Transfection of DG44 Cells

Introduction

The OptiCHO™ Express Kit is designed for easy cloning and expression of recombinant proteins in dihydrofolate reductase (DHFR)-deficient, Chinese hamster ovary (CHO)-derived DG44 cells in suspension culture. The OptiCHO™ Express Kit provides reagents for cloning the gene encoding your secreted protein of interest, high efficiency transfection of the DNA construct into DG44 cells, and generation of stable cell lines producing your protein of interest in a chemically defined medium.
 
The OptiCHO™ Express Kit contains the pOptiVEC™-TOPO® TA Cloning Kit, FreeStyle™ MAX transfection reagent, DG44 cells, growth and selection media, and additional reagents for complete transfection of DG44 cells in a serum-free environment and subsequent stable cell line development.
 
Components of the OptiCHO™ Express Kit

The OptiCHO™ Express Kit includes the following major components:

  • pOptiVEC™-TOPO® TA Cloning Kit: A TOPO®-adapted bicistronic plasmid and reagents for cloning of a PCR product containing a mammalian secretion signal and the gene of interest.
  • DG44 cells: DHFR-negative, CHO-derived cells adapted to high density, serum-free suspension culture in CD DG44 Medium that are capable of producing high levels of secreted, recombinant protein.
  • CD DG44 Medium: Defined, serum-free medium supplemented with hypoxanthine and thymidine to allow growth of DHFR-negative DG44 cells.
  • FreeStyle™ MAX Reagent: A proprietary, animal origin-free formulation for high transfection efficiency of plasmid DNA into DG44 cells.
  • CD OptiCHO™ Medium: Defined, serum-free medium formulated for selection and growth of DG44 cells expressing DHFR and the recombinant protein of interest.
  • The kit also contains OptiPro™ serum free medium (SFM) for optimal DNA:lipid complex formulation, L-glutamine provided separately for increased media stability, and the surfactant Pluronic® F-68 to control shear forces in suspension culture.

Advantages of the OptiCHO™ Express Kit

Using the OptiCHO™ Express Kit for protein production in mammalian cells provides the following advantages:

  • DHFR-deficient DG44 cells derived from CHO cells (Urlaub et al., 1983), which provide stable and accurate glycosylation (Sheeley et al., 1997; Werner et al., 1998) and are more likely to yield accurate glycoproteins.
  • FreeStyle™ MAX Reagent offers high transfection efficiency of suspension CHO cells with low cytotoxicity.
  • FreeStyle™ MAX Reagent, CD DG44 Medium and CD Opti CHO™ Medium are animal-origin free.
 
pOptiVEC™-TOPO® TA Cloning Kit

The OptiCHO™ Express Kit contains the pOptiVEC™-TOPO® TA Cloning Kit (also available separately). The pOptiVEC™-TOPO® TA Cloning Kit contains the pOptiVEC™-TOPO® vector, a TOPO®-adapted bicistronic plasmid that allows rapid cloning of a PCR product containing a mammalian secretion signal and the gene of interest downstream of the CMV promoter. In the pOptiVEC™-TOPO® vector, the transcription of the gene of interest is separated from the dihydrofolate reductase (DHFR) auxotrophic selection marker by an internal ribosome entry site (IRES), allowing transcription of the gene of interest and the selection marker on the same mRNA.
 
Using the reagents provided in the pOptiVEC™-TOPO® TA Cloning Kit, you will TOPO® clone your gene of interest into the pOptiVEC™-TOPO® vector, transform the plasmid into One Shot® TOP10 E. coli, and select the appropriate clone prior to transfection.
 
Experimental Outline

This manual provides instructions and guidelines to perform the following steps:

1
Thaw and propagate DG44 cells in CD DG44 Medium
2
Transfect your pOptiVEC® -TOPO® plasmid construct containing your gene of interest into DG44 cells using FreeStyle MAX Reagent
3
Select for a pool of stably transfected cells using CD OptiCHO Medium
4
Perform genomic amplification by selection with MTX
5
Using limiting dilution, select a clonal cell line that expresses high amounts of your protein of interest
6
Scale up your high-producing clonal cell line to suit your bioproduction needs

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Materials

Shipping/Storage

The components of the OptiCHO™ Express Kit are shipped and should be stored as listed below.
 
TOPO® TA  Cloning Reagents

The pOptiVEC™-TOPO®  TA cloning reagents are shipped on dry ice in two boxes, containing the cloning reagents and the One Shot®  TOP10 Chemically Competent E. coli kit. Refer to the pOptiVEC™-TOPO® TA Cloning Kit manual for detailed description of the kit contents.
 
Store the TOPO® reagents at -20ºC and the One Shot®  TOP10 Chemically Competent E. coli kit at -80°C.
 
DG44 cells
 
DG44 cells are provided as one vial shipped on dry ice containing 1 x 107 cells in 1 ml freezing medium containing 10% DMSO.
 
Important:   Upon receipt, immediately store in liquid nitrogen.
 
OptiCHO™ Express Kit Components

The OptiCHO™ Express Kit components are shipped under various conditions as listed below.
 
Store components as indicated.

Item
Amount
Shipping
Storage
FreeStyle MAX Reagent
1 ml
Blue ice
+4ºC, Do not Freeze
OptiPro SFM
100 ml
Room temperature
+4ºC
CD DG44 Medium
1000 ml
Room temperature
+4ºC, in the dark
CD OptiCHO Medium
1000 ml
Room temperature
+4ºC, in the dark
L-glutamine, 200 mM
100 ml
Dry ice
-20ºC
Pluronic® F-68, 10%
100 ml
Room temperature
Room temperature
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Ordering Information

Sku Name Size Price Qty
12744017 pOptiVEC™-TOPO® TA Cloning® Kit 1 kit USD 610.00
16447100 FreeStyle™ MAX Reagent 1 mL USD 476.00
12309050 1X OptiPRO™ SFM 100 mL USD 20.50
12610010 CD DG44 Medium 1,000 mL USD 87.00
12681011 1X CD OptiCHO™ Medium, liquid 1,000 mL USD 76.00
12604013 TrypLE™ Express Enzyme (1X), no phenol red 100 mL USD 21.90
25030081 200 mM L-Glutamine 100 mL USD 23.40

Charateristics of DG44 Cells

The DG44 cell line is a dihydrofolate reductase (DHFR)-deficient cell line derived from suspension Chinese hamster ovary (CHO-S) cells (Urlaub et al., 1983). DG44 cells are adapted to suspension culture in CD DG44 Medium. Frozen cells are supplied at 1 x 107 cells/ml and may be thawed directly into CD DG44 Medium (see Thawing and Subculturing Cells).
 
Parental Cell Line

The CHO-S cell line is a stable aneuploid cell line established from the ovary of an adult Chinese hamster (Puck, 1958). CHO cells are commonly used cell lines for transfection, expression, and large-scale production of recombinant proteins.
 
DHFR

DHFR catalyzes the reduction of 5, 6-dihydrofolate to 5, 6, 7, 8-tetrahydrofolate, which is essential for DNA synthesis. CHO-derived DG44 cells lack DHFR activity and must be propagated in medium containing the purine precursors hypoxanthine and thymidine (HT) unless the cells are stably transfected with a vector that expresses DHFR. Auxotrophic selection eliminates the need to maintain selection pressure using antibiotics, which could potentially contaminate your protein of interest.
 
DHFR also functions as a genomic amplification marker for your gene of interest using methotrexate (MTX) selection (Kaufman et al., 1985; Tanaka et al., 2002). See genomic amplification using MTX for more details.
 
Characteristics of DG44 Cells

The DG44 cell line exhibits the following characteristics:

  • Adapted to serum-free suspension growth in CD DG44 Medium containing hypoxanthine and thymidine.
  • Demonstrates high transfection efficiencies with FreeStyle™ MAX Reagent.
  • Suspension cultures may be transfected in CD DG44 Medium.

