title: Fluorescent labeling for clonal selection of Marc 145 cells secreting high levels of recombinant protein PBD-1.
abstract: Despite the powerful impact gene expression markers like the green fluorescent protein (GFP) or enhanced GFP (EGFP) exert on linking the expression of recombinant protein for selection of high producers in recent years, there is still a strong incentive to develop more economical and efficient methods for isolating mammalian cell clones secreting high levels of recombinant proteins. Here we present a new method based on the co-expression of EGFP that allows clonal selection in standard 96-well cell culture plates. The genes encoding the EGFP protein and the related protein are linked by an internal ribosome entry site and thus are transcribed into the same mRNA in an independent translation process. Since both proteins arise from a common mRNA, the EGFP expression level correlates with the expression level of the therapeutic protein in each clone. By expressing recombinant porcine β-defensin 1 in Marc 145 cells, we demonstrate the robustness and performance of this technique. The method can be served as an alternative to identify high-producer clones with various cell sorting methods.
authors.affiliation: Department of Animal Biotechnology and Cell Engineering, Wuhan Institute of Animal Husbandry and Veterinary Science, Wuhan, 430208, People's Republic of China. hygene_005@yahoo.com.cn.|Wuhan Municipal Bureau of Agriculture, Wuhan, 430023, People's Republic of China. hygene_005@yahoo.com.cn.
authors.forename: Hai-Jun
authors.initials: HJ
authors.lastname: Huang
authors.affiliation: Department of Animal Biotechnology and Cell Engineering, Wuhan Institute of Animal Husbandry and Veterinary Science, Wuhan, 430208, People's Republic of China.
authors.forename: Xia
authors.initials: X
authors.lastname: Peng
authors.affiliation: Department of Animal Biotechnology and Cell Engineering, Wuhan Institute of Animal Husbandry and Veterinary Science, Wuhan, 430208, People's Republic of China.
authors.forename: Bing
authors.initials: B
authors.lastname: Deng
authors.affiliation: Key Laboratory of Swine Breeding and Genetics, Ministry of Agriculture and Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction (Huazhong Agricultural University), Ministry of Education, Wuhan, 430070, People's Republic of China.
authors.forename: Cong
authors.initials: C
authors.lastname: Huang
authors.affiliation: Department of Animal Biotechnology and Cell Engineering, Wuhan Institute of Animal Husbandry and Veterinary Science, Wuhan, 430208, People's Republic of China.
authors.forename: Jie
authors.initials: J
authors.lastname: Li
authors.affiliation: Department of Animal Biotechnology and Cell Engineering, Wuhan Institute of Animal Husbandry and Veterinary Science, Wuhan, 430208, People's Republic of China.
authors.forename: Yun-Guo
authors.initials: YG
authors.lastname: Qian
authors.affiliation: Department of Animal Biotechnology and Cell Engineering, Wuhan Institute of Animal Husbandry and Veterinary Science, Wuhan, 430208, People's Republic of China.
authors.forename: Qi-Shuang
authors.initials: QS
authors.lastname: Gao
authors.affiliation: Department of Animal Biotechnology and Cell Engineering, Wuhan Institute of Animal Husbandry and Veterinary Science, Wuhan, 430208, People's Republic of China.
authors.forename: Min
authors.initials: M
authors.lastname: Xiang
authors.affiliation: Department of Animal Biotechnology and Cell Engineering, Wuhan Institute of Animal Husbandry and Veterinary Science, Wuhan, 430208, People's Republic of China.
authors.forename: Shun
authors.initials: S
authors.lastname: Lu
authors.affiliation: Department of Animal Biotechnology and Cell Engineering, Wuhan Institute of Animal Husbandry and Veterinary Science, Wuhan, 430208, People's Republic of China.
authors.forename: Zhi-Hua
authors.initials: ZH
authors.lastname: Chen
authors.affiliation: Department of Animal Biotechnology and Cell Engineering, Wuhan Institute of Animal Husbandry and Veterinary Science, Wuhan, 430208, People's Republic of China.
authors.forename: Cai-Yao
authors.initials: CY
authors.lastname: Zhan
authors.affiliation: Department of Animal Biotechnology and Cell Engineering, Wuhan Institute of Animal Husbandry and Veterinary Science, Wuhan, 430208, People's Republic of China.
authors.forename: Li
authors.initials: L
authors.lastname: Zhou
authors.affiliation: Department of Animal Biotechnology and Cell Engineering, Wuhan Institute of Animal Husbandry and Veterinary Science, Wuhan, 430208, People's Republic of China.
authors.forename: Bi-Fei
authors.initials: BF
authors.lastname: Tao
authors.affiliation: Department of Animal Biotechnology and Cell Engineering, Wuhan Institute of Animal Husbandry and Veterinary Science, Wuhan, 430208, People's Republic of China.|Key Laboratory of Swine Breeding and Genetics, Ministry of Agriculture and Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction (Huazhong Agricultural University), Ministry of Education, Wuhan, 430070, People's Republic of China.
authors.forename: Jie
authors.initials: J
authors.lastname: Liu
authors.affiliation: Wuhan Municipal Bureau of Agriculture, Wuhan, 430023, People's Republic of China.
authors.forename: Ben-Zhong
authors.initials: BZ
authors.lastname: Tan
keywords: High producer; Mammalian cell culture; Porcine β-defensin l; Recombinant protein