lacI shuttle vector

EnvironmentalMutagenesis

ELSEVIER

Mutation Research 334 (1995) 161-165

Development of a rat cell line containing stably integrated copies of a lambda/lacI shuttle vector Denise L. Wyborski a,, Sergei Malkhosyan b, Jane Moores a, Manuel Perucho h, Jay M. Short c a Stratagene, 11011 N. Torrey Pines Road, La Jolla, CA 92037, USA b California Institute of Biological Research, 11099 N. Torrey Pines Road, La Jolla, CA 92037, USA c Industrial BioCatalysis, Inc., 505 Coast Blvd., La Jolla, CA 92037, USA

Received 14 June 1994; revision received 25 July 1994; accepted 25 July 1994

Abstract

A rat embryo cultured cell line was generated that carries stably integrated copies of a lambda/lacI shuttle vector, containing the lacI gene as a mutational target. After the desired treatment of the cells, this vector can be rapidly and efficiently recovered from the cell D N A by in vitro packaging and then screened for mutations in the lacI gene, using bacterial detection systems. The vector is identical to that integrated into the Big Blue transgenic mouse, which was developed for in vivo mutation analysis. Characterization of the cell line by fluorescence in situ hybridization showed that the phage D N A is integrated at two distinct sites on separate chromosomes at approximately 50-70 copies per cell and the cell line is polyploid. The rescue efficiency is approximately 100 000 pfu/tzg of genomic DNA. To examine the ability of the cell line to detect mutations in the lacI gene, the cells were treated with 100 tzg/ml of the direct-acting alkylating agent N-methyl-Nnitrosourea (MNU) for 30 rain at 37°C and grown to confluence. The shuttle vector was rescued from untreated and mutagen treated cells, and spontaneous and induced mutant frequencies were determined to be 4.0 x 10 -5 and 92.7 x 10 5, respectively. The cell line can be used to detect mutations in the lacl gene, followed by recovery of mutants for sequence analysis. The cell line may be valuable for short-term in vitro mutagenesis studies, oncogene and tumor suppressor studies, and D N A repair studies. Keywords: Shuttle vector; Lacl; Rat2 cell line

1. I n t r o d u c t i o n

V a r i o u s assays exist for the d e t e r m i n a t i o n o f g e n e t i c toxicity in vitro, i n c l u d i n g r e v e r s e m u t a tion assays, g e n e m u t a t i o n assays, c y t o g e n e t i c

* Corresponding author. Tel. (619) 535-5400; Fax (619) 5350071.

Elsevier Science B.V. SSDI0165-1161(94)00071-9

tests, a n d D N A d a m a g e a n d r e p a i r studies. R e c o m b i n a n t shuttle v e c t o r s have p r o v e d useful in t h e study o f m u t a t i o n in e u k a r y o t i c systems d u e to t h e i r ability to b e e x p o s e d to m u t a g e n i c a g e n t s in m a m m a l i a n cells a n d t h e n t r a n s f e r r e d to bact e r i a for r a p i d analysis o f m u t a t i o n s ( r e v i e w e d by D u B r i d g e a n d Calos, 1988). T h e use of b a c t e r i o p h a g e l a m b d a s e q u e n c e s in t h e shuttle v e c t o r allows t h e stable i n t e g r a t i o n of t h e v e c t o r into t h e

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mammalian cell genome, followed b~, the rescue of the vector DNA by in vitro lambda packaging extracts after exposure to potential mutagens (Glazer et al., 1986; Summers et al., 1989). This permits exposure of the shuttle vector sequences to the same parameters as the chromosomal DNA, followed by rapid and unambiguous scoring of mutations in bacteria. The lacI gene is a particularly useful mutational target due to the extensive pool of sequence information available (Farabaugh, 1978; Miller, 1978; Miller and Schmeissner, 1979; Schaaper et al., 1986; Gordon et al., 1988; Horsfall and Glickman, 1989; Schaaper and Dunn, 1991; Kohler et al., 1991a; Provost and Short, 1994). We have constructed a cell line which contains stably integrated copies of a lambda/lacl shuttle vector in which the lacI gene is used as a mutational target (Fig. 1). This vector is successfully being used in the Big Blue in vivo mouse and rat mutagenesis systems (Provost et al., 1993; Kohler et al., 1991a,b; Mirsalis et al., 1993; Provost and Short, 1994; Dycaico et al., 1994). The following paper describes the Rat2 lambda/lacl cell line, and examines the mutant frequencies fQllowing exposure to N-methyl-Nnitrosourea (MNU).

