Production of Recombinant Proteins in Mammalian Cells

Su Chen1, David Gray1, Johnny Ma1, Shyamsundar Subramanian1

1 Chiron Corporation, Emeryville, California
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 5.10
DOI:  10.1002/0471140864.ps0510s12
Online Posting Date:  May, 2001
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

The best strategy for consistent production of larger quantities of pure protein is stable expression. Popular hosts for stable expression are Chinese hamster ovary (CHO) cells, baby hamster kidney (BHK‐21) cells, myeloma cells, and the transformed kidney cell line 293. Protocols for stable production in CHO cells are described in this unit. Typical methods for transfection using commercially available plasmid expression vectors are described, along with methods to select for stable expression and methods for amplifying the expression level in the transfected cell. Following this, procedures are presented for efficient cell growth to obtain significant amounts of protein product. Support protocols describe freezing of cells, determination of growth rates, determination of specific productivity of cells, preparing samples for assay, and setting up a 10‐day shaker‐flask growth curve.

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Basic Protocol 1: Plasmid Purification by Alkaline‐Lysis/Anion‐Exchange Capture
  • Basic Protocol 2: Transfection of Cells by Lipofection
  • Alternate Protocol 1: Transfection of Cells by Electroporation
  • Basic Protocol 3: Selection for Neomycin Phosphotransferase (NPTII) with Geneticin (G418)
  • Support Protocol 1: Determination of Background Sensitivity to G418
  • Alternate Protocol 2: Screening of Green Fluorescent Protein by Flow Cytometry
  • Support Protocol 2: Single‐Cell Cloning
  • Basic Protocol 4: Amplification of Dihydrofolate Reductase (DHFR) with Methotrexate (MTX)
  • Basic Protocol 5: Adaptation of Suspension Cells to Production Medium Through Multiple Passaging
  • Basic Protocol 6: Growth of Cells in Batch Mode in Large‐Scale Spinner Culture Vessels
  • Basic Protocol 7: Growth of Cells in Large‐Scale Batch Reactors
  • Alternate Protocol 3: Growth of Cells in Continuous Culture in Bioreactors
  • Harvesting of Protein Product from Batch Mode Spinner Cultures and Large‐Scale Batch Reactors
  • Basic Protocol 8: Harvesting a Secreted Product
  • Basic Protocol 9: Harvesting a Cell‐Associated Product
  • Support Protocol 3: Freezing Cells for Produciton of Cell Banks
  • Support Protocol 4: Numerical Determination of Specific Growth Rate of Cells
  • Support Protocol 5: Graphical Determination of Specific Growth Rate of Cells
  • Support Protocol 6: Numerical Determination of Specific Productivity of Cells Producing Heterologous Protein
  • Support Protocol 7: Graphical Determination of Specific Productivity of Cells Producing Heterologous Protein
  • Support Protocol 8: Preparation of Samples for Product Assay
  • Support Protocol 9: Determination of Shaker Flask Growth Curve
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Plasmid Purification by Alkaline‐Lysis/Anion‐Exchange Capture

  Materials
  • cDNA containing gene of interest
  • Plasmid with appropriate cis‐acting elements (Fig. and unit 5.2)
  • Competent E. coli cells (e.g., strain DH5‐α; Life Technologies)
  • LB medium (unit 5.2) containing antibiotics for selection (e.g, 100 µg/ml ampicillin, 40 µg/ml kanamycin, or 25 µg/ml tetracycline, depending on plasmid kit; all antibiotics available from Sigma)
  • recipeResuspension buffer (see recipe)
  • Lysis solution: 0.2 M NaOH/1% (w/v) SDS
  • Neutralization solution: 1.3 to 3.0 M potassium acetate, pH 4.8 to 5.5
  • Isopropanol
  • TE buffer ( appendix 2E)
  • 80% ethanol
  • Sterile glass test tubes for bacterial culture
  • Environmental shaker (New Brunswick Scientific)
  • 3‐liter Erlenmeyer flask
  • Beckman J2‐21 centrifuge with JA‐10 rotor or Sorvall RC‐5B centrifuge with GS‐3 rotor (or equivalent) and appropriate polycarbonate centrifuge bottles
  • UV/visible light spectrophotometer (e.g., Perkin‐Elmer)
  • Additional reagents and equipment for ligation of linkers to cDNA (Klickstein and Neve, ), plasmid selection and insertion of DNA into plasmid vectors (unit 5.2), transformation of E. coli (unit 5.2), and purification of DNA by anion‐exchange chromatography (Budelier and Schorr, )
NOTE: Several DNA plasmid preparation kits are available commercially—e.g., Plasmid Maxi Kit (Qiagen), Wizard Maxipreps (Promega), and FlexiPrep Kit (Pharmacia Biotech). Kits include resuspension buffer, lysis solution, and neutralization solution as described in the protocol.

Basic Protocol 2: Transfection of Cells by Lipofection

  Materials
  • Chinese hamster ovary (CHO) cells (Table 97.80.4711)
  • recipeαMEM medium with and without 10% FBS (see recipe)
  • Lipofectamine (Life Technologies; store up to 6 months at 4°C)
  • Plasmid DNA to be transfected (see protocol 1)
  • 100‐mm tissue culture dishes or T‐75 flasks
  • Polystyrene tubes
  • Additional reagents and equipment for growing mammalian cells in culture ( appendix 3C)

Alternate Protocol 1: Transfection of Cells by Electroporation

  • Electroporator and electroporation cuvettes with 0.4‐cm electrode gap (Bio‐Rad)
  • Additional reagents and equipment for growing mammalian cells in culture, trypsinizing cells, and counting viable cells by trypan blue exclusion ( appendix 3C)

Basic Protocol 3: Selection for Neomycin Phosphotransferase (NPTII) with Geneticin (G418)

  Materials
  • Culture of transformed cells at 50% to 70% confluency, in T‐25 or T‐75 flasks (24 to 48 hr post‐transfection; see protocol 2 or protocol 3)
  • Selective medium: recipeαMEM medium (see recipe) containing 5% to 10% FBS and 0.1 to 1.0 mg/ml G418 (Geneticin, Life Technologies; add from 50 mg/ml stock prepared in 100 mM HEPES, pH 7.0 to 7.2; see protocol 5 for optimization of concentration)
  • 96‐well flat‐bottom tissue culture plates
  • Inverted microscope
  • T‐75 tissue culture flasks
  • Additional reagents and equipment for growing mammalian cells in culture, trypsinizing cells, and counting viable cells by trypan blue exclusion ( appendix 3C)

Support Protocol 1: Determination of Background Sensitivity to G418

  • Selective media containing various concentrations (100 to 1000 µg/ml in 100‐µg/ml increments) of G418 (add from 50 mg/ml stock prepared in 100 mM HEPES, pH 7.0 to 7.2)
  • 100‐mm tissue culture dishes

Alternate Protocol 2: Screening of Green Fluorescent Protein by Flow Cytometry

  Materials
  • Cells transformed with plasmid (see protocol 2 or protocol 3) containing GFP reporter gene (pIRES1‐EGFP from Clontech or pTracer‐CMV from Invitrogen) at 50% to 70% confluency in T‐25 or T‐75 flasks
  • 1% (w/v) bovine serum albumin (BSA; Sigma) in PBS ( appendix 2E)
  • 10× (0.05 mg/ml) propidium iodide stock (PI; Molecular Probes; optional)
  • Appropriate culture medium
  • Flow cytometer with cell sorting capability (FACS Vantage and FACSort from Becton Dickinson Immunocytometry Systems or EPICS Elite ESP cell sorter from Coulter Electronics or equivalent)
  • T‐25 tissue culture flasks
  • Additional reagents and equipment for trypsinizing cells, counting viable cells by trypan blue exclusion, and growing mammalian cells in culture ( appendix 3C) and flow cytometry (Robinson et al., )

