Difference between revisions of "20.109(S07): Lipofection"

Revision as of 00:20, 26 December 2006

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

1. set up in advance 2 slides for each pair

Transfection

In anticipation of your lipofection experiment, one of the teaching faculty plated 1x10⁵ cells in each well of a pregelatinized 24-well dish 24 hours ago. Special media without antibiotics has been used.

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.

For each lipofection you will need

DNA:	 	0.1 ug in 50 ul OptiMEM 
Carrier: 	2.5 ul Lipofectamine 2000 in 50ul OptiMEM 

• 1. Dilute enough carrier for 16.5 lipofections. Let the dilution sit in the hood undisturbed for at least 5 minutes but not more than 30.
• 2. For the lipofections to be done just once, you’ll need 50 ul of DNA in OptiMEM. We will distribute to you a table indicating the appropriate volumes to use in your experiment.

• 3. For the lipofections to be done in triplicate, you’ll need a total of 150 ul of DNA diluted in OptiMEM. By making one lipofection cocktail that is later divide between replicates, you can be confident that each replicate was treated identically.

• 4. You should now have eight eppendorf tubes, four with 50 ul of OptiMEM +/- DNA and four with...Add an equal volume of diluted lipofectamine to each eppendorf (i.e., 50 ul if the tube has 50 ul). Pipet up and down to mix.
• 5. Incubate the DNA and lipofectamine cocktails at room temperature for 20 minutes. To allow the DNA/carrier complexes to form, it is important that you do not disturb the tubes during this incubation. During this time, aspirate the media from the cells in your 24-well dish, wash the wells with 0.5 ml PBS, then put 0.5 ml of fresh media on the cells. The PBS and media can be aliquoted with a 5 ml pipet.
• 6. After the 20 minute incubation is over, use your P200 to add 95 ul of the appropriate DNA:lipofectamine complexes to each well. Since the carrier is quite toxic to the cells it’s a good idea to gently rock the plate back and forth after each addition.
• 7. Return the plate to the 37°C incubator.
• 8. Tomorrow, one of the teaching faculty will remove the lipofection media from your cells and will replace it with 1 ml of fresh media. Approximately 48 hours after performing the lipofection, you and your partner will collect the cells and analyze their fluorescence. Before you leave today, see one of the teaching faculty to schedule your microscope time.

DONE!

For next time

Reagents list