The green fluorescent protein (GFP) is a protein that exhibits bright green fluorescence when exposed to the blue to ultraviolet light. GFP was first isolated from the jellyfish Aequorea Victoria by Japanese scientist Osamu Shimomura (he won the Nobel Prize in Chemistry in 2008 for the discovery and development of GFP) in 1961. GFP had been widely used in labeling the organisms for identification purposes. When the GFP is delivered inside the cell, the cell will fluoresce under fluorescence microscopy. In this instructable I will use gWiz GFP.
Transfection is the process of putting nucleic acids into cells to block or express some functions of the cell. Transfection can be divided into chemical-based transfection (Lipofectamine 2000 by Life Technologies is the most common one), non-chemical transfection (electroporation is the most common way), and particle-based transfection such as magnetofection. It is very difficult to transfect fully polarized or fully differentiated cells using chemical-based method. Chemical-based trasnfection and particle-based transfection will introduce extra stuff (such as lipid, polymers, and magnetic nanoparticles) besides the wanted nucleic acids into the cells during transfection, and those extra stuffs might create unwanted or unexpected result after transfection. In electrodeporation, the cell membrane will open lots of tiny holes under the electrode force, and nucleic acids can get into the cell via those holes. Traditional electroporation applies a very high voltage (hundreds to thousand volts) in cell suspension. It kills lots of cells, and for adherent cells their characteristics are different in cell suspension and in cell layer. For adherent cells, in situ transfection will provide the best result since the cells are in physiologically relevant postmeitotic state. In this instructable I will transfect fully differentiated MDCK-G (Madin-Darby Canine Kidney Cells) cells in situ using the electroporation system called iPorator by primax biosciences. This machine uses a small current instead of a high voltage during transfection to reduce the cell damage. During transfection, cells are growing on the porous membrane insert (no re-suspension required).This system is best for adherent cells. However, this system does not work for suspension cells.
Step 1: Procedure:
1. Seed the cells:
Harvest the MDCK-G cells from the flask. Here I skip the harvest procedure because it is a pretty standard procedure in every biology lab. Seed the cell into Millicell 24-well cell culture inserts (Millipore PIRP12L04). For MDCK-G cell, I put 75K cells/200uL in each insert and 900ul of culture medium into each bottom feeder well. This insert has 1um PET porous membrane (that means there are lots of tiny holes on the membrane). Cells can get nutrition from both sides.
24-hours after seeding, the MDCK-G cells will cover the entire membrane and form a mono-layer.
Dilute the gWiz-GFP using OPTI-MEM (Gibco 31985-070). The GFP concentration I use is 20ug/mL. Remove the culture medium out of the insert and add 150uL of diluted GFP inside the insert. The iPorator can transfect four 24-well inserts (or two 6-well insrts) at once. So I prepare four inserts with GFP.
iPorator requires special electrodes during transfection (Primax Bioscience, E24A1108). Fill bottom electrode chamber with 1mL of Opti-MEM. Transfer the inserts with GFP into bottom electrode chamber and put in the top electrode.
Next step, put the electrode set with inserts into the iPorator.
Click “Transfection” in the user interface.
The program has a build-in MDCK-G DNA transfection protocol, so I just select that protocol and click “Start”. It takes about 4 minutes to finish the transfection.
After transfection, take the electrode set out of the machine and transfer the inserts back to the original feeder plate. Leave the inserts at room temperature for about 10 minutes with GFP then replace the GFP with 200ul fresh culture medium. Put the inserts back to cell culture incubator for 24 hours to let the GFP express. For the gWiz-GFP, you can see some expression 2 hours after transfection, but you need to wait at least over night for it to fully express.
Step 2: Check Result
24 hours after transfection, take the inserts out of the incubator and check it under the fluorescence microscopy. You can see a beautiful cell layer is transfected with GFP and exhibits bright green fluorescence. The fluorescence can last more than a week under proper culturing.
Step 3: More Images and Image Taking Tips:
Fluorescence image taking tips:
1. Try to block all the normal night.
2. Increase the exposure time.
3.put the insert on top of a glass slide will increase the sharpness of the cells.
4.focus the cell layer using shorter exposure time setting. The camera response time will be very long under long exposure time.