MTX NEON 10 A User's Guide

USER GUIDEFor research use only. Not for use in diagnostic procedures.Freedom™ DG44 Kit For transfection of CHO DG44 Cells (cGMP banked) and developme

CHO DG44 cells (cGMP banked) Introduction The CHO DG44 cell line is a dihydrofolate reductase (DHFR)-deficient cell line derived from suspension Chin

CD DG44 Medium Introduction CD DG44 Medium is a defined, serum-free medium containing hypoxanthine and thymidine for high-density suspension culture

FreeStyle™ MAX Reagent and OptiPRO™ SFM FreeStyle™ MAX Reagent FreeStyle™ MAX Reagent is a proprietary, animal origin-free formulation for the highly

CD OptiCHO™ Medium Introduction CD OptiCHO™ Medium is a chemically defined, serum-free medium for selection and high-density suspension culture of s

Methods Creating expression plasmids for the Freedom™ DG44 Kit Introduction The Freedom™ DG44 Kit contains two vectors, pOptiVEC™-TOPO® and pcDNA™3.3

Thawing and subculturing CHO DG44 Cells (cGMP banked) Introduction Follow the protocol below to thaw CHO DG44 Cells (cGMP banked). The cells are supp

Thawing and subculturing CHO DG44 Cells (cGMP banked), continued Thawing procedure 1. Remove the cryovial of cells from the liquid nitrogen and thaw

Thawing and subculturing CHO DG44 Cells (cGMP banked), continued Subculturing cells Passage the cells every 2–3 days into fresh medium. When passagin

Freezing CHO DG44 Cells Introduction You may freeze CHO DG44 Cells (cGMP banked) directly in CD DG44 Medium with 10% DMSO. We recommend that you free

Establishing sensitivity to Geneticin® Selective Antibiotic (G-418) Geneticin® Selective Antibiotic (G-418) The pcDNA™3.3-TOPO TA vector contains th

For research use only. Not for use in diagnostic procedures. The information in this guide is subject to change without notice. DISCLAIMER LIFE TECH

Methods for two-subunit protein expression One-page flowcharts Introduction The following pages contain flowcharts to aid you in your expression expe

Optimizing vectors for two-subunit protein expression Introduction Prior to making stable transfectants in CHO DG44 Cells (cGMP banked), you may perf

Expressing two-subunit proteins without gene amplification Introduction The flowchart below depicts the major steps to express your two-subunit prote

Genomic amplification by MTX addition Introduction The flowchart below depicts the major steps to amplify the copy number of your gene of interest (G

Performing clonal selection by limiting dilution (two-subunit protein) Introduction The flowchart below depicts the major steps to obtain a clonal c

Transiently transfecting FreeStyle™ CHO-S™ cells Recommendation We recommend FreeStyle™ CHO-S™ cells and transfection with FreeStyle™ MAX Reagent for

Transfecting CHO DG44 Cells with FreeStyle™ MAX Reagent for two-subunit protein expression Introduction After determining which combination of pOpti

Transfecting CHO DG44 Cells with FreeStyle™ MAX Reagent for two-subunit protein expression, continued Optimal transfection conditions To transfect su

Transfecting CHO DG44 Cells with Neon® for two-subunit protein expression (optional) Recommendation Calculate the number of CHO DG44 Cells (cGMP ban

Transfecting CHO DG44 Cells with Neon® for two-subunit protein expression, continued Preparing cells 1. 24 hours before transfection, passage CHO DG

Contents Product information ... 5 Kit contents a

Transfecting CHO DG44 Cells with Neon® for two-subunit protein expression, continued Transfection procedure for expression of two-subunit protein, c

Selecting stable transfectants for two-subunit protein expression Introduction To obtain cell lines that produce high levels of your protein, first s

Selecting stable transfectants for two-subunit protein expression, continued Selecting stable transfectants for two-subunit protein expression 48 hou

Assessing productivity Protein production To check for production of your protein during stable cell establishment, you may take an aliquot of growth

Assessing productivity, continued Points to consider When choosing a workflow, consider the amount of protein you wish to produce, your available res

Genomic amplification by MTX addition Introduction Methotrexate (MTX) is a folic acid antagonist that is actively transported into cells by the folat

Genomic amplification by MTX addition, continued One round of MTX amplification The productivity of each clone depends upon the integration locus of

Clonal selection by limiting dilution Introduction Development of a CHO cell line for commercial production of a recombinant protein requires clonal

Clonal selection by limiting dilution, continued Preparing cloning medium The procedure described below uses CD FortiCHO™ Medium as the cloning medi

Clonal selection by limiting dilution, continued Cell counting and dilution 1. Label five 50-mL conical tubes “1” through “5”. 2. Pipette 5 to 10 mL

Appendix C: Ordering information ... 70 Accessory products ...

