How to select for positively transfected cells if I am using px330

[Sapna Shah]

Hi!

I am using px330 to knockout my GOI in a mouse multiple myeloma cell line. However, px330 does not have a selection marker so I am wondering how people figured out which cells were transfected and which were not.

Should I order the plasmids with puromycin or GFP instead? Any suggestions on which is a better selection marker? Puromycin selection gave better knockdown than sorting for GFP+ cells did when I used shRNA so I am wondering if such a discrepancy exists in CRISPR as well.

Thanks!

-Sapna

[Marc Morgan]

Hi Shapna,

I had sucess in mouse ESCs by co transfecting a plasmid with a Puro resistance gene. I used a 2.5 : 1 ratio of CRISPR plasmid to Puro plasmid (25ug / 10ug).

All the best,

Marc

[Sapna Shah]

Hi Marc,

Thanks for the feedback! I thought about doing that but isn't it possible that the cells you select with puromycin have the puro plasmid only and not the CRISPR plasmid? I'm sorry about the multiple questions, I'm just a little new to this field!

Best,
Sapna

[p.pri...@gmail.com]

I'm having a similar issue and would like to know what the forum comes up with.

Phil

[c...@lumc.nl]

We have validated our CRISPR (pX330 plasmid) by Lipofectamine transfection to mouse ES cells without using selection; 4 days after selection we harvested the cells as a pool and could easily detect activity (we reach 80-90% transfection efficiencies when using GFP as control, so we figured that selection was not needed). I did cloned the best performing CRISPR form the Yang/Jeanisch paper (Tet2) as a positive control.

 

Good luck,

 

Cor

[SF]

   As long as you use lipofection, the co-transfection  would not likely to produce a cell with one of each vector transfection.  When you use multiple plasmids, each cells within a population may have different expression level or intake of the plasmids amount, but the RATIO being transfected into a single cell should remain the same.  That is because even if you added a very small amount of plasmids ( for exampe 100:1 ratio), as log as it is ug or ng order, there is a number of each plasmid copies in some molecular level, and they are transferred into a cell as a group. If you want to see this you can co-transfect GFP and RFP and do FACS analysis.   But, most people do cotrasfection and IP or luciferase reporter assay with dual color system.  I have never heard any problem with those study, so essentially it will not happen.

 Anyway, using cas9-2A-GFP or -puro is very easy and excellent way, but I experienced that for some cells with low transfection efficiency, GFP or maybe Cas9 expression seems to become inefficient.  I suppose this is because the larger plasmid or protein tends to be transcribed less efficiently. So, for some types of cells it may be better to co-transfect rather than single all-in one vector or -2A system. 

Now, I would like to know what others experienced or any suggestion.

SF

[Chengchao Lin]

Hi all,

Will it be an issue to select Cas9-puro and keep expressing it in cells especially there might be some off-target events?

Cheng-Chao

On Tuesday, January 7, 2014 2:16:57 PM UTC-5, Sapna Shah wrote:

[mdc...@gmail.com]

Could you share your protocol? Thanks