Lipofectamine and FuGene 6 transfections - (Jun/21/2006 )
u have to make lentivirus and then infect ur cells with the virus. But its tedious, u will spend a few months before getting the virus preps that suit u. It been like 6 months and only now I am close to what I need with the lentivirus preps.
I know it takes similar amount of time even with adenovirus. So b careful if u want to try these out.
I know it takes similar amount of time even with adenovirus. So b careful if u want to try these out.
Scolix's comment is right for the amount of work involved to make recombinant viral particles, particularly if you are not experienced.
But there is really no better way to transfect T cells as far as I know. Either electroporation and adenovirus for transient or lentivirus for stable transfections.
GenePorter works to certain degree for Jukit cells, still the transfection level on this cell line is much less than 293, Cos and HeLa cells.
There is something about fugene not working if it touches the sides of the tube ie plastic. I have no clue as to why?? so apparently it is better if fugene is added to a tube already containing the media. basically do not add fugene to an empty tube.
They say that if Fugene6 gets in contact with the plastic walls, it will stick to them and will not be removed anymore, therefore you will compromise the transfection. It is mandatory to add the Fugene6 directly to the medium, the best is to take the fugene withth etip and imerge the tip into the medium and release the Fugene6
No one knows exactly the chemical structure of Fugene. But I think it is a "lipo something", in otherwords, containing lipid chains, and will coat hydrophobic surface like plastic.
I know it takes similar amount of time even with adenovirus. So b careful if u want to try these out.
Hi scolix,
I had no experience with lentivirus and am going to try it. To my knowledge, i will have to cotransfect gag-pol ,VSVG and the target gene between LTRs into HEK293T cell. Then use the supernatant to infect target cell. The procedures seems easy. Why would it take so long? What might be the major problems? Thank you a lot!
The procedure is easy. main problem is getting the right titres for ur experiment.
When I started with lentivirus, I had no idea how it was going to b. It took me 3 months by the time I got good titres and around a month or so before I could concentrate it efiiciently and now with more modifications to the lenti vecotr, I seem to b getting the right titres.
things I had to optimise.
transfecting 293s: I use 293FT and used lipofectamine and got more than 90% cells transfected. This is important. If u r going to use calcium phosphate u need to optimise it. I tried it but my PI wasnt keen on calcium phosphate bcoz it could have taken more time.
transfecting conditions: There r many additives people use to get better virus production. for eg. sodium butyrate or cholesterol. U have to c which works for u. This needs optimisation. I use cholesterol. I want to try out sodium butyrate as well.
vector: each lentivirus is different and can give u high or low titers. U need to get one which gives high titres. I had to remove some sequences and add others to get good titres. my original vector is from invitrogen.
If u need protocols for this, let me know, I can help u with this.
good luck!!!
Thank you scolix,
Please send me your protocols. That would be great help!
My e. mail address,
g895401@yahoo.com.tw
Thank you again
Check ur email. Let me know if u dont understand something.
Malik,
Have noticed that 293 cell changes its properties after certain passages? My start to show difference after 10 passages. Transfection efficiency seems to drop a little than that of early passages.