After positive selection in a few passages, we get a clone stably expressing the gene of interest. But what does that actually mean? Does that mean that the plasmid is integrated into the genome of the cells?
Yes sir it does.
Remember, most expression plasmids cannot replicate in eukaryotic cells (only in prokaryotic, which is how we grow up mass quantities of plasmid in bacteria). Because of this, even if the cell has been appropriately transfected and selected for, if the plasmid has not integrated into the genome it will eventually be washed out as the cells divide.
The reason you pick clones is because the integration is random, and sometimes may integrate in a gene of interest, a survival gene, or tumor suppressor gene in the cell which can transform the cells inappropriately. Additionally, the plasmid may integrate only part of its sequence, thus allowing for your gene of interest but not the selection marker, and vice versa. This is why you may pick a clone that is resistant to your selection marker, but when characterized is not expressing your gene of interest. So you should screen numerous clones and find the one that is 1) resistant to the selection marker, 2) has a high/medium/low expression of your gene of interest (depending on your study) and 3) retains most of the parental phenotype (unless of course your gene of interest changes the cells that dramatically).
I hope this helps clear things up!
Good luck,
~Adam
Yes sir it does.
Remember, most expression plasmids cannot replicate in eukaryotic cells (only in prokaryotic, which is how we grow up mass quantities of plasmid in bacteria). Because of this, even if the cell has been appropriately transfected and selected for, if the plasmid has not integrated into the genome it will eventually be washed out as the cells divide.
The reason you pick clones is because the integration is random, and sometimes may integrate in a gene of interest, a survival gene, or tumor suppressor gene in the cell which can transform the cells inappropriately. Additionally, the plasmid may integrate only part of its sequence, thus allowing for your gene of interest but not the selection marker, and vice versa. This is why you may pick a clone that is resistant to your selection marker, but when characterized is not expressing your gene of interest. So you should screen numerous clones and find the one that is 1) resistant to the selection marker, 2) has a high/medium/low expression of your gene of interest (depending on your study) and 3) retains most of the parental phenotype (unless of course your gene of interest changes the cells that dramatically).
I hope this helps clear things up!
Good luck,
~Adam
@Adam Thank you. That's clear. But I'm just thinking how a plasmid will get in to genome? Also is there any difference in the susceptibility of cells for such integration based on its type?
Hi Everybody!
I would like a very nice and deep bibliographic support for these facts which have been transmitted during decades. Someone possibly had published the molecular mechanistic for which the plasmids are randomly inserted into the genomes, do you know some refs?
Thanks a lot in advance!!
Hi Juantxo,
See if this helps:
http://www.nature.com/gt/journal/v10/n21/full/3302074a.html
If you find anything better let me know. I am looking for it as well.
and check out The mechanism of naked DNA uptake and expression by Wolff & Budker, chapter V. ENTRY OF NUCLEIC ACID INTO THE NUCLEUS, http://www.ncbi.nlm.nih.gov/pubmed/16096005,
a few words in Mechanism of chromosomal integration of microinjected DNA by Bishop & Smith, http://www.ncbi.nlm.nih.gov/pubmed/2695741 + Chromosomal insertion of foreign DNA by Bishop, http://www.ncbi.nlm.nih.gov/pubmed/9021872
also, concerning the recombination only Theoretical mechanisms in targeted and random integration of transgene DNA by Smith K., http://rnd.edpsciences.org/articles/rnd/abs/2001/06/01/01.html
- Badges
- Science topic