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why linearize plasmids make transfection stable?


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#1 truongthuthuy

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Posted 16 March 2005 - 06:42 AM

what really makes transfection stable or transient?
Thanks if someone answer me.

#2 fred_33

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Posted 16 March 2005 - 06:49 AM

transient transfection is process by which you deliver plasmid in cells and analyse the situation two or three days later.

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...

#3 tzemlo

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Posted 16 March 2005 - 11:25 AM

To follow-up further with this discussion, the size of a plasmid can influence transfection efficiency, which has implications for creating stable cell lines.

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.


#4 truongthuthuy

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Posted 20 March 2005 - 09:21 AM

Thanks so much! Though I linearized plasmids before transfection into embryonic stem cells, many clones obtained just died at passages No 3-10. I took one clone and split into two cultures, one with and one without selection antibiotic (puromycin). The culture without puromycin quickly proliferated but the one with puromycin died away. So i don't know why even I have linearized plasmids, most of transfectants are not stable? Is it possible that my cells try to get rid off the intergrated genes?

Thanks.

#5 fred_33

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Posted 21 March 2005 - 01:00 AM

hi
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)?

#6 truongthuthuy

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Posted 21 March 2005 - 05:12 AM

I did transfection by electroporation and 1 day later added selectin medium. After 7days visible clones appeared and I took these clones for normal ES cell culture in order to take samples for analysis and to keep the cells in freezer. More clones died when I selected with high puromycin concentration! By the way i have another question. Can increased selection pressure have sth to do with higher expression of POI? As I can detect mRNA of GOI but cannot detect protein by WB, someone advised me to increase puromycin concentration. I don't understand the rationale behind this.

Thanks so much for regular answers!
Thuy

#7 fred_33

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Posted 21 March 2005 - 06:13 AM

mmmh well, i hope GOI and POI are for gene / protein of interest :(

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?




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