by calcium phosphate co-transfection of the rat embryo cell line Rat2 (ATCC No. CRL 1764) (Topp, 1981) with lambda bacteriophage DNA containing the lambda/lacI shuttle vector, and the plasmid pSV2Neo, which provided an antibiotic selection marker. Stable, geneticin resistant clones were screened for the presence of the lacI vector by phage rescue using Transpack TM in vitro packaging extract (Stratagene) (Kohler et al., 1991a). One of the geneticin resistant clones exhibiting high rescue efficiency was chosen for further study. DNA hybridization experiments involving titration of genomic DNA prepared from the stable clone (Rat2AlacI) against a standard DNA curve were used to calculate the number of copies of the shuttle vector that were integrated into the chromosomes of each cell (Hogan et al., 1986). To determine the locations of the integrated vector sequences, fluorescence in situ hybridizations (FISH) were performed using the Prime-it Fluor fluorescence labeling kit and In Situ Hybridization kit from Stratagene (Ransom et al., 1992) according to the protocols provided.

2. Materials and methods

cells were grown to 30-40% confluence in Dulbecco's Modified Eagle medium (DMEM, Gibco) containing 10% fetal calf serum (ICN/Flow) and 200/~g/ml geneticin (Gibco). MNU (100/xg/ml, Sigma), a direct-acting alkylating agent, was added

Detection of mutations in the lacI gene To test the chosen Rat2AlacI cell line for the ability to detect mutations in the lacI gene, the

Integration of shuttle vector into Rat2 cell chromosomes The Rat2 lambda/lacI cell line was generated

~

COS I ll

4480bp

~ A

COS ~- J

IIII

ci857

SL

~..

45.5kb

Fig. 1. Lambda/lacl shuttle vector. The cos sites are separated by approximately45 kb. The expanded region represents the portion of the vector that is excised as a phagemidwith M13 helper phage. The entire phagemid region is flanked by partial fl filamentous phage origins. The alacZ (750 bp) flanks the lacl target gene (1100 bp). The ColE1 origin is present for plasmid replication, and the ampicillin resistance gene (900 bp) is present for selection of colonies carrying the plasmid to be used for sequencing the lacI target. The S gene contains the mutation SL (Kohler et al., 1990).

D.L. Wyborski et al. / Mutation Research 334 (1995) 161-165

to the medium, and the cells were incubated for 30 min at 37°C. A control group of' cells was grown in the absence of mutagen. The cells were washed with phosphate-buffered saline, fresh medium was added, and the cells were grown to confluence, then harvested by scraping. Genomic D N A was prepared using Stratagene's Genomic D N A Isolation kit, and the shuttle vector was recovered by mixing the D N A with Transpack T M packaging extract. The resulting phage stock was used to infect Escherichia coli SCS-8 cells (Stratagene, Kohler et al., 1991a), and the cells were plated on 25 x 25 cm assay trays in the presence of the chromogenic substrate 5bromo-4-chloro-3-indolyl-fl-D-galactopyranoside (X-gal, Stratagene).

163

3. Results and discussion Transfection of the Rat2 cell line with the l a m b d a / l a c I shuttle vector generated stable geneticin resistant clones that were screened for the presence of the shuttle vector by phage rescue. The clone selected for further analysis had undergone numerous ( > 10) passages and exhibits rescue efficiencies of approximately 100000 pfu //xg of genomic DNA. This clone contains approximately 50-70 copies of the shuttle vector integrated into the chromosomes of each cell. FISH analysis revealed the presence of the shuttle vector at two distinct sites on separate chromosomes, and also that the cell chromosome number was polyploid (Fig. 2, Table 1). The ap-

Fig. 2. FISH showing integration of shuttle vector into Rat2 chromosomes. Chromosome spreads were made from the Rat2Alacl cell line according to established procedures (Verma and Babu, 1989). Standard techniques of colcemid and hypotonictreatments and methanol/acetic acid fixation were used. A fluorescent probe was generated from lambda/lacI shuttle vector DNA and fluorescence in situ hybridizationwas performed as described in Materials and methods. The chromosomeswere observed using an inverted epifluorescence microscope with a 450-490-nm excitation wavelength and 520-nm emission wavelength filter.

