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Extraordinarily High Copy Numbers of the Plasmid THOMAS J Proc. Nati. Acad. Sci. USA Vol. 89, pp. 5725-5729, July 1992 Biochemistry Stable transformation of a mosquito cell line results in extraordinarily high copy numbers of the plasmid THOMAS J. MONROE*t, MARIA C. MUHLMANN-DIAZt, MARGARET J. KOVACH*, JONATHAN 0. CARLSON*§, JOEL S. BEDFORDt, AND BARRY J. BEATY* Departments of *Microbiology and tRadiological Health Sciences, Colorado State University, Fort Collins, CO 80523 Communicated by Fotis C. Kafatos, March 18, 1992 ABSTRACT Stable incorporation of high copy numbers nous plasmid DNA. In one line the multiple copies ofplasmid (>10,000 per cell) of a plasmid vector containing a gene are organized in an array that resembles a chromosome. conferring resistance to the antibiotic hygromycin was achieved in a cell line derived from the Aedes albopictus mosquito. MATERIALS AND METHODS Plasmid sequences were readily observed by ethidium bromide Recombinant DNA. The plasmid pUChshyg (Fig. 1) was staining of cellular DNA after restriction endonuclease diges- constructed from the P-element vector pUChsneo (21) by tion and agarose gel electrophoresis. The plasmid was demon- replacement of the neo gene with the hygromycin phospho- strated by in situ hybridization to be present in large arrays transferase gene (hyg). The hyg gene together with the intron integrated in metaphase chromosomes and in minute and from the simian virus 40 small tumor antigen gene and double-minute replicating elements. In one subclone, -60,000 poly(A) signal was isolated as a 2.2-kb HindIII-BamHI copies of the plasmid were organized in a large array that fragment, and the hsp70 heat shock promoter was isolated as resembles a chromosome, morphologically and in the segrega- a 0.35-kb HindIII-Xba I fragment from pCHA2-3 (22). These tion of its chromatids during anaphase. The original as well as fragments were cloned into Xba I-BamHI-cut pUC18, modified versions of the plasmid were rescued by transforma- thereby placing the heat shock promoter upstream from the tion ofEscherichia coli using total cellular DNA. Southern blot hyg gene. The resulting hshyg transcription unit was excised analyses of recovered plasmids indicate the presence of mos- from this plasmid as a 2.5-kb Xho I-Sma I fragment and quito-derived sequences. ligated to the 4.2-kb Xho I-Nae I fragment ofpUChsneo. The P-element inverted repeats in pUChshyg do not affect the The development of efficient procedures for the introduction transformation process in mosquito cells either with or with- of new genes into organisms has dramatically advanced our out a helper construct carrying the P transposase gene understanding of the molecular basis of many life processes. (T.J.M. and M.J.K., data not shown). Heat shock does not For example, studies of gene regulation and development in significantly affect transformation efficiency. Drosophila have been greatly facilitated by the use of effi- Cell Culture and DNA Isolation. A. albopictus cell line C6/36 cient transformation vectors derived from the P-element (23) was grown in Leibovitz's L-15 medium (GIBCO) supple- transposon (1-4). Research on other insects of medical and mented with 10% fetal bovine serum at 28°C. Transformation agricultural significance would undoubtedly benefit from ofthe C6/36 cell line with pUChshyg was performed using the similar studies. Attempts to transform mosquitoes by P-ele- Lipofectin reagent (BRL) according to the manufacturer's ment-mediated transformation have been unsuccessful; rare specifications. Twenty-four hours prior to transfection, 25- integration events were attributed to illegitimate recombina- cm2 flasks were seeded with 2 x 106 C6/36 cells. A lipofectin- (5-7). During DNA complex was made by incubating 10 ,ug ofplasmid DNA tion that was not related to P-element activity with 50 1ul of lipofectin reagent in 3 ml of serum-free L-15 the course of investigating the introduction of genes into medium for 15 min at room temperature. After rinsing the cells mosquito cells, we have detected transformation events in three times with sterile phosphate-buffered saline (PBS; 137 which the plasmid DNA is organized in such a way that it mM NaCl/27 mM KCI/10 mM Na2HPO4/1.8 mM KH2PO4), resembles gene amplification of endogenous genes in other 3 ml of the lipofectin-DNA complex was added to each flask. systems. Following an 8-hr incubation at room temperature, 3 ml of Gene amplification in vivo and in vitro can confer resis- L-15 medium supplemented with 20% fetal bovine serum was tance to a wide range of drugs (8-10) and may contribute to added to each flask. After 24-48 hr, the medium was replaced the malignant progression in certain forms of cancer (11-15). with selective medium containing 300 units of hygromycin B Amplification of gene copy number is also the basis of per ml (Calbiochem). Stable transformants were selected by resistance to certain pesticides in some plants (16) and insects growth in L-15 medium supplemented with 10% fetal bovine (17). In Culex pipiens mosquitoes the development of high serum and 300 ,ug of hygromycin B per ml. After 4-6 weeks levels of resistance to organophosphate insecticides was ofselection, individual clones were picked and expanded. The accompanied by amplification of chromosomal esterase doubling time of these clones was about 2 days in selective genes (18). Selection of methotrexate-resistant Aedes al- medium. Clones were recovered at a frequency of 10-5 to 10-6 bopictus cells resulted in cells of altered karyotype, presum- by this procedure. Subclones were isolated by inoculating -50 ably because of amplification of the chromosomal dihydro- cells in each of several 100-mm Petri dishes. After visible folate reductase gene (19). Gene amplification is frequently colonies had formed, they were isolated by means of stainless manifested in two forms ofkaryotypic abnormalities (12, 20): steel cloning cylinders. homogeneously staining regions or double-minute chromo- Total cellular DNA was isolated by lysing cells in 10 mM somes. Both ofthese manifestations have now been observed Tris.HCl/10 mM EDTA/0.6% SDS, pH 7.4. Proteinase K in an A. albopictus cell line transformed stably with exoge- was added to a concentration of 0.5 mg/ml and incubated at The publication costs of this article were defrayed in part by page charge tPresent address: The Upjohn Company, Unit 7252-25-12, 301 Hen- payment. This article must therefore be hereby marked "advertisement" rietta Street, Kalamazoo, MI 49001. in accordance with 18 U.S.C. §1734 solely to indicate this fact. §To whom reprint requests should be addressed. 5725 Downloaded by guest on September 24, 2021 5726 Biochemistry: Monroe et al. Proc. Natl. Acad Sci. USA 89 (1992) Hind!!! HindIl The cells were viewed by fluorescence microscopy, and BamHI - chromosomes were photographed at a magnification ofeither / fi'>nab~~~~~~H,?III x600 or x 1000 using Kodacolor Gold 100 print film. Hind!/ZHi~~~~~~~e? iPstI ~ ~ ~ ~ ~ c ./' t \ BsBxm.MI~~i'mH!1 In Situ Nick-Translation. The sensitivity of metaphase SV4O0 EcoRI chromosomes to digestion by pancreatic DNase I was deter- In tron/poly .4 mined by in situ nick-translation using a procedure similar to that described by Kerem et al. (25). Briefly, fresh slides were II treated for 10 min at room temperature with 100 p.l of nick-translation mixture under a coverslip. The nick- translation mixture contained 50 mM Tris HCl (pH 7.9), 5 Hpi pUChshyg mM MgCl2, 10 mM 2-mercaptoethanol, 50 tug of bovine ,!- n6.7 kb serum albumin per ml, 10 units of DNA polymerase I per ml, AicX-H;Sygromycin 20 ng of pancreatic DNase I per ml, 4 juM (each) dATP, \. resistance 7 dGTP, and dCTP, and 0.3 p.M biotin-11-dUTP. The reaction was terminated by rinsing the slide in running deionized water for 1 min. Slides were then stained as described above to reveal biotin incorporation by fluorescein isothiocyanate- Pst! labeled avidin and counterstained with propidium iodide. EcoR! Flow Cytometric Cellular DNA Measurements. The cells HindIII were fixed with ethanol and stained with chromomycin A3 PstIr Xbol according to methods described previously (26). Flow cytom- etry was carried out using a Coulter Epics V cell sorter with FIG. 1. Plasmid pUChshyg. The position of the Drosophila the Argon ion laser melanogaster hsp70 promoter and the direction of transcription are tuned to 458 nm to excite the chromomycin indicated by the large arrow. The hygromycin-resistance gene and A3 dye, which binds quantitatively to DNA. The resulting the simian virus 40 (SV40) RNA processing signals are indicated by DNA per cell histograms were analyzed by a curve-fitting dark and light stippling, respectively. The P-element inverted repeats procedure (Multicycle; Phoenix Flow Systems, San Diego). are indicated by short darkly shaded arrows. The pUC8 plasmid Southern Blot Analysis. Ten micrograms of C6/36 DNA replicon is shown as a thin line. kb, Kilobases. was digested with HindIII, separated according to molecular weight by 0.8% agarose gel electrophoresis, denatured in situ least 4 hr at 370C. The lysate was extracted three times with with 0.4 M sodium hydroxide/0.6 M NaCl/0.5 M Tris HCI, equal volumes of phenol/chloroform (1:1) and precipitated pH 7.4, and blotted onto GeneScreenPlus (NEN/DuPont) with 2 volumes of ethanol. The DNA was dissolved in 10 mM nylon membrane. Blots were prehybridized in 50%6 deionized Tris-HCl/1 mM EDTA, pH 7.4 (TE buffer), incubated with formamide/1% SDS/1 M NaCI/10% dextran sulfate for 30 0.1 mg of RNase A per ml at 370C for 1 hr, phenol/chloroform min at 42°C. The blots were then hybridized with 32P-labeled extracted, ethanol precipitated, and redissolved in TE buffer.
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