Efficient selection of high-producing subclones during gene amplification of recombinant Chinese hamster ovary cells by flow cytometry and cell sorting.
The screening procedure for high-producing cell lines is extremely time- and labor-intensive and costly, and is at present guided by an empirical approach based on individual experience. Flow cytometry and cell sorting, with its ability to analyze and separate single cells, an ideal method in the selection of such rare cells. The isolation of recombinant cell lines is especially difficult due to repeated gene amplification, which introduces high mutational variation into the population. We have established and evaluated a modification of a previous method that traps secreted product on the surface of the secreting cell, thus allowing direct analysis of single cell specific production rates. This method was used to select for high-producing subclones of a recombinant Chinese hamster ovary (CHO) cell line producing a human antibody against HIV-1 by repeated rounds of gene amplification and cell sorting. This cell line has been amplified in previous investigations, so that the amount of work and testing required by traditional methods can be compared with the protocol described herein. Forty-five 96-well plates were necessary to obtain a high-producing subclone by limited dilution methods, whereas only five plates were required when cell sorting was used. The specific production rate of the best clone obtained by sorting, however, was five times that of the clone obtained by traditional methods. In contrast to the clones obtained by limited dilution, which consisted of several populations of low- and high-producing cells even at high methotrexate concentrations (6.4 microM), the clones isolated by sorting were already homogeneous at 0.8 microM methotrexate.
Borth
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N
Institute for Applied Microbiology, University of Agriculture and Forestry, Muthgasse 18, 1190 Vienna, Austria. borth@mail.boku.ac.at
Zeyda
M
M
Kunert
R
R
Katinger
H
H
D000818:Animals; D000914:Antibodies, Viral / Q000096:biosynthesis; D016466:CHO Cells; D002469:Cell Separation; D006224:Cricetinae; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D005784:Gene Amplification; D015497:HIV-1 / Q000276:immunology; D011995:Recombination, Genetic
D000914:Antibodies, Viral