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parent similarity between the two chromosomes, their integration sites, and copy number suggests that these chromosomes may be homologous and that the chromosome duplicated following a single integration event of concatemers of the exogenous shuttle vector DNA molecules (Perucho et al., 1980; Robins et al., 1981). No changes were observed in the integration sites or distribution of chromosome number when the hybridizations were repeated after the cells had been passaged an additional five times. If a mutation in the lad gene inactivates the Lac repressor protein, transcription will occur in the alacZ gene. Complementation between the alacZ present in the shuttle vector and the carboxyl portion of lacZ present in the host bacteria generates fl-galactosidase, which cleaves X-gal and thereby generates a blue plaque. Wild-type phage with no mutations produce active Lac repressor protein, which blocks lacZ transcription, resulting in a colorless plaque. To determine mutant frequencies for both MNU-treated and untreated cells, the blue plaques were counted and compared to the total number of phage plated. Spontaneous and induced mutant frequencies from several packaging reactions are shown in Table 2. Treatment of the cell line with the MNU mutagen increased the mutant frequency 23-fold above the control. These data confirm the presence of stably integrated copies of the lambda/lacl shuttle vector in the transfected Rat2 cell line, and demonstrate that this cell line can be used successfully to detect mutations in the lad gene. The cell line permits measurement of mutations and recovery of mutants for sequence analysis. The cell line is useful for a variety of applications, including Table 1 Karyology Chromosome frequency distribution in 25 cells Total number of ceils Total number of chromosomes

1

2

4

5

6

2

1

3

1

73

74

75

76

77

78

79

80

81

Twenty-five well-spread metaphase cells were scored to determine the modal number of chromosomes. Species of origin 2n = 42.

Table 2 Spontaneous and MNU-induced mutant frequency (MF)

Spontaneous MNU-induced

Total plaques

Mutant plaques

MF× 10 -5

1714 435 1702720

68 1578

4.0 92.7

Genomic DNA prepared from treated and untreated cultured cells was packaged and plated. 150000 cells yielded approximately 1 /zg of genomic DNA. Mutant frequencies are expressed as the ratio of blue mutant plaques to total plaques rescued.

short-term in vitro mutagenesis studies, DNA repair studies, various toxicology applications, and studies of the effects of oncogenes, tumor suppressor genes and mutator genes on mutant frequencies.

Acknowledgements This work was supported in part by SBIR Grant 2R44 ES04484-02. We wish to thank G. Scott Provost for technical advice.

References DuBridge, R.B. and M.P. Calos (1988) Recombinant shuttle vectors for the study of mutation in mammalian cells, Mutagenesis, 3, 1-9. Dycaico, M.J., G.S. Provost, P.L. Kretz, S.L. Ransom, J.C. Moores and J.M. Short (1994) The use of shuttle vectors for mutation analysis in transgenic mice and rats, Mutation Res., 307, 461-478. Farabaugh, P.J. (1978) Sequence of the l a d gene, Nature, 274, 765-769. Glazer, P.M., S.N. Sarkar and W.C. Summers (1986) Detection and analysis of UV-induced mutations in mammalian cell DNA using a phage shuttle vector, Proc. Natl. Acad. Sci. USA, 83, 1041-1044. Gordon, A.J.E., P.A. Burns, D.F. Fix, F. Yatogai, F.L. Allen, M.J. Horsfall, J.A. Halliday, J. Gray, C. Bernelot-Moen and B.W. Glickman (1988) Missense mutations in the l a d gene of Escherichia coli, J. Mol. Biol., 200, 239-251. Hogan, B., F. Costantini and E. Lacy (1986) Analysis of foreign DNA in transgenic mice, in: B. Hogan, F. Costantini and E. Lacy (Eds.), Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp. 180-181. Horsfall, M.J. and B.W. Glickman (1989) Mutational specificities of environmental carcinogens in the l a d gene of

