Combination of FACS and homologous recombination for the generation of stable and high-expression engineered cell lines.

ABSTRACT: Traditionally, cell line generation requires several months and involves screening of over several hundred cell clones for high productivity before dozens are selected as candidate cell lines. Here, we have designed a new strategy for the generation of stable and high-expression cell lines by combining homologous recombination (HR) and fluorescence-activated cell sorting (FACS). High expression was indicated by the expression of secreted green fluorescent protein (SEGFP). Parental cell lines with the highest expression of SEGFP were then selected by FACS and identified by stability analysis. Consequently, HR vectors were constructed using the cassette for SEGFP as the HR region. After transfecting the HR vector, the cells with negative SEGFP expression were enriched by FACS. The complete e

SUBMITTER: Shi L 

PROVIDER: S-EPMC3960159 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature