I encounter a problem when I try to establish a stable endothelial cell line (Ea.hy926 cells) expressing siRNA.
I transfectd cells with the vectors expressing target or negative control siRNA using electroporation and start the selection with G418 (200 ug/ml). I did find clones in cells transfected with target or negative control siRNA expression vector. But when I transferred the clones into 48 well plate and cultured them, even the negative control cells stopped growing and gradually died!!!
Does anyone have the experence with siRNA expression vectors? Thanks!
Note:
vector: pSilencer 3.1-H1 neo vector provided by Ambion
negative control siRNA template: also provided by Ambion -boduolige-
Dear colleague,
Did you considered that :
1- your siRNA expression vector was actually efficiently transfected? try using a GFP expression plasmid as reference for transfection efficiency
2- how long did you performed the incubation before adding the selection of G418? at least leave 24-48 hours for cells to recuperate from the electroporation
3- did you adjusted the cell density after transfection (1:5-1:10) from the original eletroporation density? and also be sure to exclude any other antibiotics (strepto/Penicillin) in the culture media, as it may be toxic to cells during transfection.
Adrian J. Calderon, PhDc
University of Puerto Rico, School of Medicine
acalderon@stu.rcm.upr.edu -Adrian_Calderon-
I agree with Adrian_Calderon!
Good luck, boduolige! -geness-
HI, Thank Adrian_Calderon and geness
I did go through the three points you mentioned, but it didn't work. I found that the transfection efficiency is quite low (.1-2%). So I switched to viral vector, and that works great. Now i've got my cells.
Boduolige -boduolige-
Hi. I磎 new. I磎 working in a Pharmaceutical Company in M閤ico. I磎 Biochemist with 12 years in this field, particulary in Analysis adn Protein purification.
We need an expertise in Molecular Biology field, in order to hire him for our company. Our work is based in superior cells. Do you know where I could find this kind of person?
And, another question. Where can I find new and cheap cell lines for pharmaceutical products?
Thanks a lot.
Cookies -Cookies-
Hi Buodolige, hi everyone
i am also trying to get a permanent stable RNAi cell line using vector approach, but at the moment didn磘 work. I have try many different things, controls, efficiencies, protocols....and now i am thinking in using a retrovirus approach. Which kind did you use, from which company?
and also, has someone experience with pSM2 RNAi vectors from Openbiosystems?
Juan -juan-berlin-
上一篇：Can shRNA be transfected without vector...??-siRNA, micr 下一篇：RNAi实验原理与方法
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