20.109(S14):Cell preparation for DNA repair assays (Day5)

Introduction

Today's the big day! The first of three pretty big days, actually: transfecting your cells, measuring their fluorescence, and beginning to analyze relative repair rates.

F13 general to start with and revise:

DNA can be put into mammalian cells in a process called transfection. 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. The promoter tells the cell that the EGFP sequence should be transcribed by RNA polymerase. The polyadenylation sequence assists in the export and stability of the mRNA so that it gets translated by the ribosome. The coding sequence tells the ribosome which amino acids should be joined together.

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 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.

Protocols

Part 1: Transfection of cells with plasmid reporters

All manipulations are to be done with sterile technique in the TC facility.

Timing is important for this experiment, so calculate all dilutions and be sure of all manipulations before you begin.

In anticipation of your lipofection experiment, one of the teaching faculty plated xyzx10^4or5 cells in a 24-well dish xyz hours ago.

A schematic for your experiment is shown below.

Lipofection sample schematic (top half of a 24-well plate.)

Please adhere to the schematic exactly, as some wells have been pre-treated with inhibitor C401.

Part 2: There is no part 2!

For next time

1. Your Module 1 microbiota summary revision is due by 11 AM on Friday, submitted to Stellar and using a TeamColor_LabSection_Mod1-REV.doc naming scheme.
2. Your second reflection, written individually about lessons learned from the report revision process, is also nominally due Friday. We are happy to accept these within about 24 hours after you submit your report (no late penalty), but don't want to delay further than that as you may start to forget about the revision experience.

Reagent list

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