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Amplification Using CHO Cell Expression Vectors

Robert E. Kingston¹, Randal J. Kaufman², C.R. Bebbington³, M.R. Rolfe³

¹Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts
²University of Michigan, Ann Arbor, Michigan
³Celltech Ltd., Slough, United Kingdom

Publication Name:

Current Protocols in Molecular Biology

Unit Number:

Unit 16.23

DOI:

10.1002/0471142727.mb1623s60

Online Posting Date:

November, 2002

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Abstract

The ability to select for integration of plasmid DNA into the host chromosome allows the generation of stably transfected cell lines. With transfection of a selectable marker linked to a nonselectable target gene (or by cotransfection of the two unlinked genes), high-level expression of the desired gene is obtained by selecting for amplification of the selectable marker. This unit presents two systems for gene amplification and expression. The first describes the dihydrofolate reductase (DHFR) selection system while the second is based on selection of the glutamine synthetase (GS) gene. The DHFR system is probably more widely used, and results in very high levels of amplification and expression; however, the DHFR amplification process is lengthy and may require several months to isolate and characterize a stable, amplified line. In contrast, the GS system typically requires only a single round of selection for amplification to achieve maximal expression levels.

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Unit Introduction
Basic Protocol 1: Amplification Using Dihydrofolate Reductase
Alternate Protocol: Amplification by Cloning at Each Selective Step
Basic Protocol 2: Amplification Using Glutamine Synthetase
Reagents and Solutions
Commentary
Bibliography
Figures
Tables

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Materials

Basic Protocol 1: Amplification Using Dihydrofolate Reductase

Materials

pED (Kaufman et al., 1991) expressing appropriate cDNA; or pCVSVEII-DHFR or pAd26SV(A) (Kingston et al., 1984; Kaufman and Sharp, 1982a) and a separate vector expressing appropriate cDNA
CHO DXB11 or CHO DG44 cell lines (available from Lawrence Chasin, Columbia University) or CHO GRA (available from Randal Kaufman, University of Michigan)
Complete ADT medium (see recipe)
10% glycerol
Dialyzed fetal bovine serum (FBS; see recipe)
Complete ^– medium (Life Technologies) with 10% dialyzed FBS
Sterile vacuum grease
0.05% trypsin/0.6 mM EDTA in PBS (see APPENDIX 2 for PBS), 37°C
2% methylene blue in 50% ethanol (optional)
5 mM methotrexate (see recipe)
Cloning cylinders (see recipe)

Additional reagents and equipment for subcloning (UNIT 3.16), and either CaPO₄-mediated transfection (UNIT 9.1), electroporation (UNIT 9.3), or liposome-mediated transfection (UNIT 9.4)

NOTE: All tissue culture incubations are performed in a humidified 37°C, 5% CO₂ incubator unless otherwise indicated.

Basic Protocol 2: Amplification Using Glutamine Synthetase

Materials

Plasmid vector pEE14 (Celltech)
Complete Glasgow modified Eagle medium containing 10% dialyzed FBS (complete GMEM-10; see recipe)
CHO K1 cell line (ATCC #CCL61)
100 mM L-methionine sulfoximine (MSX; Sigma) prepared in PBS (see APPENDIX 2 for PBS; filter sterilize MSX solution and store in aliquots at –20°C; handle carefully)

Additional reagents and equipment for subcloning (UNIT 3.16), CaPO₄-mediated transfection and glycerol shock (UNIT 9.1), and cloning by limiting dilution (UNIT 11.8)

NOTE: All tissue culture incubations are performed in a humidified, 37°C, 5% CO₂ incubator unless otherwise indicated.

