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Development of a Highly Efficient Expression Cdnacloning System

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Development of a Highly Efficient Expression Cdnacloning System Proc. Nati. Acad. Sci. USA Vol. 88, pp. 5167-5171, June 1991 Cell Biology Development of a highly efficient expression cDNA cloning system: Application to oncogene isolation (mouse hepatoma/stable expression/plasmid rescue/B-raf gene/in vivo activation) TORU MIKI*, TIMOTHY P. FLEMING*, MARCO CRESCENZI*t, CHRISTOPHER J. MOLLOY*t, SHELLEY B. BLAM*§, STEVEN H. REYNOLDS¶, AND STUART A. AARONSON*JI *Laboratory of Cellular and Molecular Biology, National Cancer Institute, Building 37, Room 1E24, Bethesda, MD 20892; and sNational Institute of Environmental Health Sciences, Biological Risk Assessment Program, Research Triangle Park, NC 27709 Communicated by G. D. Aurbach, March 19, 1991 (receivedfor review October 16, 1990) ABSTRACT We developed an expression cDNA cloning Cells were maintained in Dulbecco's modified Eagle's me- system capable of generating high-complexity libraries with dium (DMEM)/5% calf serum. unidirectionally inserted cDNA fragments and allowing effi- Plasmid Rescue. Genomic DNA (1.22g) was cleaved byXho cient plasmid rescue. As an application of this system, a cDNA I and ligated at the concentration of3 ng/,ul. Four aliquots (100 library was constructed from an NIH 3T3 transformant in- Al each) of PLK-F' competent cells (Stratagene) were trans- duced by mouse hepatocellular carcinoma DNA. Transfection formed by the ligated DNA, as directed by the manufacturer. of NIH 3T3 cells by the library DNA led to the detection of After heat shock, the cells were diluted 10-fold with S.O.C. several transformed foci from which identical plasmids with medium (BRL) containing 1 mM isopropyl /8-D-thiogalacto- transforming ability could be rescued. Structure and sequence pyranoside to induce expression ofthe neo gene driven by the analysis of the cDNA clones revealed that the oncogene was tac promoter. The culture was incubated for 2 hr with shaking created by recombinational events involving an unknown gene and plated on NZY hard agar (8) containing ampicillin (100 and the mouse homologue of the B-raf protooncogene. Detec- ,ug/ml), kanamycin (25 ,ug/ml), and isopropyl /B-D-thiogalac- tion ofthe same genetic rearrangement in independent primary topyranoside (100 MM). transformants implied that generation of the oncogene oc- Recombinant DNA Techniques. Preparation of A and plas- curred within the tumor rather than during DNA transfection mid DNA was performed as described (1). Genomic DNA or cDNA library construction. The high frequency at which was extracted by the standard procedure (8). Total RNA was clones were identified and the large sizes of some of the isolated and poly(A)-selected as described (1). DNA frag- ments were isolated by Geneclean (Bio 101 Inc., La Jolla, transforming cDNA inserts isolated suggest wide applicability CA) and labeled by random priming using an Oligo labeling of this mammalian expression cloning system for isolating kit (Pharmacia). Hybridizations were performed at 420C in cDNAs of biologic interest. 50% (wt/vol) formamide/5 x standard saline citrate (8)/2.5x Denhardt's solution (8)/7 mM Tris hydrochloride, pH 7.5; In bacterial and yeast systems, genes can be cloned by denatured calfthymus DNA at 0.1 mg/ml/tRNA at 0.1 mg/mi. complementing mutant alleles with introduced genes. Simi- Nucleotide sequence was determined by the dideoxynucleo- larly, in mammalian systems, introduction of cDNAs placed tide chain-termination method (11). under a suitable promoter can complement mutations in recipient cells or allow for selection of any identifiable RESULTS phenotype. We have recently reported the development ofan efficient system to construct unidirectional cDNA libraries Development of an Efflicient Expression cDNA Cloning Sys- (1), which has made it possible to isolate full-length coding tem. The ApCEV27 system was developed to clone cDNAs sequences of several genes (1-6). In this report, we refined by means of stable phenotypic changes induced by a specific the vector to allow for high-level cDNA expression in mam- cDNA. Use ofa A-plasmid composite vector made it possible malian cells and the ability to perform efficient plasmid to generate high-complexity cDNA libraries and to efficiently rescue. We tested the potential of this expression cloning excise the plasmid from the stably integrated phagemid DNA. This phagemid vector (Fig. 1) contained several features system by cloning an oncogene cDNA from a mouse hepato- including two Sfi I sites for construction ofcDNA libraries by cellular carcinoma (7).