Did you get a chance to try low voltage electroportation?

Hi,

the Lonza Nucleofector might be an option. At least the have nice data showing it works with neuronal cells.

Regarding lentivirus: Perhaps another site at your campus has the capability to use lentivirus. The reason for saying this is that the cells itself are not rated as S2 only the virus, so after some culture time you could retunr your cells into a standrad S1 lab. Perhaps this is an option, too (still this needs the regulatory stuff depending on the location you are working).

Good luck

Sven

Nagarjun Kasaraneni we have used both, electroporation and Lipofectamine. Both didn‘t work well

Hi,

Have you tried nanoparticle transfection?

Minyu Chan i‘m not familiar with nanoparticle transfection. Could you please provide a link or describe that in more detail?

Hi Benjamin Jurek ,

Here are 2 papers that talks about nanoparticle transfection: https://pubs.rsc.org/en/content/articlehtml/2016/ra/c5ra25333k

Article Comparison of nanoparticle-mediated transfection methods for...

Hope this helps.

Minyu Chan thanks Minyu, i will read into it

Hello. The paper about nanoparticles is interesting but not very relevant for the neuronal cell line of Benjamin. Nanoparticle reagents refer here to polymer-based reagents forming particles at the nanoscale. Our Viromers - as modified PEI - are also classified into this category. For me, the problem here is your cell line. You say neuronal cell line, is it neurons or something else? If they are very differentiated, then the proceeding of DNA once into the cytosol is the hurdle (if cell uptake is effective). To have positive cells, you need that the DNA reaches the nucleus. if your cells do not divide enough, the nucleus pores are not opened enough to let the DNA penetrating...

You said you need a stable transfection. I imagine that if you have at least 10-20% of transfected it will be enough??

If you want to try our Viromers (Viromer RED or Viromer PLASMID), I would be happy to provide you with free samples. We are also in Germany, it's very easy.

See for example how getting a stable PC12 cell line in this paper:

https://www.researchgate.net/project/VIROMERR-an-innovative-technology-for-transfection/update/5c52d6f0cfe4a781a57b8c11

Article Characterization of Cells Interactions with Patterned Azopol...

You can write me at [email protected]

Best, Sandra

Hi Benjamin ,

You might be able to use sleeping beauty vector. You can use it in S1 lab and it integrates into the genome. You need to choose a sensitive antibiotic for your cell line for the selection process. The main problem is not the transfection. Even if you can get 100% transfected cells with the normal plasmid ,they will be gone after few days.

low level transfection is not a problem for clone selection. even it is an advantage if you got some clone. of course, you should go with higher cell number in start. might be your POI has some issue in the cell line, better you check it after transient transfection.

Sandra Lagauzère i happen to already have a sample of the red viromer. I will give it a try and let you know if it worked.

we also have the Lonza Nucleofector and tried several protocols with it. However, the transfection efficiency was too low

if you "still" have problem with the POI, try an inducible system.

Attila Lehotzky we have used an inducible promoter in the piggyBac System of VectorBuilder, however, the level of expressed protein was identical to cells that were not treated with tetracyclin. So that didn‘t work either.

Mohsen Honarpisheh where can I get a sleeping beauty vector? Sounds interesting, thanks for the suggestion!

Benjamin Jurek Here is the link to Addgene website:

https://www.addgene.org/browse/article/9749/

They have a very nice collection of the plasmids with different selection markers.

Your welcome!

Good luck!

thanks Mohsen! I will look into it.

Benjamin Jurek Your welcome Benjamin.

Hi, Ben

I've ever heard that some of the overexpression promoters, such as CAG/CMV/Ef1alpha and so on, have been strongly supressed by negative feedback in host cell.

In the case of I have heard, he changed promoter to pGK, and get successed.

I'm not sure, weather this is same problem of you, but I think you can try.

Hope you good luck,

Yuichi Hiraoka thanks for that valuable information, I will keep that in mind when designing the next plasmid.

best,

Ben