Fluorescent labeling in semi-solid medium for selection of mammalian cells secreting high-levels of recombinant proteins
1 Institut de Recherche en Biotechnologie, Conseil National de Recherches du Canada, 6100 Royalmount Avenue, Montréal, QC H4P 2R2, Canada
2 Chemical Engineering Department, Université Laval, Québec, QC, G1K 7P4, Canada
3 Département de Microbiologie et Immunologie, Faculté de Médecine, Université de Montréal, Montreal, QC, H3C 3J7, Canada
4 INRS-IAF, Université du Québec, Laval, QC, H7N 4Z3, Canada
5 Neuromuscular research group, Montreal Neurological Institute, Montreal, QC, H3A 2B4, Canada
Citation and License
BMC Biotechnology 2009, 9:42 doi:10.1186/1472-6750-9-42Published: 11 May 2009
Despite the powerful impact in recent years of gene expression markers like the green fluorescent protein (GFP) to link the expression of recombinant protein for selection of high producers, there is a strong incentive to develop rapid and efficient methods for isolating mammalian cell clones secreting high levels of marker-free recombinant proteins. Recently, a method combining cell colony growth in methylcellulose-based medium with detection by a fluorescently labeled secondary antibody or antigen has shown promise for the selection of Chinese Hamster Ovary (CHO) cell lines secreting recombinant antibodies. Here we report an extension of this method referred to as fluorescent labeling in semi-solid medium (FLSSM) to detect recombinant proteins significantly smaller than antibodies, such as IGF-E5, a 25 kDa insulin-like growth factor derivative.
CHO cell clones, expressing 300 μg/ml IGF-E5 in batch culture, were isolated more easily and quickly compared to the classic limiting dilution method. The intensity of the detected fluorescent signal was found to be proportional to the amount of IGF-E5 secreted, thus allowing the highest producers in the population to be identified and picked. CHO clones producing up to 9.5 μg/ml of Tissue-Plasminogen Activator (tPA, 67 kDa) were also generated using FLSSM. In addition, IGF-E5 high-producers were isolated from 293SF transfectants, showing that cell selection in semi-solid medium is not limited to CHO and lymphoid cells. The best positive clones were collected with a micromanipulator as well as with an automated colony picker, thus demonstrating the method's high throughput potential.
FLSSM allows rapid visualization of the high secretors from transfected pools prior to picking, thus eliminating the tedious task of screening a high number of cell isolates. Because of its rapidity and its simplicity, FLSSM is a versatile method for the screening of high producers for research and industry.