Support Protocol 2: Single‐Cell Cloning

  Materials
  • Cells expressing gene product of interest (see protocol 4 or protocol 3) growing in tissue culture flasks or dishes at 70% to 90% confluency
  • Appropriate culture medium
  • Coulter Counter or equivalent
  • 96‐well tissue culture plates
  • Additional reagents and equipment for growing mammalian cells in culture, trypsinizing cells, and counting viable cells by trypan blue exclusion ( appendix 3C)

Basic Protocol 4: Amplification of Dihydrofolate Reductase (DHFR) with Methotrexate (MTX)

  Materials
  • Culture of transformed cells at 50% to 70% confluency, in T‐25 or T‐75 flasks (24 to 48 hr post‐transfection; see protocol 2 or protocol 3)
  • Nucleoside‐free recipeαMEM medium (see recipe but omit nucleosides) containing 10% FBS that has been dialyzed to remove nucleosides (HyClone)
  • 10 mM methotrexate (MTX) stock solution (Sigma; store up to 1 year at −20°C; thaw just before use)
  • 96‐well flat‐bottom tissue culture plates
  • 24‐well tissue culture plates
  • T‐25 tissue culture flasks
  • 100‐mm tissue culture dishes
  • Additional reagents and equipment for growing mammalian cells in culture, trypsinizing cells, and counting viable cells by trypan blue exclusion ( appendix 3C)

Basic Protocol 5: Adaptation of Suspension Cells to Production Medium Through Multiple Passaging

  Materials
  • Mid–log phase culture of stable‐transfected cells in T‐75 flask (or thawed 1‐ml aliquot of cryopreserved cells; see protocol 7), produced by DHFR selection/MTX amplification (see protocol 8) or other selection/amplification procedure
  • recipeNucleoside‐free αMEM medium (see recipe but omit nucleosides) containing 10% FBS that has been dialyzed to remove nucleosides (for DHFR selection/MTX amplification cells) or reciperegular αMEM medium (see recipe) containing 10% FBS (prepare as appropriate for cells produced by other selection/amplification procedures)
  • 10 mM methotrexate (MTX) stock solution (Sigma; store up to 1 year at −20°C; thaw just before use)
  • Dispase solution (Boehringer Mannheim)
  • recipeDMEM/F12 custom‐modified medium (see recipe) with 10%, 1%, and 0.5% dialyzed FBS
  • CD CHO medium (for DHFR systems; Life Technologies), Super CHO (for non‐DHFR systems; Bio‐Whittaker), or CHO‐S‐SFM (for non‐DHFR systems; Life Technologies)—optional, for adaptation to serum‐free medium
  • 1 g/liter Nucellin‐Zn salt (recombinant insulin; Eli Lilly)—optional, for adaptation to serum‐free medium
  • T‐175 flasks
  • Sterile 45‐ml polycarbonate screw‐cap tubes
  • Inverted phase‐contrast microscope
  • 250‐ml shaker flasks
  • Platform shaker (New Brunswick Scientific)
  • Additional reagents and equipment for growing mammalian cells in culture, trypsinizing cells, and counting viable cells by trypan blue exclusion ( appendix 3C), determining specific growth rate of cells (see protocol 16Support Protocols 4, protocol 175, and protocol 219), determining specific productivity of cells producing heterologous protein (see protocol 18Support Protocols 6 and protocol 197), and freezing cells (see protocol 7)

Basic Protocol 6: Growth of Cells in Batch Mode in Large‐Scale Spinner Culture Vessels

