title: 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 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.
authors.affiliation: Key Laboratory of Cell Proliferation and Regulation Biology, Ministry of Education, College of Life Sciences, Beijing Normal University, Beijing, China; R&D Department, AutekBio, Inc., Beijing, China.
authors.forename: Lei
authors.initials: L
authors.lastname: Shi
authors.affiliation: Key Laboratory of Cell Proliferation and Regulation Biology, Ministry of Education, College of Life Sciences, Beijing Normal University, Beijing, China.
authors.forename: Xuesi
authors.initials: X
authors.lastname: Chen
authors.affiliation: R&D Department, AutekBio, Inc., Beijing, China.
authors.forename: Wenying
authors.initials: W
authors.lastname: Tang
authors.affiliation: R&D Department, AutekBio, Inc., Beijing, China.
authors.forename: Zhenyi
authors.initials: Z
authors.lastname: Li
authors.affiliation: Key Laboratory of Cell Proliferation and Regulation Biology, Ministry of Education, College of Life Sciences, Beijing Normal University, Beijing, China.
authors.forename: Jin
authors.initials: J
authors.lastname: Liu
authors.affiliation: Key Laboratory of Cell Proliferation and Regulation Biology, Ministry of Education, College of Life Sciences, Beijing Normal University, Beijing, China; R&D Department, AutekBio, Inc., Beijing, China.
authors.forename: Feng
authors.initials: F
authors.lastname: Gao
authors.affiliation: Key Laboratory of Cell Proliferation and Regulation Biology, Ministry of Education, College of Life Sciences, Beijing Normal University, Beijing, China.
authors.forename: Jianli
authors.initials: J
authors.lastname: Sang
mesh_terms: D000818:Animals; D016466:CHO Cells; D060846:Cell Engineering / Q000379:methods*; D002999:Clone Cells; D003412:Cricetulus; D018274:Electroporation; D000068800:Etanercept; D005434:Flow Cytometry; D018703:Founder Effect; D015870:Gene Expression; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics* / Q000378:metabolism; D059765:Homologous Recombination; D007074:Immunoglobulin G / Q000096:biosynthesis / Q000235:genetics*; D018124:Receptors, Tumor Necrosis Factor / Q000096:biosynthesis / Q000235:genetics*; D012641:Selection, Genetic; D014162:Transfection; D019076:Transgenes
chemical_list: D007074:Immunoglobulin G; D018124:Receptors, Tumor Necrosis Factor; D049452:Green Fluorescent Proteins; D000068800:Etanercept