As with other human cell lines, when working with DG44 cells, handle as potentially biohazardous material under at least Biosafety Level 2 containment.
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Thawing and Subculturing DG44 Cells

Follow the protocol below to thaw DG44 cells. Cells are supplied in a vial containing 1 ml of cells at 1 x 107 viable cells/ml in 90% freezing medium and 10% DMSO. Thaw DG44 cells directly into CD DG44 Medium supplemented with 8 mM L-glutamine and 18 ml/L Pluronic® F-68.
 
Do not thaw and grow DG44 cells in CD OptiCHO™ Medium. Parental or untransfected DG44 cells are DHFR-negative and require supplementary hypoxanthine and thymidine, present in CD DG44 Medium.
 
Preparing Complete CD DG44 Medium

  • All solutions and equipment that come in contact with the cells must be sterile. Always use proper sterile technique and work in a laminar flow hood.
  • Supplement CD DG44 Medium to a final concentration of 8 mM L-glutamine and 18 ml of 10% Pluronic® F-68 per liter before use.
  • Addition of antibiotics is not recommended.
  • CD DG44 Medium is light sensitive. For optimal results, store medium at 4ºC, protected from light.
 
Materials Needed

You should have the following reagents and materials before beginning:

  • Frozen DG44 cells (supplied with the kit; store frozen cells in liquid nitrogen until ready to use)
  • Complete CD DG44 Medium (prepared as above; pre-warm at 37°C before use).
  • 125 ml polycarbonate, disposable, sterile Erlenmeyer flasks with vented caps (available from VWR, West Chester PA, cat. no. 30180-036)
  • Orbital shaker set at 130-135 rpm in 37°C incubator with a humidified atmosphere of 8% CO2

Thawing Procedure

To thaw and establish cells:

  1. Remove the cryovial of cells from the liquid nitrogen and thaw quickly (<1 minute) in a 37°C water bath.
  2. Decontaminate the outside of the vial with 70% ethanol. Gently break up any clumps with a sterile pipette tip and aseptically transfer the entire contents of the cryovial into a disposable, sterile polycarbonate 125-ml Erlenmeyer shaker flask containing 30 ml of pre-warmed complete CD DG44 Medium.
  3. Incubate cells in a 37°C incubator containing a humidified atmosphere of 8% CO2 in air on an orbital shaker platform rotating at 130-135 rpm.
  4. After 24-48 hours in culture, determine the cell density and viability using the protocol described below.
  5. Once the culture has reached >1.2 x 106 viable cells/ml, expand cultures using the subculturing protocol below.
 
Determining Cell Density and Viability

Follow the procedure below to determine viable and total cell counts.

  1. Transfer a small aliquot of the cell suspension to a microcentrifuge tube.
  2. Determine viability using trypan blue dye exclusion.
  3. Determine cell density electronically using a Coulter Counter or manually using a hemacytometer and an inverted microscope.
 
Subculturing Cells

Passage the cells once the culture has reached >1.2 x 106 viable cells/ml. When passaging DG44 cells, use disposable, sterile polycarbonate 125-ml Erlenmeyer shaker flasks with vented caps containing 30 ml of pre-warmed complete CD DG44 Medium.

  1. Determine viable and total cell counts.
  2. Using the cell density determined in Step 1, calculate the split ratio needed to seed each new shaker flask at 2 x 105 viable cells/ml by dilution.
  3. Dilute the cells in 30 ml of pre-warmed complete CD DG44 Medium to give a final cell density of 2 x 105 viable cells/ml.
  4. Incubate flasks in a 37°C incubator containing a humidified atmosphere of 8% CO2 in air on an orbital shaker platform rotating at 130-135 rpm.
  5. Repeat Steps 1-5 as necessary to maintain or expand cells.
 
