I am facing some problems understanding the mechanincs of stable transfection.
Stable transfection requires the transfected DNA to integrate itno the cell's chromosomes. This may happen quickly or take some time. I do stable transfections with Lipofectamine, so that is not a problem. It has been said that linearized plasmids integrate better than supercoiled plasmids, but supercoiled plasmids give better rates of transfection. For transient transfections, I would use supercoiled, and for stable I use ilinear. I cut the plasmid with an enzyme that will not interrupt either the expression cassette (promoter and cDNA, etc.) or the antibiotic resistance cassette. You need a selective marker, such as a neomycin, puromycin, hygromycin or bleomycin resistance gene for most cells. Other options include thymidine kinase for a TK negative cell line, for example. Neomycin resistance is commonly used, as a kanamycin/neomycin resistance cassette is present in many plasmids used for transfection. Determine the amount of Geneticin (also known as G418) that will inhibit growth of the normal cell line (50 micrograms/ml is usually effective). Linearize the DNA , clean it up and determine its concentration. Perform the transfection as usual. After a day or so, add Geneticin to the culture. Maintain the cells in medium with Geneticin. Passage the cells as usual, until you get a point where proliferation is lower and dead cells are common. At this point cells that have not integrated the DNA will die if they do not receive a copy during mitosis. This will happen all the time at a low rate, but at a few weeks you will notice a change, after which only stable transfectants will be found. They will be derived from numerous insertion events, so there will be a variety of clones with varying expression levels.
You can study expression using any method you want. It depends on the inserted gene. You can do Western blots, immunofluorescence, look at expression of flourescent proteins with a microscope, etc. It all depends on what you put into the cells. A good negative control can be the normal cell line, or cells transfected with the plasmid vector without the gene of interest. Bottom line, the method for studying the expression depends on what the gene is and what you have to detect it, not the fact that it is transfected, unless you have some type of alternative splicing that works only in certain cell types or something that depends on the cell line itself. In that case, you have to test it as you would in different tissues that allows you to determine which way htings are processed.
I hope that helps.
Stable transfection requires the transfected DNA to integrate itno the cell's chromosomes. This may happen quickly or take some time. I do stable transfections with Lipofectamine, so that is not a problem. It has been said that linearized plasmids integrate better than supercoiled plasmids, but supercoiled plasmids give better rates of transfection. For transient transfections, I would use supercoiled, and for stable I use ilinear. I cut the plasmid with an enzyme that will not interrupt either the expression cassette (promoter and cDNA, etc.) or the antibiotic resistance cassette. You need a selective marker, such as a neomycin, puromycin, hygromycin or bleomycin resistance gene for most cells. Other options include thymidine kinase for a TK negative cell line, for example. Neomycin resistance is commonly used, as a kanamycin/neomycin resistance cassette is present in many plasmids used for transfection. Determine the amount of Geneticin (also known as G418) that will inhibit growth of the normal cell line (50 micrograms/ml is usually effective). Linearize the DNA , clean it up and determine its concentration. Perform the transfection as usual. After a day or so, add Geneticin to the culture. Maintain the cells in medium with Geneticin. Passage the cells as usual, until you get a point where proliferation is lower and dead cells are common. At this point cells that have not integrated the DNA will die if they do not receive a copy during mitosis. This will happen all the time at a low rate, but at a few weeks you will notice a change, after which only stable transfectants will be found. They will be derived from numerous insertion events, so there will be a variety of clones with varying expression levels.
You can study expression using any method you want. It depends on the inserted gene. You can do Western blots, immunofluorescence, look at expression of flourescent proteins with a microscope, etc. It all depends on what you put into the cells. A good negative control can be the normal cell line, or cells transfected with the plasmid vector without the gene of interest. Bottom line, the method for studying the expression depends on what the gene is and what you have to detect it, not the fact that it is transfected, unless you have some type of alternative splicing that works only in certain cell types or something that depends on the cell line itself. In that case, you have to test it as you would in different tissues that allows you to determine which way htings are processed.
I hope that helps.
Yes Lipofec can be used for stable transfection. As mentioned by Michael, anti biotic selection is usualy used. you can also use the plamids with other markers like GFP, in addition to anti-biotic.
Also, to get a good stably transfected cells, atleast after one week of transfection with selection media, select a single colony and propagate, so that u have a similar clones of cells with the protein of interest.
@ Michael: Does the linearized plasmid really integrate into the genome, I guess at locations where double strand breaks occur, or is the plasmid lost when you withdrawl the antibiotic selection form the transfected cell culture?
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