Genomic integration of siRNA expression cassette
Posted 26 March 2005 - 01:04 AM
I´m currently working with a shRNA expression vector and probably generated stable clones.
In my cells tested I found differences in the expression level of the targeted protein (it´s maybe decreased for 50% which could be better of course), which is in a way not surprising, but I´m not 100% sure about the reason for these differences.
Is it for example a common explanation that in a few cells more copies of the vector integrated than in other cells? And how can one compare these clones with each other?
I would be grateful for every suggestion made.
Thanks a lot for response
Posted 29 March 2005 - 12:27 AM
for differential expression levels, they are two major explanations. First is about the number of copies integrated in the genome and the other one is the part of the genome where the cassette integrated. In major cases, cassettes are integrated in a good region due to the fact these regions are unpacked. And if they're unpacked, genome activity is correct. But on the other had, integration can be done is regions with less activity and therefre less transcription of the cassette.
If we're considering the expression of targeted protein, it could depend on both these factors. But it may depend on the efficiency of shRNA sequence too.
In order to test several clones, the best method is to separate cells in a 96 well playte by flow cytometry. But if you can't get through this, you can make limit-dilutions in a 96well to.
I will try to explain it. Please forgive me to be a little hard to understand.
start from a well and pick half of it. Go in the second well and put the volume of well 1 in an equal volume in well2. Pick up half of the volume and put it in an equal volume in the third well. And again and again.
After few days, you'll get cell expansion in several well. The one of interest is the last dilution that gives cell expansion. Yopu can admit that there were very very few cells. ANd probably it will be a single clone.
Posted 29 March 2005 - 02:06 AM
in case you come back once again:
thanks for the nice suggestions made.
To be honest I haven´t thought about the genome structure and its imnplication for integration and transcription of plasmid DNA yet; but surely this is an argument!
Anyway what I considered so far were just two things:
- Are there any possibilities to control integration of plasmid DNA is ~ equal between different clones? Or can I just assume that anyway?
- Where should I know from (without having stacks of work to do) that the integration into genome does not destroy important features of my cells? And by the way, are the phenotypic (if there are any) changes due to the knock-down or due to the integration of the plasmid into other genes etc... ?
Thanks for the suggestions made concerning the 96-well plates. I already got through this and could show that I got stable clones but I´m not sure about their reliability considering discussions about their phenotype. Are such clones comparable anyway?
One last point:
Guess what? All these knock-down things are a part of my master thesis, so for a discussion it would be the finest to quote a bit literature so my big boss believes my discussions... .
I was not able to find literature discussing the mentioned points (maybe I searched the wrong journals...), so if you (or anybody else of course) have some nice references/literatures etc. to share I would be very grateful.
Thats it for now
Thanks very much once again
Posted 29 March 2005 - 02:43 AM
Are there any possibilities to control integration of plasmid DNA is ~ equal between different clones? Or can I just assume that anyway?
you can't control where dna will integrate in mammalian cells (or i don't know how... ) if you can afford it, chromosome printing would show where is your cassette. But what can be done with the information??? moreover it's a hard an expensive technique. So let our cells do correct stuff and reserve us analytical job
Where should I know from (without having stacks of work to do) that the integration into genome does not destroy important features of my cells? And by the way, are the phenotypic (if there are any) changes due to the knock-down or due to the integration of the plasmid into other genes etc... ?
that's an interesting question. But the answer is not easy. In fact, despite of a great change in cell, you won't be able to detect anything. But according to the fact in mammals only 3% of genome is active, it would be a hard lck to integrate in a gene. Regarding this fact, it is admitted that integration does not occur in a gene or active sequence.The only phenotypic change you reasonably could wait is the evel of your targeted mRNA/protein.
once again if you want to save time and get gretaer results, try to do a separation with a facs. you will be zable to test without great effort many different clones. When i did so, only 10 of the 96 wells did not resulted in stable clones
and you'll appreciate that a machine do the seeding for you. I've done it too and think it's quite amazing.
PS : my name is fred. 33 is from my department in france