A GFP-based screen for growth-arrested, recombinant protein-producing cells.
The growth of anchorage-dependent Chinese hamster ovary (CHO) cells is arrested upon serum deprivation; however, a portion of these cells remain viable for extended time periods in serum-free culture. This work presents a strategy to both rapidly generate a heterogeneous population of CHO cells as well as to select for subpopulations that remain robust and continue to produce recombinant protein when their growth is arrested. Stable expression of recombinant proteins in mammalian cells is often a tedious and time-consuming process because only a small percentage of transfected cells will express sufficient quantities of protein. To overcome the limitations associated with standard transformation and selection methods, bicistronic retroviral expression technology was used. First, bicistronic retroviral constructs encoding for both interferon gamma (IFN-gamma), the model therapeutic protein, and green fluorescent protein (GFP), the quantitative selectable marker, were generated. Next, recombinant retroviruses were obtained from transient transfection of a helper-cell line and were used to infect susceptible CHO cells. Cells with the bicistronic expression module stably integrated into their genome fluoresce green and could thereby be easily isolated by fluorescence-activated cell sorting. Upon subjecting successfully infected cells to serum withdrawal, significant declines in cell viability and GFP expression occurred. After imposing this selection pressure on the cells for 8 days, GFP producers were isolated from the survivors by fluorescence-activated cell sorting and expanded. To evaluate the effectiveness of the screening process, the selected cells were exposed to a second round of serum deprivation. Unlike the original cell population from which it was derived, the subpopulation remained robust and continued to stably express both GFP and IFN-gamma throughout the extended period of serum-free culture. Within 2 weeks, cells selected for recombinant protein production under serum-free conditions were successfully generated and isolated.
Yuk
Inn H Y
IH
Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
Wildt
Stefan
S
Jolicoeur
Mario
M
Wang
Daniel I C
DI
Stephanopoulos
Gregory
G
D000818:Animals; D001681:Biological Assay / Q000379:methods*; D016466:CHO Cells; D002469:Cell Separation / Q000379:methods*; D006224:Cricetinae; D003412:Cricetulus; D005434:Flow Cytometry / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000378:metabolism*
D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins