Editing Epi293 Protein Production (section)

== Transfection and Expression ==

<br>Follow the procedure below to transfect suspension Expi293F Cells in a 30 mL volume. For each 30 mL transfection, you will need 7.5 × 107 cells in 25.5 mL of Expi293 Expression Medium.

<br>1. The day before transfection (day –1), seed Expi293F Cells at a density of 2.0 × 10^6 viable cells/mL in Expi293 Expression Medium to transfect them on the following day (day 0).

2. Incubate the cells overnight in a 37°C incubator with a humidified atmosphere of 8% CO2 in air on an orbital shaker rotating at 125 rpm.

3. On the day of transfection (day 0), determine cell viability using an automated cell counter or the trypan blue dye exclusion method.<br>Important: Cell should not be clumping and viability of cells must be over 95% to proceed with transfection.

4. Add the appropriate volume of cell suspension containing 7.5 × 10^7 cells (see Note on page 1) into a sterile, disposable 125-mL Erlenmeyer shaker flask, and bring up the volume to 25.5 mL with fresh, pre-warmed Expi293 Expression Medium. Return the cells to the incubator.

5. Prepare lipid-DNA complexes by performing the following:

a. Dilute 30 μg (i.e., 30 μL) of Antibody Expressing Positive Control Vector in Opti-MEM® I medium to a total volume of 1.5 mL. Mix gently.

b. Dilute 80 μL of ExpiFectamine 293 Reagent in Opti-MEM I medium to a total volume of 1.5 mL. Mix gently and incubate for 5 minutes at room temperature.<br>Note: Longer incubation times may result in decreased activity.

c. After the 5 minutes, add the diluted control vector DNA to the diluted ExpiFectamine 293 Reagent to obtain a total volume of 3 mL. Mix<br>gently.

d. Incubate DNA-ExpiFectamine 293 Reagent mixture for 20–30 minutes at room temperature to allow the DNA-ExpiFectamine 293 Reagent complexes to form.

6. After the DNA-ExpiFectamine 293 Reagent complex incubation is complete, add the DNA-ExpiFectamine 293 Reagent complex to the cells from Step 4. The flask should contain a total volume of 28.5 mL.

7. Incubate the cells in a 37°C incubator with a humidified atmosphere of 8% CO2 in air on an orbital shaker rotating at 125 rpm.

8. Approximately 16–18 hours post-transfection, add 150 μL of ExpiFectamine 293 Transfection Enhancer 1 and 1.5 mL of ExpiFectamine 293 Transfection Enhancer 2 to each flask. The final volume should be approximately 30 mL in each 125-mL flask.

9. The rabbit IgG expressed by the control will be secreted into the Expi293 Expression Medium. Expression can be detected by day 3 post-transfection;<br>however maximum yield is obtained 6–7 days post-transfection. The expected yield of the control IgG is approximately 200–300 mg IgG per liter of culture on day 7.<br><br>

[[Category:Protocols]]