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BMC Biotechnology

, 9:42

First Online: 11 May 2009Received: 09 September 2008Accepted: 11 May 2009

Abstract

BackgroundDespite 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.

ResultsCHO 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.

ConclusionFLSSM 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.

Electronic supplementary materialThe online version of this article doi:10.1186-1472-6750-9-42 contains supplementary material, which is available to authorized users.

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Fuente: https://link.springer.com/article/10.1186/1472-6750-9-42



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