After 25 passages, you should thaw a new vial of cells. To maintain sufficient stocks of low-passage cells (i.e., under 25 passages), be sure to freeze aliquots of DG44 cells in liquid nitrogen. See the next section for instructions on cryopreserving cells.
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Freezing DG44 Cells

Introduction

You may freeze DG44 cells directly in CD DG44 Medium with 10% DMSO. We recommend that you freeze cells at a density of ≥1 x 107 viable cells/ml. Guidelines to prepare freezing medium and to freeze cells are provided in this section.
 
Materials Needed

You will need the following reagents and equipment before starting:

  • Complete CD DG44 Medium
  • Tissue culture grade DMSO
  • Reagents and equipment to determine viable and total cell counts
  • Sterile, labeled cryovials
  • Sterile, 15 ml-conical tubes
  • Automated or manual controlled-rate freezing apparatus
 
Preparing Freezing Medium

Prepare freezing medium immediately before use.

  1.   In a sterile, conical centrifuge tube, mix together the following reagents for every 1 ml of freezing medium needed:
 
Complete CD DG44 Medium
0.9 ml
DMSO
0.1 ml
                         
  1. Filter-sterilize the freezing medium through a 0.22 µm filter and place the tube on ice until use. Discard any remaining freezing medium after use.
 
Freezing Cells

  1. Grow the desired quantity of DG44 cells in shaker flasks, harvesting when the cell density reaches 1 x 106 viable cells/ml. Transfer cells to a sterile, conical centrifuge tube.
  2. Determine the viable and total cell counts and calculate the volume of freezing medium required to yield a final cell density of 1 x 107 viable cells/ml.
  3. Centrifuge cells at 1,200 rpm for 5 minutes at room temperature and carefully aspirate the medium.
  4. Resuspend the cells in the pre-determined volume of chilled freezing medium.
  5. Place cryovials in a microcentrifuge rack and aliquot 1 ml of the cell suspension into each cryovial. 
  6. Freeze cells in an automated or manual controlled-rate freezing apparatus following standard procedures. For ideal cryopreservation, the freezing rate should be a decrease of 1°C per minute.
  7. Transfer frozen vials to liquid nitrogen for long-term storage.
 
Note:   You may check the viability and recovery of frozen cells 24 hours after storing cryovials in liquid nitrogen by following the thawing procedure.

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Transfecting DG44 Cells with FreeStyle™ MAX Reagent

You will use FreeStyle™ MAX Reagent to transfect suspension DG44 cells with your pOptiVEC™-TOPO® construct. Refer to the pOptiVEC™-TOPO® TA Cloning Kit manual supplied wit the kit for instructions to clone your gene of interest into the pOptiVEC™-TOPO® vector.
 
Plasmid Preparation

The pOptiVEC™-TOPO® plasmid must be clean, sterile, and free from contamination with phenol and sodium chloride for transfection into DG44 cells. Contaminants may kill the cells, and salt will interfere with lipid complexing, decreasing transfection efficiency. We recommend isolating plasmid DNA using the S.N.A.P. Midiprep DNA Isolation Kit.
 
Linearizing the Plasmid

Prior to using the OptiCHO™ Express Kit to transfect DG44 cells with your pOptiVEC™ construct, you may linearize the plasmid. While linearizing your vector may not improve transfection efficiency, it increases the chance that the vector will integrate into the host cell genome without disrupting the gene of interest or other elements required for expression in mammalian cells.
 
We suggest using Pvu I, which cuts once in the ampicillin resistance gene. Other unique restriction sites are possible. A complete restriction map of pOptiVEC™-TOPO® is available at www.invitrogen.com. Be sure that your insert does not contain the restriction enzyme site you use to linearize your vector.
 
After digestion, precipitate the DNA, resuspend pellet in sterile water, and re-quantify using your method of choice.
 
Note:    If an appropriate linearization site is not present, you may transfect the circular plasmid. Transfection efficiency will not be affected.
 
Positive Control

If you performed the control TOPO® cloning reaction to clone the LacZα fragment into the pOptiVEC™-TOPO® plasmid, you may use this plasmid construct as a positive control for transfection. The DHFR gene in the plasmid will allow generation of stably-transfected DG44 cell lines in HT-deficient CD OptiCHO™ medium.
 
