Editing 293E Protein Production (section)

=== <br>'''MATERIALS'''  ===

'''Reagents'''<br> 

*293fectin (Invitrogen)<br> 
*293-6E HEK293-EBNA1 cells, maintenance (in shake flask). The presence of G418 does not interfere with transfection.<br> 
*Culture medium for 293-6E cells (e.g., F17 culture medium [Invitrogen] or HyQ SFM4Transfx-293 [HyClone])<br>

Add 10 mL of Pluronic F-68 stock solution per liter of culture medium to a final concentration of 0.1% (w/v) prior to use.<br> 

*Opti-MEM I medium (Invitrogen)<br> 
*Plasmid DNA of interest, purified<br> 
*Plasmid, fluorescent protein-expressing <br>

Adding a plasmid encoding a fluorescent protein (e.g., green fluorescent protein [GFP]) to 5% in the transfection mixture does not significantly alter expression of the gene of interest and allows visual (or quantitative, if using flow cytometry) confirmation of transfection efficiency. A few GFP-positive cells can be detected as early as 3-4 h post-transfection using a fluorescence microscope.<br> 

*Pluronic F-68, 10% stock solution (w/v) (Invitrogen)TN1 medium, prewarmed (e.g., FreeStyle medium or LC-SFM containing 20% [w/v] Tryptone N1 [Organotechnie]) (optional; see Step 12)Supplement with 0.1% (w/v) Pluronic F-68 and filter-sterilize prior to use.

<br> '''Equipment'''<br> 

Containers, plastic (e.g., Tupperware)<br> 

Flasks, Erlenmeyer, shake, plastic disposable 125-mL (Corning)<br> 

Hemocytometer<br> 

Incubator preset to 37°C, humidified, 5% CO2Microscope, fluorescence <br> 

Paper towels<br> 

Plates, six-well, disposable (Nunc)<br> 

Shaker, orbital<br> 

Tubes, 1.5- or 15-mL<br> 

Vortex mixer<br><br>'''METHOD'''<br> 

'''Small-Scale Transfection in Six-Well Plates or 125-mL Shake Flasks using 293fectin'''<br> 

Although the protocol described here uses a DNA:293fectin ratio of 1.5:1.3, the ratio selected for production should be determined by testing various ratios in six-well plate experiments. These small-scale tests must include regular samplings to determine the optimal harvest periods. The flask procedure is scaleable to 650 mL in a 2-L shake flask. <br> 

1. One or two days prior to transfection, dilute the cells to'''2.5 × 105 cells/mL (for 48 h prior) or 5 × 10<sup>5</sup> cells/mL (for 24 h prior)''' with cell culture medium. Optimal cell density should be reached on the day of transfection. '''This avoids having to centrifuge the cells, which can have a detrimental effect on transfection efficiency if performed shortly prior to transfection.''' <br> 

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2. On the day of transfection, warm the Opti-MEM I medium to 25°C-37°C. Remove the 293fectin from the refrigerator. Remove the DNA from the freezer to thaw. <br> 

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3. From a maintenance flask, determine the cell density and viability using a hemocytometer. Cell density at transfection should range from 8 × 10<sup>5</sup> to 1.2 × 10<sup>6</sup> cells/mL, provided the doubling time is 24 h and viability is greater than 95%. <br> 

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4. Add 1.8 mL of cells to each of the six wells (or 18 mL to a new 125-mL shake flask). Transfer to an incubator. <br> 

To minimize medium evaporation, put the plates in a plastic container lined with a wet paper towel to preserve humidity. “Trap” some CO2 in the container before closing the lid. <br> 

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5. Add 100 μL of Opti-MEM I to each of six 1.5-mL tubes (or 1.0 mL to a 15-mL tube for a 125-mL shake flask). <br> 

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6. Add the required DNA volume corresponding to 3 μg to each tube (or 30 μg to the 15-mL tube). Vortex. <br> 

To evaluate transfection efficiency, add a GFP expression plasmid at 5% of the final DNA content used. A reasonable indicator of successful transfection is when the percentage of GFP-positive cells is between 30% and 50% by 48-72 h post-transfection. <br> 

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7. To another 1.5-mL tube, add 682 μL of Opti-MEM I and 18 μL of 293fectin (or to another 15-mL tube, add 1.0 mL Opti-MEM I and 26 μL 293fectin). Vortex gently. <br> 

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8. Incubate for 5 min at room temperature. <br> 

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9. Add 100 μL of diluted 293fectin to each DNA-containing tube (or the entire 1.0 mL of the 293fectin dilution to the 15-mL DNA tube). Vortex immediately after addition. <br> 

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10. Incubate the mixtures for 20 min at room temperature. <br> 

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11. Remove the culture (from Step 4) from the incubator. Add the DNA/293fectin mixture(s) and swirl. Return the culture to the incubator. <br> 

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12. For secreted protein production, add 50 μL of prewarmed TN1 medium at 24-48 h post-transfection to each of the six wells (or 500 μL for the 125-mL shaker flask). Return the culture to the incubator. The final TN1 concentration in the culture(s) should be 0.5% (w/v). TN1 supplementation is not necessary for intracellular protein production. <br> 

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13. Harvest the r-proteins: <br> 

For intracellular r-proteins <br> 

*i. Harvest the cells at 48-72 h post-transfection. Establish the best harvesting time by performing an expression kinetics study. For secreted r-proteins <br> 
*ii. Harvest the supernatants between 96 and 168 h post-transfection as long as the culture viability is high and the product titer is increasing. Since culture viability decreases with time, it is important to harvest when the viability is relatively high (i.e., greater than 75%) to protect the r-protein from proteolytic degradation. <br>

<br> '''TROUBLESHOOTING'''<br> 

''Problem'': The transfection does not work.[Step 13]<br> 

''Solution'': Consider the following: <br> 

*1. Make sure the cells are in exponential growth phase for transfections. If the culture grows to higher-than-prescribed transfection densities, medium nutrient depletion and metabolic by-products will compromise the transfection. <br> 
*2. Anti-clumping agents such as dextran sulfate can inhibit transfection. <br> 
*3. DNA plasmid quality may be inadequate. Check for contaminants by A260/A280 ratio readings; acceptable values range between 1.80 and 1.95. In addition, the DNA plasmid can be digested and run on an agarose gel to verify its integrity (it should be mostly in a supercoiled form) and/or to detect RNA contamination. 
*4. Monitor cell line productivity occasionally by transfecting the cells with an easily measured protein such as secreted alkaline phosphatase or GFP. Low transfection efficiencies are readily indicated when the percentage of GFP-positive cells is below 30% by 48 h post-transfection. Also, for each new medium or reagent lot, monitor transfection efficiencies using six-well plate assays.