Pearl Satyr
posted 8 months ago

Gene knockout protocol from HCC cancer cell lines using px459-v2?

I would be highly appreciated if someone could share a detailed protocol (worked) for knocking out genes from any HCC cancer cell lines using px459-v2.

Thank you

White Pegasus8 months ago

Are you doing a CRISPR screen? I have not used the px459-v2 plasmid specifically, but I've used similar plasmids (LentiCRISPRv2 and TLCV2) to perform a CRISPR screen in LN229 glioblastoma cells in vitro.

It looks like the px459-v2 plasmid has a puromycin resistance gene, so the first thing you want to do is determine the minimal concentration of puromycin that will kill regular (i.e. - non-transduced) HCC cancer cells. You'll need to seed some HCC cancer cells in a 6, 12, or 24 well plate and then test the ability of 0, 1, 2.5, 5, 7.5, and 10 ug/mL of puromycin to kill these cells. You can determine cell counts using a hemacytometer, or with something like an MTT assay with a plate-reader (if you have one). You'll have to note the lowest concentration of puromycin that kills 100% of your HCC cancer cells. You will use this dose to select your transduced cells in subsequent steps.

Next you need to titer your virus (assuming you have virus and not simply your plasmid. If you only have your plasmid, you'll need to get an easily transfectable cell line, like the LentiX HEK 293T cells:, as well as the LentiX single pack shots: to produce virus. Let me know if you need further instructions on how to produce virus). To titer your virus, plate some HCC cells in a 6, 12, or 24 well plate and add polybrene at 8 ug/mL. Then add 0.5, 1, 2, 4, and 8 uLs of your virus to the cells in quadruplicate. Incubate at 37 C and 5% CO2 overnight and replace the polybrene media with fresh media the next day. The day after, add the dose of puromycin you determined above in your puromycin "kill curve" to two of the four wells of each dose of virus you're testing. After 4 or so days, count the number of cells in each well. Divide the average number of cells in the puromycin treated wells by the average number of cells in the non-puromycin treated wells for each dose of virus. This will yield the percentage of cells that were transduced for each dose of virus. Determine the amount of virus that transduced approximately 30% of cells and use this in your calculations to determine how much virus to add to your cells for your actual transduction (be sure to scale up the amount of virus you used based on the amount of cells you plated - for example, if you plated 20,000 cells per well and 1 uL virus transduced 30% of these, then if you plate 2,000,000 cells in your actual transduction, you will need to add 100 uL virus to transduce 30% of these). You want to use an amount of virus that will transduce ~30% of your cells because this ensures that most cells are infected with only 1 virus and therefore only have 1 gene knocked out.

When you perform the actual transduction, you want to use an amount of virus that will transduce ~30% of cells (based on the results from the above). Then you will want to puromycin select the transformed ones. And once you have a pure population of transduced cells, you can do you subsequent experiments.

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