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Figures

Figure 16.23.1

Map of dicistronic mRNA expression vector pED. The components of the 5360-bp pED expression vector in the pUC18 background are indicated as follows: SV40, HindIII-PvuII fragment containing the SV40 origin of replication and enhancer element; MLP, adenovirus major late promoter fron the XhoI site (15.83 map units, m.u.) to the 5¢ cap site (16.55 m.u.); TPL, 180 bp of the first two and 2/3 of the third leaders from adenovirus major late mRNAs; IVS, a hybrid intron composed of the 5¢ splice site from the first leader of adenovirus major late mRNAs and a 3¢ splice site from an immunoglobin gene; PstI and EcoRI unique cloning sites; EMC-L, the 5¢ untranslated leader from EMC virus (nucleotides 260-827); DHFR, a murine DHFR coding region; SV40-polyA, the SV40 early polyadenylation signal; VA, the adenovirus VAI RNA gene from the HpaI (28.02 m.u.) to the BalI (29.62 m.u.); and -lactamase, a selectable gene for propagation in E. coli. Below is indicated the sequence junction of the EMC-L and DHFR as compared to the context of the AUG 11 which is initiation codon for the EMC virus polyprotein. A unique XhoI restriction site is available for insertion of other coding regions to be translated from the EMC virus leader. Adapted with permission from IRL Press.

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Figure 16.23.2

Placement of cloning cylinder around CHO colony.

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Figure 16.23.3

Map of pEE14 GS expression vector. pEE14 (~9.4 kb in length) contains a GS minigene as the selectable marker which has a single intron and GS polyadenylation signals and is driven from an SV40 late promoter. The hCMV-MIE promoter-enhancer and 5¢ untranslated region are used to express the gene of interest and the remainder of the plasmid contains an ampicillin-resistance gene and replication origin for replication in E. coli. The plasmid was constructed as follows. A 900-bp E coRI fragment from the cDNA clone gs1.1 (Hayward et al., 1986) was assembled with a 3.4-kb E coRI-SacI hamster GS genomic fragment from pGS1 (Sanders and Wilson, 1984), which provides the 3¢ end of the minigene. (The SacI site was converted to a BamHI site to facilitate vector construction.) The E coRI site within the GS coding sequence was destroyed by site-directed mutagenesis without altering the amino acid sequence and a HindIII site in GS 3¢-flanking DNA was destroyed by digestion with HindIII, filling in the single-stranded ends, and religation. A 340-bp SV40 late promoter (Cockett et al., 1990) was added to the 5¢ end as a BamHI-EcoRI fragment and the EcoRI site between the promoter and the GS sequences was destroyed by filling in. The resulting 4.5-kb BamHI fragment was inserted into pEE6hCMV (Stephens and Cockett, 1989) at a single BglII site upstream of the hCMV enhancer (hence destroying the BglII and BamHI sites) to form pEE14. The resulting SV40-GS minigene in pEE14 is functionally equivalent to that in pSVLGS.1 (Bebbington and Hentschel, 1987) but has been deleted of EcoRI and HindIII sites. Polylinker sequence of pEE14 is shown below.

