Difference between revisions of "20.109(F11): Mod 1 Day 6 Lipofection & lab practical"
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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. | 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 called 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 | + | The most popular chemical approach for getting DNA into cells is called 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 several DNA samples into your mouse embryonic stem cells. As a positive control, you will transfect one sample with a plasmid encoding full-length EGFP. This plasmid will cause any transfected cells to fluoresce green. Next time we will measure fluorescence of your positive control to assess the success rate of the transfection. | Today you will lipofect several DNA samples into your mouse embryonic stem cells. As a positive control, you will transfect one sample with a plasmid encoding full-length EGFP. This plasmid will cause any transfected cells to fluoresce green. Next time we will measure fluorescence of your positive control to assess the success rate of the transfection. | ||
− | You will also be transfecting two experimental plasmids, one of which you have been constructing for weeks. The EGFP coding sequence on these plasmids is truncated at either the | + | You will also be transfecting two experimental plasmids, one of which you have been constructing for weeks. The EGFP coding sequence on these plasmids is truncated at either the 5 or 3 end of the gene. Cells expressing these truncated EGFPs should not fluoresce green. The plasmids provide a wonderful tool for studying recombination since a cell will fluoresce green if it has been transfected with both plasmids and has recombined the genes to regenerate a full-length EGFP. Finally, you will be using the truncated EGFP plasmids to investigate the effect of double strand breaks on the frequency of recombination. |
Elaborate mechanisms for coping with DNA breaks have evolved since these forms of DNA damage are so dangerous for the cell. You should (re)familiarize yourself with these mechanisms by (re)reading the excellent review by Thomas Helleday that you can find in the References section of the [http://openwetware.org/wiki/20.109%28F10%29:Module_1 Module 1 frontpage]. You should also check out the animations of repair mechanisms that are linked there. These animations were made by Justin Lo, a class of '08 Course 20 student and a former UROP student in Professor Engelward's laboratory. | Elaborate mechanisms for coping with DNA breaks have evolved since these forms of DNA damage are so dangerous for the cell. You should (re)familiarize yourself with these mechanisms by (re)reading the excellent review by Thomas Helleday that you can find in the References section of the [http://openwetware.org/wiki/20.109%28F10%29:Module_1 Module 1 frontpage]. You should also check out the animations of repair mechanisms that are linked there. These animations were made by Justin Lo, a class of '08 Course 20 student and a former UROP student in Professor Engelward's laboratory. | ||
− | One kind of DNA damage with particularly catastrophic consequences for the cell is double stranded breaks. The broken ends of the DNA must be correctly repaired (literally here re-paired) without loss of any encoded information. The model for such repair is called to the Szostak model, named after the person who first described it. According to this model, illustrated below, a gap in one chromosome is repaired through an interaction with its homologous chromosome. The repair first requires | + | One kind of DNA damage with particularly catastrophic consequences for the cell is double stranded breaks. The broken ends of the DNA must be correctly repaired (literally here re-paired) without loss of any encoded information. The model for such repair is called to the Szostak model, named after the person who first described it. According to this model, illustrated below, a gap in one chromosome is repaired through an interaction with its homologous chromosome. The repair first requires |