20.109(S07): Lipofection
Introduction
DNA can be put into mammalian cells in a process called transfection. Mammalian cells can be transiently or stably transfected. For transient transfection, DNA is put into a cell and the transgene is expressed, but eventually the DNA is degraded and transgene expression is lost ("transgene" is used to describe any gene that is introduced into a cell). For stable transfection, the DNA is introduced in such a way that it is maintained indefinitely. Today you will be transiently transfecting your cultures of mouse embryonic stem cells.
There are several approaches that researchers have used to introduce DNA into a cell's nucleus. At one extreme there is ballistics. In essence, a small gun is used to shoot the DNA into the cell. This is both technically difficult and inefficient, and so we won't be using this approach! More common approaches are electroporation and lipofection. During electroporation, mammalian cells are mixed with DNA and subjected to a brief pulse of electrical current within a capacitor. The current causes the membranes (which are charged in a polar fashion) to momentarily flip around, making small holes in the cell membrane that the DNA can pass through.
The most popular chemical approach for getting DNA into cells is "lipofection." With this technique, a DNA sample is coated with a special kind of lipid that is able to fuse with mammalian cell membranes. When the coated DNA is mixed with the cells, they engulf it through endocytosis. The DNA stays in the cytoplasm of the cell until the next cell division at which time the cell’s nuclear membrane dissolves and the DNA has a chance to enter the nucleus.
Today you will lipofect the genetically-encoded calcium sensor into your mouse embryonic stem cells. This will cause the cells to fluoresce green. Next time we will measure fluorescence to assess the success of the transfection and to detect differences in intercellular calcium in response to agents that affect calcium flux and free calcium concentration.
Protocols
Transfection
- set up in advance 2 slides for each pair
DONE!