Calculate the number of DG44 cells that you will need for your transfection experiment and expand cells accordingly. Make sure that the cells are healthy and greater than 95% viable before proceeding to transfection.
 
Materials Needed

You should have the following reagents and equipment before beginning:

  • Suspension DG44 cells cultured in complete CD DG44 Medium at 5 x 105 cells/ml
  • Purified, linearized pOptiVEC™-TOPO® plasmid DNA containing your gene of interest
  • Optional: purified, linearized pOptiVEC™-TOPO® plasmid DNA containing the LacZα fragment, as a positive control
  • FreeStyle™ MAX Reagent (supplied with the kit; store at +4°C until use)
  • OptiPro™ SFM (supplied with the kit; pre-warmed to room temperature)
  • Disposable, sterile, 125-ml polycarbonate Erlenmeyer flasks
  • Orbital shaker in 37°C incubator with a humidified atmosphere of 8% CO2
  • Reagents and equipment to determine viable and total cell counts (e.g., Trypan Blue, hemacytometer, Coulter Counter)
  • Vortex mixer

 
Optimal Transfection Conditions

To transfect suspension DG44 cells in a 30 ml volume, we recommend using the following optimized conditions:

  • Final transfection volume: 30 ml
  • Number of cells to transfect: total of 1.5 x 107 cells (cell density at time of transfection should be 5 x 105 cells/ml)
  • Amount of plasmid DNA: 18 µg
  • FreeStyle™ MAX Reagent: 15 µl


Note:   Further optimization of culture volume or transfection conditions is not necessary for stable cell line production.
 
Transfection Procedure

Follow the procedure below to transfect DG44 cells in a 30-ml volume. We recommend including a positive control (linearized pOptiVEC™ TOPO® plasmid with LacZa) and negative controls (no DNA, no FreeStyle™ MAX Reagent) in your experiment to help you evaluate your results.

  1. At 48 hours before transfection, pass DG44 cells at 3 x 105 cells/ml in complete CD DG44 Medium. Place the flask(s) on an orbital shaker platform rotating at 130-135 rpm at 37°C, 8% CO2.
  2. At 24 hours before transfection, pass DG44 cells at 3 x 105 cells/ml in complete CD DG44 Medium. Place the flask(s) on an orbital shaker platform rotating at 130-135 rpm at 37°C, 8% CO2.
  3. On the day of transfection, perform a viable cell count. To ensure optimal transfection results, viability of cells must be over 95%.
  4. For each transfection or control, transfer 1.5 x 107 viable DG44 cells to a new 125-ml flask. Add pre-warmed, complete CD DG44 Medium to a final volume of 30 ml. Place flask in shaker until ready to transfect.

 
Note:   Do not spin cells down prior to transfection, as it will decrease transfection efficiency.
 

  1. Gently invert the tube of FreeStyle™ MAX Reagent several times to mix. Do not vortex.
  2. Add 18 µg of plasmid DNA and 15 µl of FreeStyle™ MAX Reagent into 1.2 ml of OptiPro™ SFM and mix gently.
  3. Incubate the DNA-FreeStyle™ MAX mix for 10 minutes at room temperature to allow complexes to form. Do not incubate for longer than 20 minutes.
  4. Slowly add 1.2 ml of DNA-FreeStyle™ MAX Reagent complex into the 125-ml flask containing cells while slowly swirling the flask.
  5. Incubate transfected cell cultures at 37°C, 8% CO2 on an orbital shaker platform rotating at 130-135 rpm.
  6. At 48 hours post transfection, pass cells into HT-deficient, complete CD OptiCHO™ Medium. Proceed to the next section, Selecting for Stable Transformants

 

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Selecting for Stable Transformants

To obtain cell lines that produce high levels of your protein, you will first need to select for a pool of stably-transfected cells in which the linearized pOptiVEC™ construct has integrated into the host cell genome. At 48 hours post-transfection passage your transfected cells into complete CD OptiCHO™ Medium, which does not contain hypoxanthine and thymidine, allowing growth of cells expressing DHFR (i.e., stably transfected DG44 cells).
 