  Materials
  • 1‐ml cryovial or mid–log phase culture of suspension‐adapted cells (see protocol 9)
  • Medium to which cells have been adapted (see protocol 9), supplemented with 4 mM L‐glutamine and 4.5 to 5.5 g/liter D‐glucose
  • Medical‐grade, sterile‐filtered 5% CO 2/air mixture
  • Medical‐grade, sterile‐filtered 100% O 2 (if necessary)
  • 250‐ml screw‐cap shaker flasks
  • Platform shaker (New Brunswick Scientific)
  • 1‐, 5‐, and 20‐liter borosilicate glass spinner jars (Bellco Biotechnology)
  • Flat‐plate stirrer platform (Bellco Biotechnology)
  • Peristaltic pump (head size 15, 16, 24; with 0 to 600 rpm capability; Watson‐Marlow or Cole‐Parmer)
  • Sterile silicone tubing (Pt‐cured; size 15, 16, 24; Masterflex from Cole‐Parmer)
  • Sterile 50‐ml syringes
  • Dissolved oxygen probe (Ingold)
  • pH probe (Ingold or Broadley James)
  • Additional reagents and equipment for growing mammalian cells in culture and counting viable cells by trypan blue exclusion ( appendix 3C), determining specific growth rate of cells (see protocol 16Support Protocols 4, protocol 175, and protocol 219), and determining specific productivity of cells producing heterologous protein (see protocol 18Support Protocols 6 and protocol 197)

Basic Protocol 7: Growth of Cells in Large‐Scale Batch Reactors

  Materials
  • Inoculum of mid–log phase cells in spinner jar (see protocol 10)
  • Ultrafiltered deionized water
  • Medium to which cells have been adapted (see protocol 9), supplemented with 8 mM L‐glutamine and 4.5 to 5.5 g/liter D‐glucose
  • Medical‐grade, sterile‐filtered 95% N 2/5% CO 2 and 95% air/5% CO 2 mixtures and 100% oxygen
  • 10‐ to 100‐liter bioreactor (New Brunswick Scientific, Bioengineering, Braun, New MBR, Applikon)
  • pH probe (Broadley James or Ingold), calibrated
  • Dissolved oxygen probe (Ingold), pretested
  • Autoclave large enough to accommodate bioreactor vessel (Finn Aqua)
  • Inoculum transfer bottle with bottom side port attached to sterile size 16 tubing (Bellco Biotechnology)
  • Sterile flexible disposable medium and harvest bags (Stedim Laboratories)
  • Additional reagents and equipment for counting viable cells by trypan blue exclusion ( appendix 3C), determining specific growth rate of cells (see protocol 16Support Protocols 4, protocol 175 and protocol 219) and determining specific productivity of cells producing heterologous protein (see protocol 18Support Protocols 6 and protocol 197)

Alternate Protocol 3: Growth of Cells in Continuous Culture in Bioreactors

  • Bioreactor with accessories for continuous operation (consult New Brunswick Scientific, New MBR, Bioengineering AG, Braun, or other fermenter manufacturer; for perfusion operation where retention is required, use reactors with spin filter design, e.g., New MBR or Braun)
  • Sterile bags (Stedim Laboratories) or sterilized stainless steel containers for collection of medium

Basic Protocol 8: Harvesting a Secreted Product

  Materials
  • Cells in batch culture (see protocol 10Basic Protocols 6 or protocol 117 or protocol 12)
  • Phosphate‐buffered saline (PBS; appendix 2E)
  • TE buffer or PBS ( appendix 2E)
  • Sterile bags (Stedim Laboratories)
  • Refrigerated centrifuge: Sorvall RC‐5B or RC‐3B, Beckman J2‐21 or S600, or equivalent
  • Microfiltration system with membranes for cell concentration and debris/product separation (Sartorius, Pall‐Filtron, or Millipore)
  • Depth filtration membranes for large‐scale debris separation prior to sterile filtration (Cuno io‐Cap 90 SP or equivalent products from Millipore or Sartorius)
  • Filter‐sterilizing capsules (0.2‐µm membrane; Sartorius, Pall, or Millipore)
  • Silicone tubing, autoclaved
  • Sterile polycarbonate containers
  • Ultrafiltration system with membranes for protein concentration (MWCO 10‐kDa; Sartorius, Pall‐Filtron, or Millipore)

