Horizontal Spread Assay
(on 10T1/2 mouse fibroblasts)
10T1/2 fibroblasts work well for this assay because they grow well in DME 10% IFS, are infectable with high efficiency, and can be killed off rather quickly with standard drugs. Also, for routine passaging, the cells can also be split sparsely (a 1:10 or 1:15 split is usually fine) and they can sit at confluency for several days.
¨ Add ~ 2.5 x 105 cells per 10 cm plate at least 8 hr prior to infection.
¨ Infect cells for at least 4 hr with the supernatant from the tester cell line with 5µg/ml polybrene. For supernatants from HMECs or other cells growing in serum-free media, add DME 10% IFS so that the final serum concentration is ~2% on the 10T1/2 cells.
¨ Replace the media with DME 10% IFS and let the cells recover and grow for at least 12 hour and then add the following drugs.
100% death on control plate
|Neo||1 mg/ml||Day 3 (from addition of drug)|
|Hygro||300 µg/ml||Day 4 *|
|Puro||2 µg/ml||Day 2|
|Zeo||1.2 mg/ml||Day 6 *|
|Blast||15 µg/ml||Day 3|
* These plates will probably get confluent and the cells won’t die completely. They’ll probably need to be split ~ 1:4 on Day 3 in the presence of drug to see 100% killing. The cells will take a long time to be killed with Zeo, but resistant cells are smaller and healthier while sensitive cells flatten out.
Horizontal Transfer Test (PJ Keller additions)
Document this in the HT test lab notebook
Do always for retrovirus, can spot check for lentivirus or do for high titer preps.
After you have infected your cells of interest do one of the following:
A: Selectable viral constructs (puromycin etc.)
Split infected cells into selection media and select colonies that have been infected by virus.
Post-selection, change the media to fresh media with out puro etc. and allow the cells to condition the media for at least 24 hours. Collect this media, filter it to remove any cells and apply it, with polybrene/protamine sulfate, to a test cell line such as 293T etc.
Allow cells to infect as usual and then split into selection and monitor until all cells die (if a colony survives, your virus is replication competent and has failed the HT test)
B: Non-selectable viral constructs with GFP marker (etc.)
Verify infected cells are green (etc.), usually this happens when titering virus or you can also check on the scope. Wash infected cells several times to remove any residual virus
Split infected cells onto a fresh plate and allow to condition media for at least 24 hours.
Collect the media, filter it to remove any cells and apply it, with polybrene/protamine sulfate to a test line such as 293T.
Allow cells to infect as usual and then verify the lack of GFP+ cells by flow cytomentry (preferred) or visual inspection (in this case, green cells mean that a replication competent virus was made and you failed the HT test).