Stable Transfection Portal
Here you will find information related to the generation of stable cell lines, used in transfection.
What is stable transfection?
Transfection is a combination of the words, “transfer” and “infection”. Transfection occurs when genetic material (DNA or RNA) “infects” a cell and is transferred into the cell’s own genetic material.
This is a very complex process that requires a lot of patience and scientific equipment, but today there are labs which focus on developing certain cell lines, which are available to the public. If you have a need for a certain type of transfected cell, there are many products and services at your disposal. Providing these services utilizes Cell Line Development, companies that perform this function are called Contract Research Organizations (CROs).
What is Cell Line Development?
Cell Line Development is how scientists create large numbers of identical cell types.
It’s based on a simple idea, that cells will divide by mitosis, continually producing genetically identical clone cells until they can no longer expand. Despite the simplicity of the idea, this becomes much more complex when one wants a cell to express a certain trait, that it doesn’t naturally express. This is where transfection comes in, and stable transfection is the act of creating a stable cell line of successfully transfected cells.
How are Stable Cell Lines Developed?
Cancer cell lines and primary cell types. This is the cell stock to be used that will be transfected. Using a plasmid, three things are injected into the cell. This is a relatively easy process helped by the cell’s natural ability to absorb plasmids in its environment. The three things are:
- Genetic Material
- Transfection Reagent
- DNA Encoding Antibiotic Resistance
This cocktail of microscopic molecules serves a very important 3 step process.
Generally, less than 5% of the genetic material makes its way inside of the cell wall, and then further to the inside of the nuclear envelope where the DNA is. And even then, it may never become a part of the host DNA. It’s a very unlikely event, and we don’t have a good way of directly incorporating foreign genetic material into the DNA. It’s basically a numbers game, most of it won’t work as we want it to, but all we really need is for a small number to successfully and stably become transfected.
The Transfection Reagent aids scientists in this process of successfully transfecting cells. A transfection reagent is just an extra tool used that helps the genetic material incorporate itself into the host DNA. What kind of transfection reagent you use will depend on the type of cell. There are a variety of different methods available, which can be tailored for your specific needs. Useful and common transfection reagents can be purchased from vendors with relative ease.
All right, now that we’ve infected <5% of our cells in the Petri dish, how do we extract just those cells? We can’t exactly go in and pick them out one by one (they are far too small).
This is where the antibiotic resistance comes in. To create a “pure” cell culture, a solution that is toxic to the cells is introduced to the culture. This toxic solution basically eliminates the undesired cells which were not transfected with the plasmid packet. Since successfully transfected cells should have the genetic material, as well as antibiotic resistance, they are more resistant to the solution.
It should be noted that, it usually doesn’t work as simply as just killing off untransfected cells and letting transfected cells live. The toxic solution usually will not be strong enough to kill a cell at low levels, even a medium dose will simply slow cell growth, only high dosage will kill it. That is why dosage must be carefully calculated so that it will allow normal growth to continue for cells that have antibiotic resistance (and transfected DNA), but that will cause the normal cells to significantly slow in growth. Over the course of a few weeks the normal cells will slowly disappear from the population, leaving an entirely pure cell culture after approximately five weeks.