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 Module 1 frontpage.
Finally, you will be using the truncated EGFP plasmids to investigate the effect of double strand breaks on the frequency of recombination.Įlaborate mechanisms for coping with DNA breaks have evolved since these forms of DNA damage are so dangerous for the cell. 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. Cells expressing these truncated EGFPs should not fluoresce green. The EGFP coding sequence on these plasmids is truncated at either the 5’ or 3’ end of the gene. You will also be transfecting two experimental plasmids, one of which you have been constructing for weeks. Next time we will measure fluorescence of your positive control to assess the success rate of the transfection.
This plasmid will cause any transfected cells to fluoresce green. As a positive control, you will transfect one sample with a plasmid encoding full-length EGFP. Today you will lipofect several DNA samples into your mouse embryonic stem cells. 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. When the coated DNA is mixed with the cells, they engulf it through endocytosis. With this technique, a DNA sample is coated with a special kind of lipid that is able to fuse with mammalian cell membranes. The most popular chemical approach for getting DNA into cells is called lipofection. 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. During electroporation, mammalian cells are mixed with DNA and subjected to a brief pulse of electrical current within a capacitor. This is both technically difficult and inefficient, and so we won't be using this approach! More common approaches are electroporation and lipofection. In essence, a small gun is used to shoot the DNA into the cell. There are several approaches that researchers have used to introduce DNA into a cell's nucleus. Today you will be transiently transfecting your cultures of mouse embryonic stem cells. For stable transfection, the DNA is introduced in such a way that it is maintained indefinitely. 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). Mammalian cells can be transiently or stably transfected. The coding sequence tells the ribosome which amino acids should be joined together. The polyadenylation sequence assists in the export and stability of the mRNA so that it gets translated by the ribosome. The promoter tells the cell that the EGFP sequence should be transcribed by RNA polymerase. If you wanted to make a mouse cell fluoresce green, you could transfect it with DNA carrying the EGFP open reading frame, a promoter directing transcription of EGFP and a signal sequence for polyadenylation of the mRNA. DNA can be put into mammalian cells in a process called transfection.
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