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difference between a linear and circular plasmid/DNA for stable transfections


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4 replies to this topic

#1 cotchy

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Posted 04 February 2009 - 04:28 AM

Hi all

We in our lab have been working in molecular biology for a good few months now and have got to use some very interesting techniques in developing a cell line by immortalisation/ transfection with apSV3-neo plasmid. This produces proteins that bind to p53 and pRb eventually inactivating them (although not fully as i can still see slight staining for them when immunostaining my immortalised cells for p53 and pRb)

As i know my cells have been immortalised to some degree now, as they have been growing for a number of months through many passages, while the original primary cells themselves when isolated from the mice didn't divide or increase in number at all over the 3 weeks they survived in culture (i know for a cell to be considered "immortalised" it should grow for 10 months and we are working towards this)

Having been reading some books on the topic lately which got my mind thinking, my question is in relation to this is,

If we have used a circular plasmid (pSV3-neo) to immortaliose our cells or indeed for any other purpose does the fact that it is not linear prevent it from being integrated into the genome stably? and does it just stay in the nucleus seperate to the chromosomes (in a similar way to bacterial DNA being loose in its cytoplasm) meaning it can be removed at any stage dependoing on cell passages and divsions and our cells can potentially un-immortalise?

Or can a circular piece of DNA also fully and stably integrate into the DNA of our cells?

Thanks for any advice on this area.

#2 Bomber

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Posted 04 February 2009 - 09:09 AM

Hi,

circular plasmids are also integrated in cells and otherwise usually not maintained (unless you have specifically designed for that purpose).
Linearizing a plasmid before you try to stably transfect/transform your cells has the advantage of cutting your plasmid at any site which will not destroy you selection cassette or your expressed gene. If you work with a circular plasmid you have no clue where the plasmid will be lineraized within the cell (so you might have the chance that sensitive parts are destroyed upon linearization).
However, the disadavantage of cutting your plasmid beforehand is the lower efficiency of transfection and or gene expression which is mainly due to the destruction of linearized DNA by cytosolic nucleases.
Hope this provides some help.
Cheers

#3 cotchy

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Posted 05 February 2009 - 05:57 AM

Hi,
circular plasmids are also integrated in cells and otherwise usually not maintained (unless you have specifically designed for that purpose).

Linearizing a plasmid before you try to stably transfect/transform your cells has the advantage of cutting your plasmid at any site which will not destroy you selection cassette or your expressed gene. If you work with a circular plasmid you have no clue where the plasmid will be lineraized within the cell (so you might have the chance that sensitive parts are destroyed upon linearization).
However, the disadavantage of cutting your plasmid beforehand is the lower efficiency of transfection and or gene expression which is mainly due to the destruction of linearized DNA by cytosolic nucleases.
Hope this provides some help.
Cheers


Hi bomber thanks for your reply.

It has explained alot of things to me regarding linerizing youself as apposed to allowing the cell to do it foor you.

One thing though about your explanantion, when you say "circular plasmids are also integrated in cells and otherwise usually not maintained (unless you have specifically designed for that purpose"

Do you mean that circular plasmids are integrated into the genome stably unless they are designed not to be?

#4 Bomber

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Posted 07 February 2009 - 04:52 AM

Hi,

well - so far I have not come across any plasmids that are stably maintained and not being integrated into the genome; so I cannot give you any example for this. Non-integrated plasmids are usually not replicated. I am not aware of plasmids that contain a eukaryotic origin of replication and can therefore be maintained in cells outside the genome (probably I just do not know them).
Cells tend to loose the plasmid over time in cell culture due to simple cell divisions. In very rare cases plasmids may integrate.

If you do not provide any selection/pressure to maintain the plasmids, cells will unequally distribute plasmids upon cell division and in the end your culture will be taken over by cells that have lost the plasmids or never ever received one.
Putting on selective agents to your cell culture will 'enrich' the chance of the plasmid being integrated or to identify those who have integrated the plasmid.
An alternative is to enrich and sort positive transfected cells (you might need several rounds e.g, facs sorting etc...).

Best regards

#5 genehunter

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Posted 08 February 2009 - 06:11 AM

The majority of the plasmid do get degraded after transfection. So you have plenty linear form of random plasmid inside the cells. Very few of them become integrated. Do a Southern blot with cellular genomic DNA with a RE that only cut you plasmid once will tell if it is integrated.




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