophage. This is accomplished by infecting host bacteria that constitutively express the lambda cI gene. The cl protein represses the genes responsible for lytic phage growth, thereby forcing lysogenic replication of the phage DNA (Hendrix et al., 1983). This system also relies on the expression of the a-lacZ gene from the ALIZ vector to create a functional /3-galactosidase protein by complementation with the c0-portion of LacZ protein that is created in the host cell. This /3-galactosidase protein allows the cell to utilize lactose for growth. Thus, host cells carrying phenotypically mutant lacI genes can grow on minimal medium containing lactose as the sole carbon source. The detectable endpoint in this case is the appearance of blue bacterial colonies when plated in the presence of the chromogenic substrate, X-gal. Bacteriophage that carry a wild-type lacl target gene also infect the host strain, however, the resulting cell is unable to grow due to a lack of /3-galactosidase production. This system has been shown to demonstrate selection of lacl mutants as well as induction of mutant frequencies in mutagen treated A1 lineage mice. A second selectable system under development employs lacI as the target and the E. coli groE gene as the reporter. The gene product of groE is a molecular chaperonin that is essential for lambda phage particle morphogenesis (Zeilstra-Ryalls et al., 1991). E. coli cells that are deficient in groE cause a block in lambda head assembly such that infectious lambda phage particles are not formed. In developing this selectable assay, groE was cloned such that its expression is controlled by the lacZ p r o m o t o r / o p e r a t o r region. Transcriptional terminators are used to inhibit expression of the groE gene prior to infection by lambda. When phage particles infect a host cell that carries this groE construct, a lambda protein causes antitermination of these terminators. Transcription of groE is then dependent on the status of Lacl. If the lacl target in the shuttle vector has been mutated such that it cannot repress the reporter gene, groE is expressed, which then allows the formation of intact phage patti-

cles. The detectable endpoint in this system is a blue lambda plaque. Selection for mutants and detection of induced mutant frequencies has been demonstrated using this system (Kretz et al., 1993a,b). The two selectable systems described above demonstrate techniques that utilize bacterial and bacteriophage genetics to generate a variety of assays that take advantage of the established lacl gene for mutant target gene selection. However, each assay may display a unique level of phenotypic sensitivity to mutations present in the lacl gene resulting in different mutational spectra. The minimal medium lactose system is an example, as mutants confirmed by sequencing have been isolated with this system that do not appear as mutants on the color-screening system (Lundberg et al., 1993). This suggests that the lactose system is sensitive to a wider, or perhaps different, range of lacl mutations. Additionally, control mutants that are routinely used with the color-screening system do not perform identically on each system described here. As one example, a mutant that appears "weak" in the colorscreening assay, due to a low or weak color intensity, appears as a phenotypically "strong" mutant in the lactose assay. When designing or considering the use of a selectable system, it is also important to recognize the effects on mutant frequency that may result from the dependence of the assay on two different plating methods, one for determining the total number of targets assayed and one for determining the number of mutant target genes. Yet, another issue to be taken into consideration before using these systems for large scale mutagenesis studies is the effect that selective pressure may have on the mutant frequencies and spectral data. The effects of selection on bacterial replication and mutagenesis are, as yet, not understood (Cairns et al., 1988; Hall, 1991; Foster, 1992; MacPhee, 1993). Therefore, the information available regarding selective pressure must be carefully considered when designing selectable systems as well as when interpreting data that have been obtained with such systems. In the development of any selectable system, the mutants initially detected may require confirmation at the sequence level to

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assist in ruling out possible effects imposed by selection. The potential gain through the use of systems that select for mutant target genes makes the continued development and investigation of these systems valuable.

5. Transgenics oncogenes

combining lambda/lacl and

The advent of transgenic mouse technology has led to significant advances in the field of oncogenes and tumor research. Some of these studies involve the introduction of an oncogene, a transforming allele of a proto-oncogene, into the germ line of transgenic mice, often resulting in tissue-specific expression. Alternatively, an endogenous tumor suppressor gene can be deleted or inactivated. Since cancer in both humans and mice is believed to result from a multistage process involving the mutation of several genes (reviews include Adams and Cory, 1991; Bishop, 1991; Hunter, 1991; Sugimura et al., 1992), the appropriate genetic alterations advance these mice one step in the carcinogenic process, thus predisposing them to tumor formation. These experiments not only aid in characterizing oncogenic mechanisms but provide a system for delineating the normal cellular function of the associated tumor suppressor or proto-oncogene. Another potential use for these transgenics is in the area of carcinogen testing where tumorigenesis data can be obtained much sooner than with the traditional long-term rodent bioassay. Crossing such mice with the lacI shuttle-vector mice engenders animal models that have the potential to be tested simultaneously for both mutation analysis and tumor induction. In the case of mice harboring an oncogene, this would allow the study of certain tissue-specific tumors as determined by the tissue-specific expression of the oncogenic transgene. In the case of mice possessing a damaged tumor suppressor gene, theoretically all of the tissues affected by the inactivation of that gene are subject to tumors and therefore are candidates for dual analysis. This second model is presently under exploration. Hybrids between the A1 lineage lambda/

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lacI mutagenesis model and the TSG-p53 TM mouse (GenPharm International) are now being produced for dual mutagenesis/carcinogenesis research. Informally known as the "p53 knockout" mouse (Donehower et al., 1992), the latter is a transgenic produced by the technique of gene targeting in embryonic stem cells (Mansour et al., 1988; Travis, 1992; Wilder and Rizzino, 1993). The abrogation of the endogenous p53 gene in these mice leads to the early formation of tumors. When the null allele is present in the homozygous state, the incidence of a wide variety of tumors is sharply increased in individual mice between the ages of 3 and 6 months. Heterozygous null allele mice develop tumors at a later age (usually between 9 and 12 months), but still at a higher rate relative to their wild-type counterparts. When tumor induction studies are performed on hybrid mice that contain both the p53 null allele and the lambda/lacI shuttle vector, it will be possible to examine both normal and tumor-derived tissues for mutant frequency determination and mutant sequence characterization. In addition to mutagen testing, these mice could prove to be useful for studying p53 function and the extent of its involvement in cancer and apoptosis. The lacI/p53 hybrid animal is one example of the many novel in vivo models for mutation analysis made possible through the use of transgenic technology. Crosses between mice deficient in metabolism a n d / o r DNA repair with shuttle vector-based transgenics are also of significant interest. Furthermore, it is anticipated that mutagenesis models in other species, such as the rat, will eventually result in several useful cross hybridizations. Thus, the evolution of simpler and more efficient mutagenesis assays based on advancing technologies will likely play a significant role in the investigation of mechanisms of DNA damage and the carcinogenic process.

Acknowledgments We thank Jan K. Lohse-Heideman for her generous guidance and advice in the production of our transgenic rats; Anne Boyle for generously

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providing chromosome-specific mouse probes; Dan Ardourel, Brenda Rogers, Denise Wyborski and Kelly Lundberg for excellent technical contrib u t i o n s . T h i s w o r k w a s s u p p o r t e d in p a r t b y g r a n t s and/or contracts from the NIEHS and NIH, numbers N01-ES-95252, 2R44CA57066-02 and 5R44ES04484-03.

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