Basic Protocol 9: Harvesting a Cell‐Associated Product

  Materials
  • Cells in batch culture (see protocol 10Basic Protocols 6 or protocol 117 or protocol 12)
  • Phosphate‐buffered saline (PBS; appendix 2E)
  • recipeHomogenization buffer (see recipe), 4°C
  • Cell homogenizer: e.g., Microfluidizer (Microfluidics), Tissumizer (Tekmar‐Dohrmann), Dounce homogenizer (Bellco Biotechnology), or Manton‐Gaulin‐APV homogenizer (APV‐Gaulin)
  • Additional reagents and equipment for centrifugation or cross‐flow microfiltration of cell suspension (see protocol 13)

Support Protocol 3: Freezing Cells for Produciton of Cell Banks

  Materials
  • High‐viability (>95%) log‐phase culture of cells
  • Freezing medium: complete medium used for growing cells supplemented with 10% to 20% (v/v) FBS and 5% to 10% (v/v) DMSO (Sigma), 4°C
  • 45‐ml sterile screw‐cap tubes polycarbonate centrifuge tubes (Falcon from Becton Dickinson)
  • Sorvall T‐6000B centrifuge (DuPont) or equivalent
  • 1.5‐ml Nalgene cryotubes (Nunc)
  • Additional reagents and equipment for counting viable cells by trypan blue exclusion ( appendix 3C)

Support Protocol 4: Numerical Determination of Specific Growth Rate of Cells

  Materials
  • Spreadsheet software with graphical output (e.g., Microsoft Excel or Lotus 123)

Support Protocol 5: Graphical Determination of Specific Growth Rate of Cells

  Materials
  • Spreadsheet software with graphical output (e.g., Microsoft Excel or Lotus 123)

Support Protocol 6: Numerical Determination of Specific Productivity of Cells Producing Heterologous Protein

  Materials
  • Sample of cell culture (see protocol 10Basic Protocols 6 or protocol 117 or protocol 12)
  • 0.9% NaCl, filter‐sterilized, 4°C
  • Refrigerated centrifuge
  • Ultrasonic homogenizer (Misonix)

Support Protocol 7: Graphical Determination of Specific Productivity of Cells Producing Heterologous Protein