Clonal selection by limiting dilution, continued Plating cells 1. After warming the cloning medium (step 4, page 38), remove it from the incubator

Clone scale-Up Introduction After isolating your clones of interest (previous section), transfer single-cell colonies from 96-well plates to 24-well

Methods for single-subunit protein expression One-page flowcharts Introduction The following pages contain flowcharts to aid you in your expression e

Expressing single-subunit protein using pOptiVEC™ expression construct Introduction The flowchart below depicts the major steps to transfect suspensi

Genomic amplification by MTX addition Introduction The flowchart below depicts the major steps to amplify the copy number of your gene of interest (G

Clonal selection by limiting dilution (single-subunit protein) Introduction The flowchart below depicts the major steps to obtain a clonal cell line

Transfecting CHO DG44 Cells with FreeStyle™ MAX Reagent for single-subunit protein expression Introduction You will use FreeStyle™ MAX Reagent to tr

Transfecting CHO DG44 Cells with FreeStyle™ MAX Reagent for single-subunit protein expression, continued Recommendation Calculate the number of CHO

Transfecting CHO DG44 Cells with FreeStyle™ MAX Reagent for single-subunit protein expression, continued Transfection procedure using pOptiVEC™ expre

Transfecting CHO DG44 Cells with Neon® for single-subunit protein expression (optional) Recommendation Calculate the number of CHO DG44 Cells (cGMP

Product information Kit contents and storage Introduction The components of the Freedom™ DG44 Kit and their shipping and storage conditions are liste

Transfecting CHO DG44 Cells with Neon® for single-subunit protein expression, continued Preparing cells 1. 24 hours before transfection, passage CHO

Transfecting CHO DG44 Cells with Neon® for single-subunit protein expression, continued Transfection procedure for expression of single-subunit prote

Selecting stable transfectants for single-subunit protein expression Introduction To obtain cell lines that produce high levels of your protein, firs

Assessing productivity Protein production To check for production of your protein during stable cell establishment, you may take an aliquot of growth

Assessing productivity, continued Points to consider When choosing a workflow, consider the amount of protein you wish to produce, your available res

Genomic amplification by MTX addition Introduction Methotrexate (MTX) is a folic acid antagonist that is actively transported into cells by the folat

Genomic amplification by MTX addition, continued One round of MTX amplification The productivity of each clone depends upon the integration locus of

Clonal selection by limiting dilution Introduction Development of a CHO cell line for commercial production of a recombinant protein requires clonal

Clonal selection by limiting dilution, continued Preparing cloning medium The procedure described below uses CD FortiCHO™ Medium as the cloning medi

Clonal selection by limiting dilution, continued Cell counting and dilution 1. Label five 50-mL conical tubes “1” through “5”. 2. Pipette 5 to 10 mL

Description of the system Freedom™ DG44 Kit The Freedom™ DG44 Kit is designed for easy cloning and expression of recombinant proteins in dihydrofolat

Clonal selection by limiting dilution, continued Plating cells 1. After warming the cloning medium (step 4, page 58), remove it from the incubator

Clone scale-up Introduction After isolating your clones of interest (previous section), transfer single-cell colonies from 96-well plates to 24-well

Appendix A: Troubleshooting Troubleshooting Culturing CHO DG44 Cells (cGMP banked) The table below lists some potential problems and possible soluti

Troubleshooting, continued Culturing CHO DG44 Cells (cGMP banked) The table below lists some potential problems and possible solutions that may help

Troubleshooting, continued Transfection The table below lists some potential problems and possible solutions that may help you troubleshoot your tra

Troubleshooting, continued Protein Expression The table below lists some potential problems and possible solutions that may help you troubleshoot you

Appendix B: Vectors Map and features of pOptiVEC™-TOPO vector Map The map below shows the elements of the pOptiVEC™-TOPO vector. The vector sequenc

Map and features of pOptiVEC™-TOPO® vector, continued Features The pOptiVEC™-TOPO® vector contains the following elements. Features have been functi

Map and features of pcDNA™3.3-TOPO® vector Map The map below shows the elements of the pcDNA™3.3-TOPO®vector. The vector sequence is available from w

Map and features of pcDNA™3.3-TOPO® vector, continued Features The pcDNA™3.3-TOPO® vector contains the following elements. Features have been functi

Description of the system, continued Advantages of the Freedom™ DG44 Kit The Freedom™ DG44 Kit provides the following advantages for protein producti

Appendix C: Ordering information Accessory products Freedom™ DG44 Kit Products Many of the components supplied with the Freedom™ DG44 Kit are also av

Accessory products, continued Additional products The products listed below may be used with the Freedom™ DG44 Kit. For more information, go to www.l

Appendix D: Safety Chemical safety WARNING! GENERAL CHEMICAL HANDLING. To minimize hazards, ensure laboratory personnel read and practice the gener

Biological hazard safety WARNING! BIOHAZARD. Biological samples such as tissues, body fluids, infectious agents, and blood of humans and other anim

Appendix E: Purchaser notification Limited Label License Information Limited Use Label License No. 335: Technology with CRO Rights This product and i

Limited Label License Information, continued Limited Use Label License No. 296: DG44 Cells Notice to Purchaser: The cells are sold under license from

Documentation and support Obtaining support Obtaining Certificates of Analysis The Certificate of Analysis provides detailed quality control and prod

References Andersson, S., Davis, D. L., Dahlbäck, H., Jörnvall, H., and Russell, D. W. (1989) Cloning, Structure, and Expression of the Mitochondria

Notes 78

Experimental flowchart for two-subunit protein expression Introduction The diagram below schematically depicts the steps necessary to express your tw

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Experimental flowchart for single-subunit protein expression Experimental flowchart for single-subunit protein expression The diagram below schemati