Schumer lab: HSV transduction

HSV transduction in Xiphophorus primary embryo cells Last updated April 2024

A few days before transduction:

Passage cells into the plates/flask you’d like to use and maintain until 70-80% confluency. Make sure to passage at least 24 hours prior to transduction to avoid stressing the cells as much as possible. I will try to seed around 100k cells in a few wells of a 12 well plate and let them grow for a few days before infection

Day of transduction:

1. Treat cells with 10 uM ROCKi for at least 1 hour prior to transduction ROCKi is a small molecule that greatly increases cell survival (https://www.stemcell.com/products/y-27632.html)

2. Create a waste bucket in the hood of 10% bleach for tip disposal. We also would like to lay out multiple clorox wipes under the waste bucket and underneath the cell plate to kill any drips that may accidentally happen.

3. Thaw viral lysate on ice.

4. While the virus is thawing, perform a regular media change, supplemented with 10uM ROCKi

5. Add the desired amount of virus to each well.

       When pipetting virus, it is critical to avoid blowing bubbles with the pipette tip, as this can aerosolize the virus. We can prevent bubbles by pushing the pipette down only until the first stop when dispensing virus, never fully expelling all the liquid. Similarly, do not try to draw up all the liquid inside a virus aliquot. 
       To safely dispose of a tip containing virus, place it in the 10% bleach solution while still attached to the pipette and draw up bleach. Dispense the tip while full into the bucket. Anything that touched the virus should be left fully submerged in the bleach bucket for at least 30 mins prior to disposal

6. Change media 24 hours post transduction using the same technique used to disinfect tips in bleach. All media that is removed from the plate should be carefully drawn up using a P1000 and carefully dispensed directly into the bleach. I like to do a PBS wash in between the removal of the virus and the switch back to base media but this is optional.

The cells will continue to produce more GFP over the coming days, and the signal should get stronger.

Notes: Prior to using ROCKi, most cells died during our transductions. I would highly recommend using this for best results (it also works wonders when you supplement it for the first 24 hours after passaging!) For PPE: in addition to a lab coat and nitrile gloves, we use tyvek sleeves and additional latex gloves on top (https://www.fishersci.com/shop/products/dupont-tyvek-bib-apron-sleeves/0136121) DO NOT USE THE VACUUM ASPIRATOR at any point when there is virus in the media Polybrene is commonly used in lentivirus transductions and makes the cell membrane more permeable, but we have had better success with cell survival and successful infection without using it HSV is freeze thaw sensitive, the first time it is thawed it should be aliquoted, adding 1-2 uL more than you expect to use each time to avoid bubbles when you draw up the virus next time Our current base media is L-15 + 20% FBS and 15 mM HEPES buffer