My favorite method of landing pad cell line generation is with lentiviral transduction. The original method was with site-specific knock-in using genomic disruption (Using Cas9 or TALENs), followed by homology-directed repair from a transfected circular plasmid template. It worked, but it was slow, with a fair number of false positives, including cells that had likely still expressed BFP transiently during single cell sorting, or even more annoyingly, cells where numerous landing pads had integrated per cell. I’ve since gotten some potentially helpful tips (eg. linear template supposedly degrades faster than circular plasmid), but I still don’t think this method is practical for routine transgenic knock-in. Moreover, I’m not convinced the AAVS1 locus is even that great for cell engineering.
This brings me to Lenti landing pad transduction, which I’ve developed a bunch of supported tools for. It’s easy (few steps), fast (cells can be single-cell sorted 2 – 7 days following transduction), broadly applicable (many different cell lines are easily transduced), with low false positives (low MOI transduction, due to Poisson behavior, ONLY gives single integrants). Pretty good arguments, no?
The general steps are as follows:
- Transfect HEK 293T cells with a mixture of lentiviral vector plasmids to produce lentivector particles. Do this in no dox media.
- Change media the next day, and then replace and collect supernatant over a ~ 72 hour period. Do this with no dox media. It’s fine to keep collecting into the same collection tube.
- After all of your collections are done, spin out any cells that may have been accidentally collected, and pass the supe through a 0.45 micron filter.
- Plate out your target cells, and mix with a series of volumes of the filtered supernatant. This can be in dox media.
- At least 2 days after switching into dox media, read out how many cells are fluorescent protein (often BFP) positive. Go with the well closest to but lower than ~5%. If the positive cells are incredibly few, treat cells with antibiotic (often blasticidin) to enrich for transductants.
- Using FACS, sort individual positive cells into wells of a 96 well plate. Use conditioned media if your cells may require it.
- Allow clonal lines to grow out. Transfect with a mixture of Bxb1 expression plasmid, attB-EGFP, and attB-mCherry to confirm no stable double positives exist. Cells that pass this test should be good to go for library work
Now for the flavors of lenti-landing pad that I’ve developed. It’s very choose your own adventure, based on your needs. The original (LLP) is the most conservative choice to start with in non-HEK cell lines. LLP-Int-Blast can make your life easier with easier / higher recombination rates, but requires a bit more care in its use. If you want to study a potentially toxic gene, use LLP-rEF1alpha. For everyone else, use LLP-iCasp9-Blast. The selection with AP1903 is so quick and easy that it allows you to brute force recombinations quite easily. I used to chuckle whenever anyone used the phrase “game changer”, but I totally find it appropriate for this landing pad.