Protocol

Transfected DG44 cells should be passaged in complete CD OptiCHO™ Medium. To passage cells:
 
  1. Determine viable and total cell counts using your preferred method.
  2. Dilute the cells in pre-warmed complete CD OptiCHO™ Medium to give a final cell density of 3 x 105 viable cells/ml (see Important, below).
  3. Incubate flasks in a 37°C incubator containing a humidified atmosphere of 8% CO2 on an orbital shaker platform rotating at 130-135 rpm.
  4. Cells should be passaged in fresh complete CD OptiCHO™ Medium every 2 days for 10-14 days until cell viability increases to >90%.
 
During selection with CD OptiCHO™ Medium, cell viability could drop dramatically (to <10%) due to the death of untransfected and transiently-transfected cells. To promote the optimal growth of stably transfected cells, keep the culture volume at a minimum of 30 ml.
 

Positive Control

DG44 cells transfected with the control pOptiVEC™ plasmid containing LacZα will not express your protein of interest but will express DHFR, allowing them to grow in CD OptiCHO™ Medium. Maintaining these cells in parallel with your experimental cells will help you estimate how long it will take to obtain stable cell line formation, generally about 10-14 days.
 
You can detect expression of DHFR in the positive control cells by western blot using an antibody to DHFR (Rabbit polyclonal anti-DHFR, Sigma cat. no. D0942).
 
Protein Production

To check for production of your protein during stable cell establishment, you may take an aliquot of growth media and perform SDS-PAGE, protein-specific ELISA, or the bioactivity assay of choice to determine that your cells are producing your protein of interest.
 
When you have a pool of stably-transfected cells, you should cryopreserve several aliquots of the pool.
 
Next Steps

Depending on your protein production experimental needs, refer to the next section Choosing a Workflow to determine the next steps.
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Choosing a Workflow

You will have a population of stably-transfected DG44 cells expressing your protein of interest at various levels. For most bioproduction applications, several clonally-derived cell lines producing your protein of interest are desirable for screening. However, the growth and optimization of each clone is dependent upon the integration locus of the plasmid, the response to amplification using MTX, and the nature of the protein.
 
Depending on your protein production needs, the time and effort required to generate clonal, high-producing cell lines is also variable. Several common pathways from stable pool to clone scale-up are outlined below.



Points to Consider

When choosing a workflow from the options above, you should consider the amount of protein needed for your application, your available resources, and the amount of time it will take to obtain your clonal, high-producing cell lines.
 
Additional media and other products may be purchased separately from Invitrogen.
 
Protocols

  • To perform genomic amplification using MTX selection to obtain a population of cells expressing high levels of your protein of interest.
  • To perform limiting dilution to obtain single clones expressing high levels of your protein of interest.
  • To scale up your clones for protein production, Clone Scale-Up.
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Genomic Amplification by MTX Selection

Methotrexate (MTX) is a folic acid antagonist that is actively transported into cells by the folate transporter. In the cell, it is converted to a high molecular weight polyglutamate metabolite by folylpolyglutamate synthase, which binds to DHFR and inhibits its activity.
 
If MTX is present in the medium, cells compensate by increasing the DHFR copy number in the genome to overcome inhibition by MTX. Since the gene of interest is integrated into the same genetic locus as DHFR, the gene of interest is amplified as well, leading to increased production of the protein of interest (Kaufman et al., 1985; Tanaka et al., 2002). MTX (as methotrexate hydrate) is available from Sigma (10 mg, catalog number A6770).
 
MTX is toxic to the skin, eyes, and respiratory system. Wear suitable protective clothing, gloves, and eye and face protection when working with MTX. Refer to the product MSDS for complete precautions.
 
Preparing 1 mM MTX

To prepare a 1 mM MTX stock solution:
 

  1. Dissolve 10 mg MTX in 22 ml of PBS.
  2. Filter sterilize the solution through a 0.22 µm filter.
  3. Store in 250 µl aliquots at -20ºC.

