Primary stem cells can be difficult to transfect, and 50% efficiency is quite an accomplishment. In terms of transfection reagents, having liposomes be combined with other factors to improve efficiency (see https://altogen.com/product/altofect-transfection-reagent/) can help you get better results. Plasmids usually result in low transfection efficiency, but what you got is pretty amazing, and I don't think you really need to change the protocol that much.

most people use electroporation for ES cells (if you want to introduce a targeting vector, that is). 

50% is a really good efficiency. I wouldn't expect more than 10% in a normal experiment, they're not HEK cells... 

What are you trying to do? Can you select for transfection by antiobiotic resistance or FACS sorting to enrich transfected cells? 

Agree with Thomas.  Transfection efficiency in mES is generally low, around 20-30%, especially for large plasmids. When you look at the transfection results presented by reagent providers, please note that those numbers are often obtained using much smaller plasmids.

If your gene of interest is

I used Lipofectamine 3000 or LTX with overnight transfection in ES cell medium. The plasmid was about 5 kb and the efficiency was 20-40%.

I would not recommend lentiviral vectors as they are efficiently silenced in pluripotent stem cells. I.e. in iPS cells after successful reprogramming the exogenous factors are not expressed in the majority of cases.

I also use liposomal-based transfection reagents such as Lipofectamine 2000 and LTX. Indeed transfection efficiency is low in ESCs, however the big advantage of working with ESCs is that you can seed the in a clonal fashion and select out the correctly targeted clones. We have developed a neat strategy that allows 100% targeting efficiency independent of the length incoming construct.

To do so, we generated a recombination mediated cassette exchange (RMCE)-compatible G4 ESC line (see publication). This ESC line has a truncated NeoR gene (lacks promoter and start codon). We transfect these ESCs with  a Flpe vector and a (RMCE-compatible) destination vector using Lipofectamine 2000. The Flpe-mediated cassette exchange inserted the gene of interest (GOI) into the ROSA26 locus and in addition, restored neomycin-resistance. This allows targeting efficiencies up to 100% and these targeted ESCs routinely give rise to germline-competent mice.

Article Efficient ROSA26-Based Conditional and/or Inducible Transgen...

50% efficiency is really good in mES! I am not surprised you can not reproduce it. Before putting eveything in the bin, remember that you won't need the same number of clones depending if you just want to insert your plasmid randomly in the genome or if you want it to be in a specific place (perfom homologous recombinaison, RH).

We want to have RH and keep ES with very good GLT rate (we want to make mouse line out of this). We have routinely plasmid about the same size than yours. We take advantage of the fact that is very easy to get large number of mES to perform electroporation followed by G418 selection. You let your ES grow at low density so that resistant clones can be isolated. Of course you need to adapt with your own cell line but to give you an idear, we eletroporate 5 millions of cells with 10 µg of linearized plasmid, then plate on 2 10 cm plates. We start Neo selection the following day, and pick clones about 10 days latter.

Otherwise, you can try the Nucleofector technology. Works very good (but more expensive).

good luck

HI Kylie

I recommend DreamFect Stem, for mES transfection, see here

http://www.ozbiosciences.com/transfection-dna/17-dreamfect-stem-transfection-reagent.html

If you want a free sample let us know at [email protected]

Good Luck

Hello

I agree with researchers , 50% is a good efficiency . Also , i suggest use Amaxa Nucleofector , where we found the "nucleofection" give the best results for ESC transfection .

look at links here will help you 

Good Luck

http://www.ncbi.nlm.nih.gov/pubmed/25102378#

http://www.nature.com/gt/journal/v16/n1/full/gt2008125a.html

Hi Kylie,

I routinely use Lipofectamine 2000 for my transfections in mES cells and the largest plasmid that I have used is ~9kb with ~70% efficiency. This was reproducible. In my hands, 2 rounds of transfection also produced similar efficiencies without causing too much cytotoxicity. The typical numbers were, 40,000 cells, 1ul lipofectamine 2000, 100 ul Optimem (Thermo Fisher) and 1.5-2ug plasmid (preferably maxiprep) in a 12 well dish. For double transfections, forward (seeding 24h before transfection) method was used. Good luck!