  • Thawed vial of cryopreserved cells (0.5 ml to 1 ml at 107 viable cells/ml) or aliquot of healthy cells in exponential growth phase (107 cells total in ∼25 ml)
  • Additional reagents and equipment for determination of specific growth rate (see protocol 16Support Protocols 4 and protocol 175) and specific protein productivity of cells (see protocol 18Support Protocols 6 and protocol 197)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Aitken, A. 1997. A library of concensus sequences. Methods Mol. Biol. 64:357‐367.
   Aruffo, A. 1997. Transient expression of proteins in COS cells. In Current Protocols in Molecular Biology (F.A. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhleds.) pp. 16.13.1‐16.13.7. John Wiley & Sons, New York.
   Ausubel, F.A., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., and Struhl, K., (eds.) 1998. Current Protocols in Molecular Biology. John Wiley & Sons, New York.
   Batt, B.C., Davis, R.H., and Kompala, D.S. 1990. Inclined sedimentation for selective retention of viable hybridomas in a continuous suspension bioreactor. Biotechnol. Prog. 6:458‐464.
   Brady, R.O., Murray, G.J., and Barton, N.W. 1994. Modifying exogenous glucocerebrosidase for effective replacement therapy in Gaucher Disease. J. Inherited Metab. Dis. 17:510‐519.
   Brinster, R.L., Allen, J.M., Behringer, R.R., Gelinas, R.E., and Palmiter, R.D. 1988. Introns increase transcriptional efficiency in transgenic mice. Proc. Natl. Acad. Sci. U.S.A. 85:836‐840.
   Brown, P.C., Wininger, M., Chow, R., and Cox, J. 1993. Perfusion culture of CHO cells in suspension facilitated by inclined sedimentation chambers for cell recycle. Presented at the 205th National Meeting of the ACS, Division of Biotechnology, March 31, 1993.
   Budelier, K. and Schorr, J. 1998. Purification of DNA by anion‐exchange chromatography. In Current Protocols in Molecular Biology (F.A. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 2.1.11‐2.1.18. John Wiley & Sons, New York.
   Chalfie, M., Tu, Y., Euskirchen, G., Ward, W.W., and Prasher, D.C. 1994. Green fluorescent protein as a marker of gene expression. Science 263:802‐805.
   Chalmers, J.J., Zborowski, M., Sun, L., and Moore, L. 1998. Flow‐ through immunomagnetic cell separation. Biotechnol. Prog. 14:141‐148.
   Chernajovsky, Y., Mory, Y., Chen, L., Marks, Z., Novick, D., Rubinstein, M., and Revel, M. 1984. Efficient constitutive production of himan fibroblast interferon by hamster cells transformed with the IFN‐β1 gene fused to an SV40 early promoter. DNA 3:297‐308.
   Chesnut, J.D., Baytan, A.R., Russel, M., Chang, M‐P., Bernard, A., Maxwell, I.H., and Hoeffler, J.P. 1996. Selective isolation of transiently transfected cells from a mammalian cell population with vectors expressing a membrane anchored single‐chain antibody. J. Immunol. Methods 193:17‐27.
   Cockett, M.I., Bebbington, C.R., and Yarranton, G.T. 1990. High level expression of tissue inhibitor of metaloproteinase in Chinese hamster ovary cells using glutamine synthetase gene amplification. Biotechnol. 8:662‐667.
   Earl, P. and Moss, B. 1998. Characterization of recombinant vaccinia viruses and their products. In Current Protocols in Molecular Biology (F.A. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 16.18.1‐16.18.10. John Wiley & Sons, New York.
   Earl, P., Wyatt, L., Carroll, M., and Moss, B. 1998a. Generation of recombinant vaccinia viruses. In Current Protocols in Molecular Biology (F.A. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 16.17.1‐16.17.16. John Wiley & Sons, New York.
   Earl, P.L., Cooper, N., Wyatt, L., Carroll, M., and Moss, B. 1998b. Preparation of cell cultures and vaccinia virus stocks. In Current Protocols in Molecular Biology (F.A. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 16.16.1‐16.16.7. John Wiley & Sons, New York.
   Elroy‐Stein, O. and Moss, B. 1998. Gene expression using the vaccinia virus/T7 RNA polymerase hybrid system. In Current Protocols in Molecular Biology (F.A. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 16.19.1‐16.19.9. John Wiley & Sons, New York.