D.L. Wyborski et al. / Mutation Research 334 (1995) 161-165 Escherichia coli. The direct-acting analogue N-nitroso-Nmethyl-N-a-acetoxymethylamine, Carcinogenesis, 10, 817822. Kohler, S.W., G.S. Provost, P.L. Kretz, M.J. Dycaico, J.A. Sorge and J.M. Short (1990) Development of a short-term, in vivo mutagenesis assay: the effects of methylation on the recovery of a lambda phage shuttle vector from transgenic mice, Nucleic Acids Res., 18, 3007-3013. Kohler, S.W., G.S. Provost, A. Fieck, P.L. Kretz, W.O. Bullock, J.A. Sorge, D.L. Putman and J.M. Short (1991a) Spectra of spontaneous and mutagen-induced mutations in the lacl gene in transgenic mice, Proc. Natl. Acad. Sci. USA, 88, 7958-7962. Kohler, S.W., G.S. Provost, A. Fieck, P.L. Kretz, W.O. Bullock, D.L. Putman and J.M. Short (1991b) Analysis of spontaneous and induced mutations in transgenic mice using a lambda Z A P / l a c I shuttle vector, Environ. Mol. Mutagen., 18, 316-321. Miller, J.H. (1978) The lacl gene: its role in lac operon control and its use as a genetic system, in: J.H. Miller and W.S. Reznikoff (Eds.), The Operon, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp. 31-88. Miller, J.H. and U. Schmeissner (1979) Genetic studies of the lac repressor, J. Mol. Biol., 131, 223-248. Mirsalis, J.C., G.S. Provost, C.D. Matthews, R.T. Hamner, J.E. Schindler, K.G. O'Loughlin, J.T. MacGregor and J.M. Short (1993) Induction of hepatic mutations in lacI transgenic mice, Mutagenesis, 8, 265-271. Perucho, M., D. Hanahan and M. Wigler (1980) Genetic and physical linkage of exogenous sequences in transformed cells, Cell, 22, 309.

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Provost, G.S. and J.M. Short (1994) Characterization of mutations induced by ethylnitrosourea in seminiferous tubule germ cells of transgenic B6C3F 1 mice, Proc. Natl. Acad. Sci. USA, 91, 6564-6568. Provost, G.S., P.L. Kretz, R.T. Hamner, C.D. Matthews, B.J. Rogers, K.S. Lundberg, M.J. Dycaico and J.M. Short (1993) Transgenic systems for in vivo mutation analysis, Mutation Res., 288, 133-149. Ransom, S., J. Anderson, H. Huang, J. Braman and J. Moores (1992) Fluorescence in situ hybridization using directly fluoresceinated probes, Strategies Mol. Biol., 5, 35-36. Robins, D., S. Ripley, A. Henderson and R. Axel (1981) Transforming DNA integrates into the host chromosome, Cell, 23, 29. Schaaper, R.M. and R.L. Dunn (1991) Spontaneous mutations in the Escherichia coli lacl gene, Genetics, 129, 317-326. Schaaper, R.M., B.N. Danforth and B.W. Glickman (1986) Mechanisms of spontaneous mutagenesis: An analysis of the spectrum of spontaneous mutation in the Escherichia coli lacl gene, J. Mol. Biol., 189, 273-284. Summers, W.C., P.M. Glazer and D. Malkevich (1989) Lambda phage shuttle vectors for analysis of mutations in mammalian cells in culture and in transgenic mice, Mutation Res., 220, 263-268. Topp, W.C. (1981) Normal rat cell lines deficient in nuclear thymidine kinase, Virology, 113, 408-411. Verma, R.S. and A. Babu (1989) Tissue culture techniques and chromosome preparation, in: R.S. Verma and A. Babu (Eds.), Human Chromosomes: Manual of Basic Techniques, Pergamon Press, New York, pp. 4-16.