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Literature Cited

Literature Cited
	Alt, F.W., Kellems, R.E., Bertino, J.R., and Schimke, R.T. 1978. Selective multiplication of dihydrofolate reductase genes in methotrexate-resistant variants of cultured mammalian cells. J. Biol. Chem. 253:1357-1370.
	Bebbington, C.R. and Hentschel, C.C.G. 1987. The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells. In DNA Cloning, Volume III (D. Glover, ed.). Academic Press, San Diego.
	Christman, J.K., Gerber, M., Price, P.M., Flordellis, C., Edelman, J., and Acs, G. 1982. Amplification of expression of hepatitis B surface antigen in 3T3 cells cotransfected with a dominant-acting gene and cloned viral DNA. Proc. Natl. Acad. Sci. U.S.A. 79:1815-1819.
	Cockett, M.I., Bebbington, C.R., and Yarranton, G.T. 1990. High-level expression of tissue inhibitor of metalloproteinases in Chinese hamster ovary cells using glutamine synthetase gene amplification. Bio/Technology 8:662-667.
	Davis, S.J., Ward, H.A., Puklavec, M., Willis, A.C., Williams, A.F., and Barclay, A.N. 1990. High-level expression in Chinese hamster ovary cells of soluble forms of CD4 T lymphocyte glycoprotein including glycosylation variants. J. Biol. Chem. 265:10410-10418.
	Harfst, E., Johnstone, A.P., Gout, I., Taylor, A.H., Waterfield, M.D., and Nussey, S.S. 1992. The use of amplifiable high-expression vector pEE14 to study the interactions of autoantibodies with recombinant human thyro trophin receptor. Mol. Cell Endocrinol. 83:117-123.
	Hayward, B.E., Hussain, A., Wilson, R.H., Lyons, A., Woodcock, V., McIntosh, B., and Harris, T.J.R. 1986. The cloning and nucleotide sequence of cDNA for an amplified glutamine synthetase gene from the Chinese hamster. Nucl. Acids Res. 14:999-1008.
	Kaufman, R.J. 1989. Selection and coamplification. Meth. Enzymol. 185:537-566.
	Kaufman, R.J. 1990. Strategies for expressing high-level expression in mammalian cells. Technique 2:221-236.
	Kaufman, R.J. and Sharp, P.A. 1982a. Amplification and expression of sequences cotransfected with a modular dihydrofolate reductase complementary DNA gene. J. Mol. Biol. 159:601-621.
	Kaufman, R.J. and Sharp, P.A. 1982b. Construction of a modular dihydrofolate reductase cDNA gene: Analysis of signals utilized for efficient expression. Mol. Cell. Biol. 2:1304-1319.
	Kaufman, R., Davies, M., Wasley, L., and Michnik, D. 1991. Improved vectors for stable expression of foreign genes in mammalian cells by use of internal ribosomal entry site from EMC virus. Nucl. Acids Res. 19:4485-4490.
	Kingston, R.E., Kaufman, R.J., and Sharp, P.A. 1984. Regulation of transcription of the adenovirus EII promoter by EIa gene products: Absence of sequence specificity. Mol. Cell. Biol. 4:1970-1977.
	Ringold, G., Dieckman, B., and Lee, F. 1981. Coexpression and amplification of dihydrofolate reductase cDNA and the Escherichia coli XGPRT gene in Chinese hamster ovary cells. J. Mol. Appl. Genet. 1:165-175.
	Sanders, P.G. and Wilson, R.H. 1984. Amplification and cloning of the Chinese hamster glutamine synthetase gene. EMBO J. 3:65-71.
	Stephens, P.E. and Cockett, M.I. 1989. The construction of a highly efficient and versatile set of mammalian expression vectors. Nucl. Acids Res. 17:7110.
	Urlaub, G. and Chasin, L.A. 1980. Isolation of Chinese hamster cell mutants deficient in dihydrofolate reductase activity. Proc. Natl. Acad. Sci. U.S.A. 77:4216-4220.
	Urlaub, G., Kas, E., Carothers, A.M., and Chasin, L.A. 1983. Deletion of the diploid dihydrofolate locus from cultured mammalian cells. Cell 33:405-412.
	Wigler, M., Silverstein, S., Lee, L., Pellicer, A., Cheng, Y., and Axel, R. 1977. Transfer of purified herpes virus thymidine kinase gene to cultured mouse cells. Cell 11:223-232.
	Wurm, F.W., Gwinn, K.A., and Kingston, R.E. 1986. Inducible overproduction of the mouse c-myc gene in mammalian cells. Proc. Natl. Acad. Sci. U.S.A. 83:5414-5418.
Key References
	Kaufman et al., 1991. See above.
Describes the construction and application of dicistronic DHFR vectors that allow stable, high-level expression of inserted cDNAs by selection for methotrexate resistance in both DHFR-containing and DHFR-deficient cells.
	Cockett et al., 1990. See above.
Describes the construction of vectors that provide for high-level expression using the GS gene-amplification system.