   Fukushige, S. and Sauer, B. 1992. Genomic targeting with a positive‐selection lox integration vector allows highly reproducible gene expression in mammalian cells. Proc. Natl. Acad. Sci. U.S.A. 89:7905‐7909.
   Gramer, M.J., Goochee, C.F., Chock, V.Y., Brousseau, D.T., and Sliwkowski, M.B. 1995. Removal of sialic acid from a glycoprotein in CHO cell culture supernatant by action of an extracellular CHO cell sialidase. Bio/Technology 13:692‐298.
   Gray, F., Kenney, J.S., and Dunne, J.F. 1995. Secretion capture and report web: Use of affinity derivatized agarose microdroplets for the selection of hybridoma cells. J. Immunol. Methods 182:155‐163.
   Griffiths, B. 1992. Scaling‐up of animal cell cultures. In Animal Cell Culture, 2nd ed. (R.I. Freshneyed.) pp. 47‐93. IRL Press, Oxford.
   Gu, M.B., Todd, P., and Kompala, D.S. 1993. Foreign gene expression (β‐galactosidase) during the cell cycle phases in recombinant CHO cells. Biotechnol. Bioeng. 42:1113‐1123.
   Kahana, J. and Silver, P. 1996. Use of the A. victoria green fluorescent protein to study protein dynamics in vivo. In Current Protocols in Molecular Biology (F.A. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 9.7.22‐9.77.28. John Wiley & Sons, New York.
   Kain, S. and Ganguly, S. 1996. Overview of genetic reporter systems. In Current Protocols in Molecular Biology (F.A. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 9.6.3‐9.6.12. John Wiley & Sons, New York.
   Kaufman, R.J. 1978. Quantitation of dihydrofolate reductase in individual parental and ethotrexate‐resistant murine cells. J. Biol. Chem. 16:5852‐5860.
   Kaufman, R.J. 1990. Selection and coamplification of heterologous genes in mammalian cells. Methods Enymol. 185:537‐566.
   Kaufman, R.J., Wasley, L.C., Spiliotes, A.J., Gossels, S.D., Latt, S.A., Larsen, G.R., and Kay, R.M. 1985. Coamplificaton and coexpression of human tissue‐type plasminogen activator and murine dihydrofolate reductase sequences in Chinese hamster ovary cells. Mol. Cell. Biol. 5(7):1750‐1759.
   Kaufman, R.J., Davies, M.V., Wasley, L.C., and Michnick, D. 1991. Improved vectors for stable expression of foreign genes in mammalian cells by use of the untranslated leader sequence from EMC virus. Nucl. Acids Res. 19(16):4485‐4490.
   Kingston, R.E., Kaufman, R.J., Bebbington, C.R., and Rolfe, M.R. 1993. Amplification using CHO cell expression vectors. In Current Protocols in Molecular Biology (F.A. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 16.14.1‐16.14.13. John Wiley & Sons, New York.
   Klickstein, L.B. and Neve, R.L. 1991. Ligation of linkers or adapters to double‐stranded cDNA. In Current Protocols in Molecular Biology (F.A. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 5.6.1‐5.5.10. John Wiley & Sons, New York.
   Kozak, M. 1986. Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell 44:283‐292.
   Kriegler, M. 1990. Gene Transfer and Expression, A Laboratory Manual. Stockton Press, New York.
   Liljestrom, P. and Garoff, H. 1995. Expression of proteins using Semliki Forest virus vectors. In Current Protocols in Molecular Biology (F.A. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 16.20.1‐16.20.16. John Wiley & Sons, New York.
   Lucas, B.K., Giere, L.M., DeMarco, R.A., Shen, A., Chisholm, V., and Crowley, C.W. 1996. High level expression of recombinant proteins in CHO cells using adicistronic DHFR intron expression vector. Nucl. Acids Res. 24(9):1774‐1779.
   Maiorella, B.M., Dorin, G., Carion, A., and Harano, D. 1991. Crossflow microfiltration of animal cells. Biotechnol. Bioeng. 37:121‐126.
   Michel, M‐L., Sobczak, E., Malpiece, Y., Tiollais, P., and Streeck, R.E. 1985. Expression of amplified hepatitis B surface antigen in Chinese hamster ovary cells. Bio/Technology 3:561‐566.
   Miloux, B. and Lupker, J.H. 1994. Rapid isolation of highly productive recombinant Chinese hamster ovary cell lines. Gene 149:341‐344.
   Morris, A.E., Lee, C‐C., Hodges, K., Aldrich, T.L., Krantz, C., Smidt, P.S., and Thomas, J.N. 1997. Expression augmenting sequence element (EASE) isolated from Chinese hamster ovary cells. In Animal Cell Technology (M.J.T. Carrondo, ed.) pp. 529‐534. Kluwer Academic Publishers, Boston.
   Mortensen, R., Chesnut, J.D., Hoeffler, J.P., and Kingston, R.E. 1997. Selection of transfected mammalian cells. In Current Protocols in Molecular Biology (F.A. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 16.15.1‐16.5.19. John Wiley & Sons, New York.
   Mory, Y., Ben‐Barak, J., Segev, D., Cohen, D., Novick, D., Fischer, D.G., Rubinstein, M., Kargman, S., Zilberstein, M., and Revel, M. 1986. Efficient constitutive production of human IFN‐γ in Chinese hamster ovary cells. DNA 5:181‐193.
   Moss, B. and Earl, P.L. 1998. Overview of the vaccinia virus expression system. In Current Protocols in Molecular Biology (F.A. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 16.15.1‐16.15.5. John Wiley & Sons, New York.
   Page, M.J. and Sydenham, M.A. 1991. High level expression of the humanized monoclonal antibody Campath‐1H in Chinese hamster ovary cells. Bio/Technology 9:64‐68.
   Parekh, R.B. and Patel, T.P. 1992. Comparing the glycosylation patterns of recombinant glycoproteins. Trends Biotechnol. 10(8):276‐280.
   Pendse, G.J., Karkare, S., and Bailey, J.E. 1992. Effect of cloned gene dosage on cell growth and Hepatitis B surface antigen synthesis and secretion in recombinant CHO cells. Biotechnol. Bioeng. 40:119‐129.
   Robinson, J.P., Darzynkiewicz, Z., Dean, P.N., Orfao, A., Rabinovitch, P.S., Stewart, C.C.. Tanke, H.J., and Wheeless, L.L., (eds.) 1998. Current Protocols in Cytometry. John Wiley & Sons, New York.
   Sambrook, J., Fritsch, E.F., and Maniatis, T. 1989. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
   Simonsen, C. and Levinson, A. 1983. Isolation and expression of an altered mouse dihydrofolate reductase cDNA. Proc. Natl. Acad. Sci. U.S.A. 80:2495‐2499.
   Stanley, P. 1989. Chinese hamster ovary cell mutants with multiple glycosylation defects for production of glycoproteins with minimal carbohydrate heterogeneity. Mol. Cell Biol. 9:377‐383.
   Stanley, P. and Ioffe, E. 1995. Glycosyltransferase mutants: Key to new insights in glycobiology. FASEB J. 9:1436‐1444.
   Subramanian, S. and Srienc, F. 1996. Quantitative analysis of transient gene expression in mammalian cells using green fluorescent protein. J. Biotechnol. 49:137‐151.
   Tiege, M., Weidemann, R. and Kretzmer, G. 1994. Problems with serum‐free production of antithrombin III regarding proteolytic activity and product quality. J. Biotechnol. 34:101‐105.
   Wurm, F.M., Pallavicini, M.G., and Arathoon, R. 1992. Integration and stability of CHO amplicons containing plasmid sequences. Dev. Biol. Stand. 76:69‐82.
   Wurm, F.M., Johnson, A., Ryll, T., Kohne, C., Scherthan, H., Glaab, F., Lie, Y.S., Petropoulos, C.J., and Arathoon, W.R. 1996. Gene transfer and amplification in CHO cells: Efficient methods for maximizing specific productivity and assessment of genetic consequences. Ann. N.Y. Acad. Sci. 782:70‐78.
   Yenofsky, R.L., Fine, M., and Pellow, J.W. 1990. A mutant phosphotransferase II gene reduces the resistance of transformants to antibiotic selection pressure. Proc. Natl. Acad. Sci. U.S.A. 87:3435‐3439.
   Yim, K.W. 1991. Fractionation of human recombinant tissue plasminogen activator (rtPA) glycoforms by high‐performance capillary zone electrophoresis and capillary isoelectric focusing. J. Chromatogr. 559:401‐410.
   Zettlmeissel, G., Ragg, H., and Karges, H.E. 1987. Expression of biologically active Antithrombin III in Chinese hamster ovary cells. Bio/Technology 5:720‐725.
Key References
   Butler, M. (ed.). 1991. Mammalian Cell Biotechnology. IRL Press, Oxford.
  Excellent introductory background with protocol and tips.
   Freshney, R.I. (ed.). 1992. Animal Cell Culture. (2nd ed.), IRL Press, Oxford.
  Practical guide to cell culture with useful protocols and tips.
   Kriegler, 1990. See above
  Comprehensive manual for molecular biology.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library