Editing Expression of HA NA using Baculovoirus (section)

=== <br> Rescue of recombinant baculovirus and verification of protein expression<br> <br> ===

Explanatory note: The following steps have to be performed aseptically in a laminar flow hood Perform this procedure independently for HA and NA (or any other gene of interest). <br>

Insect cells are usually grown at 28 °C without CO2. Sf9 cells for this protocol are grown in full TNM-FH insect cell media supplemented with 10% FBS, 1% Pen-Strep* and 0.1% Pluronic F68 solution, if not indicated otherwise, and passaged 1:4 twice a week. High Five cells are grown in SFX serum free media and passaged 1:15 twice a week.<br> <br>

* Addition of antibiotics should be considered optional throughout the protocol and adjusted according to the practices used in each laboratory.<br> <br>

1. Plate Sf9 insect cells in 6-well plates at a density of 2 x 105 cells/cm2 and let sit for 20 min in a 28 °C incubator (without CO2).<br> <br>

2. Mix 2 μg (conc. 0.5-2 μg/ul) of recombinant bacmid with 100 μl of TNM-FH medium (serum free, 1% Pen-Strep). Mix 6 μl of transfection agent with 100 μl of TNM-FH medium (serum free, Pen-Strep). Combine the two volumes, mix gently and let sit for 20 min at room temperature (transfection mixture). Include one mock-transfection as control. <br> <br>

3. Remove supernatant from plated Sf9 cells (cells will now stick to the plate) and replace with 2 ml of serum free TNM-FH medium containing Pen-Strep (1%). Add the complete volume of the transfection mixture (from step 1.2.2) and rock the 6-well plate carefully for 5 sec. Incubate in a 28 °C incubator without CO2 for 6 hr.<br> <br>

4. Replace media with fresh full TNM-FH media (containing 10% FBS, 1% Pen-Strep and 0.1% Pluronic F68). Incubate in a 28 °C incubator without CO2 for 6 days. Check cells occasionally under a microscope. Infected cells usually appear bigger, rounder, have enlarged nuclei, and start to detach. <br>