Combination of FACS and Homologous Recombination for the Generation of Stable and High-Expression Engineered Cell Lines

Lei Shi,Xuesi Chen,Jin Liu,Feng Gao,Wenying Tang,Zhenyi Li,Jianli Sang,Sebastian D Fugmann

doi:10.1371/journal.pone.0091712

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 exchange between SEGFP and target gene (TNFR-Fc) cassettes was demonstrated by DNA analysis. Compared with the traditional method, by integrating the cassette containing the gene of interest into the pre-selected site, the highest producing cells secreted a more than 8-fold higher titer of target protein. Hence, this new strategy can be applied to isolated stable cell lines with desirable expression of any gene of interest. The stable cell lines can rapidly produce proteins for researching protein structure and function and are even applicable in drug discovery.

Highlights

In recent years, the market for global biopharmaceuticals has widely expanded, and it is expected to exceed sales of US $166 billion by 2017 [1]
The secreted green fluorescent protein (SEGFP) expression cassette was used as the homologous sequence to construct the homologous recombination (HR) vector
The gene of interest (GOI) would be inserted in the preselected site by replacing the SEGFP expression cassette with the HR region to establish the GOI high-expression cell line

Summary

Introduction

The market for global biopharmaceuticals has widely expanded, and it is expected to exceed sales of US $166 billion by 2017 [1]. In the process of recombinant protein production, one of the critical steps is rapid selection of stable and high-expression cell lines for the gene of interest (GOI), which is a time-consuming and labor-intensive step [4]. To generate cell lines for the production of target proteins, the traditional strategy involves transfection of the target gene for random integration into genomic DNA by homologous recombination (HR). The titer of the target protein is analyzed among a large number of cell clones to select high-expression cell clones. Using this method, more than 80% of cell clones express the GOI at a very low level. In modern biopharmaceutical technology, different strategies have been developed to increase the screening throughput of cell clones and/or raise GOI expression directly

Methods

Results

Conclusion