User guide

Contents
Product information ........................................................................................................ 5
Kit contents and storage ........................................................................................................................................ 5
Description of the system ..................................................................................................................................... 6
Experimental flowchart for two-subunit protein expression .......................................................................... 8
Experimental flowchart for single-subunit protein expression ....................................................................... 9
CHO DG44 cells (cGMP banked) ....................................................................................................................... 10
CD DG44 Medium ............................................................................................................................................... 11
FreeStyle
™
MAX Reagent and OptiPRO
™
SFM ................................................................................................ 12
CD OptiCHO
™
Medium...................................................................................................................................... 13
Methods ........................................................................................................................ 14
Creating expression plasmids for the Freedom
™
DG44 Kit ........................................................................... 14
Thawing and subculturing CHO DG44 Cells (cGMP banked) ...................................................................... 15
Freezing CHO DG44 Cells .................................................................................................................................. 18
Establishing sensitivity to Geneticin
®
Selective Antibiotic (G-418) ............................................................... 19
One-page flowcharts ........................................................................................................................................... 20
Transiently transfecting FreeStyle
™
CHO-S
™
cells .......................................................................................... 25
Transfecting CHO DG44 Cells with FreeStyle
™
MAX Reagent for two-subunit protein expression ....... 26
Transfecting CHO DG44 Cells with Neon
®
for two-subunit protein expression (optional) ..................... 28
Selecting stable transfectants for two-subunit protein expression ................................................................ 31
Assessing productivity ........................................................................................................................................ 33
Genomic amplification by MTX addition ......................................................................................................... 35
Clonal selection by limiting dilution ................................................................................................................ 37
Clone scale-Up ..................................................................................................................................................... 41
One-page flowcharts ........................................................................................................................................... 42
Transfecting CHO DG44 Cells with FreeStyle
™
MAX Reagent for single-subunit protein expression ... 46
Transfecting CHO DG44 Cells with Neon
®
for single-subunit protein expression (optional) .................. 49
Selecting stable transfectants for single-subunit protein expression ............................................................ 52
Assessing productivity ........................................................................................................................................ 53
Genomic amplification by MTX addition ......................................................................................................... 55
Clonal selection by limiting dilution ................................................................................................................ 57
Clone scale-up ...................................................................................................................................................... 61
Appendix A: Troubleshooting ........................................................................................ 62
Troubleshooting ................................................................................................................................................... 62
Appendix B: Vectors ...................................................................................................... 66
Map and features of pOptiVEC
™
-TOPO

vector ............................................................................................. 66
Map and features of pcDNA
™
3.3-TOPO
®
vector ............................................................................................. 68
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