Transient transfection with a pcDNA plasmid - How long does it take for chromosomal integration??? (May/14/2008 )
Hello, I am doing transient transfections with a pcDNA3 plasmid, after having trouble with my stable cell lines. The response I see with this plasmid in a transient transfection is great, but now, I would like to know if I'm looking at chromosomal integration and not just protein expressed transiently like with a transient vector.
I transfect my cells with lipofectamine for 4 hours, I serum starve for 24, dose with compounds and on the third day I measure gene response. Was that enough time for chromosomal integration?
Is the pcDNA3 vector has the ability of transcribe a protein without integration? like a normal vector would?
Can anyone can help me?
sorry for all the questions
Thanks
Chromosomal integration of a circular (or even linear) plasmid (as opposed to viral vectors) is a rare event. That is why we need to select those very few clones with high conc antibiotics. So, most of your cell pool is expressing through extra-chromosomal plasmid.
Ok, but if this plasmid is specific for stable trsansfection, can it also express the protein outside of the chromosome? Even if you use antibiotics, if the plasmid is inside the cell and is being expressed extra-chromosomal it will still be resistant to antibiotics.
There is nothing like plasmid specific for stable transfection, any plasmid can get integrated in the genome (at a very low frequency), if the plasmid expresses an antibiotic resistance gene under eukaryotic promoter, such low level of integration can be
The extra-chromosomal plasmid will give resistant to antibiotics alright, but it will get diluted out with consecutive cell divisions, and ultimately be lost. Whereas the integrated one will replicate just like and along with the entire cell genome, so it will stay, forever.
You can also make stable using GFP, in which case, you continue to select the cells with genomic integration expressing GFP by flow.
I see, thank you so much for that explanation. I thought there plasmid used for stable transfections were specific for integration. It makes more sense now! Yes, I have donde the GFP co-transfection and sorted by flow, but apparently my cells are not expressing enough to see the response I get in the transient transfection.
There is nothing like plasmid specific for stable transfection, any plasmid can get integrated in the genome (at a very low frequency), if the plasmid expresses an antibiotic resistance gene under eukaryotic promoter, such low level of integration can be
The extra-chromosomal plasmid will give resistant to antibiotics alright, but it will get diluted out with consecutive cell divisions, and ultimately be lost. Whereas the integrated one will replicate just like and along with the entire cell genome, so it will stay, forever.
You can also make stable using GFP, in which case, you continue to select the cells with genomic integration expressing GFP by flow.