Fluorescent labeling in semi-solid medium for selection of mammalian cells secreting high-levels of recombinant proteins.
BACKGROUND
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.


RESULTS
CHO cell clones, expressing 300 microg/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 microg/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.


CONCLUSIONS
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.
Caron
Antoine W
AW
Institut de Recherche en Biotechnologie, Conseil National de Recherches du Canada, Montréal, QC, Canada. antoine.caron@cnrc-nrc.gc.ca
Nicolas
Claire
C
Gaillet
Bruno
B
Ba
Ismaïla
I
Pinard
Maxime
M
Garnier
Alain
A
Massie
Bernard
B
Gilbert
Rénald
R
D000818:Animals; D016466:CHO Cells; D018929:Cell Culture Techniques / Q000379:methods*; D002469:Cell Separation; D006224:Cricetinae; D003412:Cricetulus; D003470:Culture Media; D005456:Fluorescent Dyes; D007334:Insulin-Like Growth Factor I / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis*; D010959:Tissue Plasminogen Activator / Q000096:biosynthesis
D003470:Culture Media; D005456:Fluorescent Dyes; D011994:Recombinant Proteins; D007334:Insulin-Like Growth Factor I; D010959:Tissue Plasminogen Activator