** using the automatic directional cloning (ADC) method (1), a Moloney murine leukemia virus long terminal repeat pro- EXPERIMENTAL PROCEDURES moter suitable for cDNA expression in mammalian cells, the simian virus 40 (SV40) promoter-driven neo gene as a select- cDNA Library Construction. cDNA libraries were con- able marker, and multiple excision sites for plasmid rescue structed as reported (1), except for use of a designed vector from genomic DNA. The ApCEV27 vector also incorporated and adaptor more suitable for expression cloning (see Re- the rat pre-pro-insulin poly(A) signal downstream from the sults). The revised Sfi I adaptor consisted of two oligonucle- cDNA cloning site (Fig. 1). otides, 5'-CCAATCGCGACC-3' and 5'-GGTCGCGATTG- GTAA-3'. Amplification of the library and preparation of the Abbreviation: SV40, simian virus 40. DNA were done by standard procedures (8). tPresent address: Dipartimento di Biologia Cellulare e Dello Svi- Cell Culture and DNA Transfection. All cells used were luppo, Universita di Roma "La Sapienza", 00185 Rome, Italy. derivatives of NIH 3T3 cells (9). Calcium phosphate trans- tPresent address: Department ofCardiovascular Biochemistry, Bris- fection (10) was used to stably introduce DNA into cells. tol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ 08543-4000. §Present address: Berlex Biosciences, Alameda, CA 94501. The publication costs of this article were defrayed in part by page charge 'lTo whom reprint requests should be addressed. payment. This article must therefore be hereby marked "advertisement" **The sequence reported in this paper has been deposited in the in accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank data base (accession no. M64429). 5167 Downloaded by guest on September 27, 2021 5168 Cell Biology: Miki et al. Proc. Natl. Acad. Sci. USA 88 (1991) XpCEV27 cDNA library (-) (+) Transfection LITransfection _ :asmid _ DNA Focus A~~~~~~~~l| __ _ Soft agar assay A E. coli G-418 selection Transformation DNA extraction 17-I' 4 pNolsA ~~~~~~~Digestion and ligation FIG. 2. Strategy for expression cloning of transforming gene cDNAs. NIH 3T3 cells are transfected by ApCEV27 cDNA library DNA and scored at 14-17 days for transformed foci. Transformed FIG. 1. Structure of the cDNA cloning-expression vector cells can then be assayed for G-418 resistance and colony formation ApCEV27. Structure of the ApCEV27 vector is shown above with in soft agar. After expansion to mass culture, genomic DNA is location of A genes. Plasmid portion is enlarged and shown below as isolated and subjected to plasmid rescue by digestion with either Not circular map. The multiple excision site (MES) contains restriction I, Xho I, orMlu I, followed by ligation at low DNA concentration and sites for infrequent cutters: Not I, Xho 1, Pvu I, and Mlu I. The transformation to a suitable bacterial strain. Bacterial colonies multiple cloning site (MCS) contains restriction sites forBamHI, Sal resistant to both ampicillin and kanamycin are isolated. Plasmid I, Sfi I(A), EcoRI, Bgl II, HindIII, Sfi I(B), Sal I, and BstEII in DNA extracted from each colony is tested by transfection analysis clockwise order. The two Sfi I sites are used to insert cDNA on NIH 3T3 cells to identify the transforming cDNA clone. molecules by the automatic directional cloning method (1), and the two Sal I sites are used to release the inserts. SP6-P and T7-P Conventional approaches for molecular cloning of trans- represent phage promoters for SP6 and T7 RNA polymerases, forming genes identified by transfection analysis have gen- respectively. The trp-lac fused promoter tac and SV40 early pro- erally taken advantage of differences in repetitive sequences moter are used to express the neo structural gene in Escherichia coli between the transfecting DNA and those ofthe recipient cell. (kanamycin resistance) and eukaryotic cells (G-418 resistance), Such sequences provide molecular probes for use in identi- respectively. In contrast to ApCEV15 (1), the bonafide promoter of fying and isolating genomic fragments of the transforming the neo gene was removed so as to fuse the SV40 early promoter to directly to the neo structural gene. Thus, in ApCEV27, expression of genes. Rodent oncogenes have been particularly difficult the neo gene in E. coli was achieved by transcription from the tac molecularly clone because relatedness of their repetitive promoter, inserted upstream from the SV40 early promoter. Direc- sequences precludes discrimination of such genes from ge- tions of transcription from the promoters are shown by arrows. nomic DNA of mouse NIH 3T3, commonly used to isolate Polyadenylylation signals are labeled as poly(A). In earlier vectors oncogenes by DNA transfection. To overcome such inherent (1), replication of rescued plasmids in E. coli was unstable. In ApCEV27, the replication origin ofpUC19 was used to increase copy number. Locations ofreplication origins (ori) and ampicillin resistant gene (amp) are shown. M-MLV LTR, Moloney murine leukemia virus long terminal repeat. --~~~~~L The strategy for expression cloning of oncogene cDNAs is (kh summarized in Fig. 2. When library cDNA is used to transfect S _ mammalian cells, cDNA clones are integrated with recom- _II0I bination between A DNA and host genomic DNA. For plasmid rescue, genomic DNA extracted from the transform- ant is subjected to digestion with an enzyme that can cleave the A-plasmid junctions. The resulting DNA can then be circularized and used for bacterial transformation. For this purpose, the sites for two additional infrequent cutters, Xho I and Mlu I, were included along with the Not I site.
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