why linearize plasmids make transfection stable?
Posted 16 March 2005 - 06:42 AM
Thanks if someone answer me.
Posted 16 March 2005 - 06:49 AM
But if you want stable transfection, you need to pick cells that have INTEGRATED in their genome the plasmid. Hence, you add selection pressure in the media and cells that aren't of interest die.
Generally, people don't linearize the plasmid before transfection. But if you want more security regarding site where the plasmid is opened in the cell (step necessary for the integration in the genome, plasmid can be linearized.
honnestly i do not linearize every time before transfection...
Posted 16 March 2005 - 11:25 AM
For a good reference, please check out the Nucleic Acids Research article by Kreiss et al that investigates this phenomenon at http://nar.oupjourna...full/27/19/3792
(Note: This article also references observations regarding the level of gene expression achieved using supercoiled vs. linearized DNA.)
"The luciferase activities measured in transfected HeLa cells (Fig. 5) were dependent on the size of the plasmid DNA, either supercoiled or linearized. Therefore, the topology of the transfected DNA molecules determines the level of gene expression but small DNA molecules have higher transfection efficiencies than larger ones. The greater transfection efficiency of small plasmids relative to that of larger plasmids may be due to differences in a step of the transfection process which occurs after particle internalization. Szoka et al. (29) recently proposed that endocytosis of cationic lipid–DNA complexes destabilizes the endosomal membrane, which results in the displacement of the cationic lipid from the DNA and the subsequent release of DNA in the cytoplasm. Behr et al. (30) also postulated that DNA is released from cationic lipids in the cytoplasm through lipid mixing and competitive exchange with anionic polymers like cytoplasmic mRNA. On the basis of these reports, we suggest that DNA size may account for either the mechanism of DNA release from cationic lipids or for the intracellullar migration of DNA from the cytoplasm to the nucleus, or both. "
Edited by tzemlo, 16 March 2005 - 11:28 AM.
Posted 20 March 2005 - 09:21 AM
Posted 21 March 2005 - 01:00 AM
if your cells with puromycin die that's because they didn't have integrated the gene. Maybe you can let them integrate the gene for a longer time (two/three days)?
Posted 21 March 2005 - 05:12 AM
Thanks so much for regular answers!
Posted 21 March 2005 - 06:13 AM
as the puromycin and the GOI are not under the same promoter, there isn't a direct relashionship between the two transcriptionnal activity. By the way translations are not directly linked.
By increasing the puromycin concentration, you'll select cells that have a high expression of the puromycin gene. But you can't touch the expression of GOI. All you get is more clones dying and no effect on POI (non detected in WB, is it?)
What's your promoter?