Allen Cell Methods : RNP transfection for gene editing human iPS cells

Hi my name is Maggie Fuqua and I’m a
research associate… here at the Allen Institute for Cell Science. Today we’re going to be doing an RNP transfection… which is the method that we’ve used to
transfect all of the lines… that are available at the Allen Cell Collection. Some of the things that I’ve taken care of… ahead of time are that I’ve got my… Cas9 s. pyogenes and my CRISPR RNA duplexed with tracer RNA both at a final
concentration of 10 micromolar… and I’ll keep those on ice until I’m
ready to begin. I’ve also prepared our cell suspension here. I’ve got an unedited cell line at low passage… that had good morphology and were also about… 70% to 80% confluent at the time of passaging. I’ve also pre labeled a 6-well plate for… where the transfected cells will be living… until we’re ready to passage them for sorting. And I’ve also got enough mTeSR1 supplemented with ROCK inhibitor I’ve also got pre-labeled tubes… for my RNP complexes, and for my cells. I’ve also prepared tubes for my control wells. Just a little bit about the electroporation system that we’re using… we’re going to be using the Neon electroporator… and we have right here in our hood the
Neon electroporation chamber… as well as an electroporation cartridge… that’s been filled with 3 mLs of E buffer. We also have some resuspension buffer, or R buffer… that we’ll end up resuspending our cells in later on. We have the Neon electroporation tip. And also Neon electroporation tips themselves. So before I do anything else… I’m gonna start my RNP complexing. So I’m going to add 1.63 microliters of Cas9 protein… to 1.63 microliters of CRISPR RNA. And this should give us enough
complexed RNP for 1.3 reactions. So I’ll let the RNP complex
for a minimum of 10 minutes before I actually start. Then I’m gonna aliquot my
cell suspension… at 1.04 million cells per tube. I’ll make sure to give my cells a good
mix beforehand. I’m aliquoting now the cells to my
eppendorf tubes… where I’ll spin them down before we transfect. Now I’m going to spin down my cells… in a swinging bucket centrifuge for
3 minutes at 211 x g. So now I’ve got our pelleted cells here… and before we actually start I’m gonna… select program 8 on the Neon. So we’re gonna go ahead and aspirate the supernatant… and there should be a visible cell pellet in the bottom. So you want to get as much of the media
out as you can… but if there’s a little left it won’t
affect your transfection. I’m going to re-suspend in R buffer. Just really gently put the cells back
into suspension. And immediately transfer to our RNP complexes. Give it a mix one or two times. Then I’m going to add my plasmid. And then using the Neon pipettor I’m
going to grab a neon tip. Mix everything one final time… and be careful to avoid
getting bubbles in the tip You’re gonna want to inspect this really closely… before you electroporate… or it will affect the transfection itself. And we’ll insert the tip into the
transfection chamber. So you want to keep your eye out for… little tiny bubbles once you hit the start button… and that indicates that the
electroporation has happened. Add your cells to the well in a swirling
motion… and then rock the plate front to back, side to side. When the experiment is
complete then you can put the plate… into the incubator for incubation overnight… where you’ll change the media the next day The following day you should see a lot
of cell death but… you should also see a lot of… healthy cells starting to attach. So thank you for watching. That concludes the RMP transfection and… all of our methods can be found online.

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