Instructables

Let the cells exhibit bright green light -- in situ GFP Transfection

I searched instructable website. There is no instructable talking about transfection, so I decided to create one about this topic. In this instructable, I will show you how to deliver green fluorescent protein (GFP) into polarized MDCK-G cells to make the cells exhibit bright green fluorescence under ultraviolet light using an electroporation system called iPorator.

GFP:
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:
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.
 
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sonicase1 year ago
nice, but not too many people would have an electroporater at home... it would be good if you could figure out ways to do this for cheaper and with things you can make at home.
bbsbb (author)  sonicase1 year ago
you can not do it at home. There is no way to do the cell culturing at home. If you are working in a biology lab, you may try it.
DIY-Guy bbsbb1 year ago
BBSBB
Thank you for teaching us how to do this when we have the equpiment available. Instructables are written for a wide range of audiences and I appreciate this laboratory level information. Thankyou.