 

  • Important: To make complete CD OptiCHO™ Medium with MTX, you will need to use complete CD OptiCHO™ Medium (prepared with L-glutamine) plus the required concentration of MTX.
  • Important: Prepare media containing MTX fresh each time. Do not re-use prepared MTX media.


Preparing Media with MTX

Using the sterile, 1 mM MTX stock solution (prepared as described above), prepare media with the appropriate amount of MTX.
 
The recommended selection concentrations per round of selection are 50 nM, 100 nM, 250 nM, 500 nM, 1 µM, 2 µM and 4 µM. Be sure to make enough media containing MTX for your application.
 
Protocol

  1. For each clone, spin down cells and aspirate old medium.
  2. Seed cells at a density of 2 - 5 x 105 cells/ml in 30 ml of media containing MTX in 125-ml flasks.
  3. Incubate flasks at 37ºC/8% CO2 with shaking at 130-135 rpm for 14-21 days or until viability >90%.
  4. Optional: Repeat steps 1-3 with the next highest concentration of MTX.

 
Expected Results

In the experiment below, a pool of stable transformants were created after transfection of DG44 cells with pOptiVEC™-TOPO® vector constructs containing erythropoietin (EPO). The cells were exposed to increasing amounts of MTX, and EPO expression was determined by ELISA after each completed round of amplification (results shown on day 5 after seeding the cells at 2 x 105 cells/well).
 
The amount of EPO produced increases 50-fold after 7 rounds of genomic amplification using MTX




Next Steps

Because MTX selection produces a population of cells producing high amounts of protein, you will have to perform clonal selection by limiting dilution prior to clone scale-up.

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Clonal Selection by Limiting Dilution

To obtain a clonal cell line (i.e., derived from a single cell), you will dilute your pool of stably transfected cells or your MTX-amplified cells at 1-2 cells per well in a 96-well plate. In most cases, one of the cells will form a distinct colony that can be scaled up using the procedure for Clone Scale-Up.
 
Cloning Medium Considerations

  • To grow DG44 cells under adherent conditions, use your clone optimization medium of choice, such as Gibco® Custom formulations F17 or CD-CHO-A.
  • Because the growth and protein production of each clone are variable, you may need to optimize clonal selection conditions by adjusting the number of cells/well, the cloning medium, and various media supplements.
  • Depending on how many clones you wish to screen before scale-up, you may increase the number of limiting dilution plates accordingly. The number of clones obtained from a 96-well plate is variable, depending on the experiment.
 
Protocol

  1. Determine the number and viability of your cells.
  2. Make a 10-fold serial dilution using an appropriate volume of cloning medium such that you will obtain 1 cell per 100 µl.
  3. Pipette 200 µl of diluted cells into each well of a 96-well plate to obtain ~2 cells/well.
  4. Incubate plates without agitation at 37ºC with a humidified atmosphere of 5% CO2.
  5. Single cell clones will be readily apparent after 14 – 21 days post-plating (see Important below).
 
 
After single-cell derived clones appear in 96-well plates, let them grow undisturbed, without changing the medium, at 37ºC in a 5% CO2 atmosphere until they are >75% confluent before proceeding to scale-up. Failing to do so will result in loss of clones due to low cell density.
 

 
Next Steps


You should isolate single clones for further testing. Once individual clones have been isolated and tested for high levels of protein production using your method of choice, they can be subjected to gradual MTX amplification or clone scale-up.
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Clone Scale-Up

After isolating clone(s) by limiting dilution, you will slowly scale up the volume of cells by allowing them to become nearly confluent and then moving the cells into the next larger culture volume (i.e., 96-well, 48-well, 24-well, 12-well, and 6-well plates, then to 125-ml flasks). The total scale-up process from a 96-well plate to a 125-ml flask will take about 2 weeks to 1 month, depending on the growth rate of the clone.
 
