A platform for Chinese hamster ovary ( CHO ) cell genome engineering
نویسنده
چکیده
.....................................................................................................................................4 1.0 Introduction..........................................................................................................................5 1.1 Expression systems for bio-manufacturing......................................................................5 1.2 CHO cell line development for recombinant protein production.....................................7 1.3 New approach towards bio-manufacturing......................................................................9 1.3.1 First approach............................................................................................................9 1.3.2 Second approach......................................................................................................10 1.4 Lentivirus – a gene delivery tool....................................................................................11 1.4.1 Engineered lentiviral vectors...................................................................................12 1.4.2 Multiplicity of Infection (MOI)...............................................................................13 1.5 Landing pad design using site-specific recombination (SSR)........................................14 1.6 Genome walking – integration site analysis...................................................................16 1.7 Promoter engineering.....................................................................................................17 2.0 Materials and Methods.......................................................................................................19 2.1 Plasmid construction......................................................................................................19 2.2 Cell culture and transfection...........................................................................................19 2.3 Fluorescence assisted cell sorting (FACS).....................................................................19 2.4 Data analysis...................................................................................................................20 2.5 Glycerol stock.................................................................................................................20 2.6 Cryopreservation of mammalian cells............................................................................20 2.7 Suspension adaptation of CHO-K1 adherent cells.........................................................20 2.8 Landing pad design.........................................................................................................21 2.9 Lentivirus production and titration.................................................................................22 2.10 Lentiviral transduction of CHO-K1 S cells..................................................................23 2.11 Bxb1 mediated cassette exchange................................................................................24 2.12 Lentiviral transduction of CHO-K1 cells.....................................................................24 2.13 Integration site analysis................................................................................................24 2.14 Synthetic promoter design............................................................................................25 3.0 Results................................................................................................................................26 3.1 Landing pad integration using lentivirus in CHO-K1 S cells.........................................26
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