Materials Needed

You will need the following materials before beginning:

  • Clones in 96 well plates that are >75% confluent
  • Cloning medium of choice
  • Complete CD OptiCHO™ Medium
  • Sterile tissue culture dishes (96-well, 48-well, 24-well, 12-well, and 6-well) and sterile 125-ml polycarbonate flasks.
  • Optional: TrypLE™ Express, if cells do not detach with gentle pipetting.

Protocol

  1. Add 100 µl fresh cloning medium to wells with single colonies and pipette gently until cells in the colony detach.

    Optional: If cells do not detach easily with gentle pipetting, aspirate media from wells, add 50 µl of TrypLE™ Express and incubate for 5 minutes at 37ºC. Add 50 µl of fresh cloning medium and pipet up and down until cells detach.

  2. Transfer the entire amount of cells to 48 well plates and add an additional 250 µl of fresh cloning medium to each well.
  3. Incubate plates undisturbed at 37ºC, 5% CO2 until they are 80-90% confluent; usually 2-3 days depending on the clone growth.
  4. Proceed to the next volume (i.e. 24-well).
  5. Repeat Steps 1-5, transferring cells into a larger format tissue culture plate with each successive round.
  6. Once cells are cultured in a 125 ml flask, switch medium to complete CD OptiCHO™ and incubate cells at 37ºC 8% CO2, with shaking at 130-135 rpm.

Note: 
  If MTX selection has been performed, include the same amount of MTX used in the final selection (e.g. Include 1 µM MTX if 4 rounds of selection were performed with a final concentration of 1 µM MTX)
 
Next Steps

Prior to optimizing protein production, the appropriate cell stocks should be frozen down. Protein production experiments can then be optimized using different culture conditions, or scale-up can be continued to meet your bioproduction needs.
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Troubleshooting

Culturing DG44 Cells

The table below lists some potential problems and possible solutions that may help you troubleshoot your cell culture experiment.

Problem
Reason
Solution
No viable cells after thawing original vial
Cells not stored correctly
Order new cell stock and store in liquid nitrogen. Keep in liquid nitrogen until thawing.
 
Incorrect thawing medium or method
  • Use pre-warmed CD DG44 Medium supplemented with 8 mM L-glutamine and
    18 ml/L Pluronic® F-68.
    DO NOT USE CD OptiCHO Medium to propagate DHFR-negative DG44 cells.
  • Do not add antibiotics to media as this may negatively impact cell growth.
  • Incubate cultures on an orbital shaker set at 130-135 rpm in a 37°C incubator with a humidified atmosphere of 8% CO2.
No viable cells after thawing stocks
Cells not frozen correctly
Follow protocol to freeze cells.
 
Incorrect thawing medium
  • Use pre-warmed, complete, CD DG44 Medium supplemented with 8 mM L-glutamine and 18 ml/L Pluronic® F-68.
    DO NOT USE CD OptiCHO Medium to propagate DHFR-negative DG44 cells.
  • Do not add antibiotics to media as this may negatively impact cell growth.
  • Incubate cultures on an orbital shaker set at 130-135 rpm in a 37°C incubator with a humidified atmosphere of 8% CO2
Cells grow slowly
Incorrect growth medium
  • Use pre-warmed CD DG44 Medium supplemented with 8 mM L-glutamine and
    18 ml/L Pluronic® F-68.
  • DO NOT USE CD OptiCHO Medium to propagate DHFR-negative DG44 cells.
 
Shaker not set up properly
Shake on an orbital shaker at 130-135 rpm in 37°C incubator with a humidified atmosphere of 8% CO2.
 
Medium is foamy
Keep shaker speed at 130-135 rpm.
 
Cells too old
Use healthy DG44 cells under passage 25; do not overgrow.
 
Cell culture clumpy
Provide agitation of the culture, a regular and frequent cell passage schedule, and maintenance of cells at recommended densities.
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References

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  • Tanaka, H., Tapscott, S., Trask, B., and Yao, M.-C. (2002) Short inverted repeats initiate gene amplification through the formation of a large DNA palindrome in mammalian cells. PNAS 99, 8772-8777.

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