Monday, March 31, 2014

Protoplasts!

One of the procedures I am learning is how to generate and transform protoplasts. Protoplasts are really cool. They are single plant cells that have had the cell wall removed but the membrane stays in tact so the cells are still alive. The cell wall of plants are mostly cellulose and resistant to environmental changes to protect the cells. Since cell walls are hard to penetrate it makes it difficult to add any DNA I want into the cells with the cell wall in tact. Which means I have to get the cells away from the cell wall and turn them into protoplasts. So how do we get them out?

 First we use the tape-sandwich method by Wu et al 2009. I put Fisher tape on the top of the leaves and then regular old Scotch tape on the bottom, press the Scotch tape on carefully, then rip that puppy off like I'm waxing eyebrows! It's soo much fun :) As you can see in the above image, the Scotch tape removes the bottom epidermal layer of cells which are very waxy and have to be removed to expose the more sensitive cell walls. The newly exposed leaves are put into a special solution full of enzymes that break down cellulose to remove the cell wall that connects the individual cells. They will fall out into the enzyme solution and I can see them under a microscope.


As you can see in the above picture, there are pretty round protoplast cells.. but there is also a lot debris. Without their cell walls, the protoplast are very sensitive to environmental conditions and shaking.. even pipetting can cause them to burst. They have to be handled extremely carefully and even with great care I still get some loss. Once they are isolated though, I can use chemicals to weaken the plasma membrane slightly to allow the introduction of plasmid DNA to the cells. Then leave them overnight so they can grow and make the gene product that I want. In this learning phase, I'm using simple reporter proteins GFP/GUS.

GFP = Green fluorescent protein.. aka glows green! Under a special microscope I can visualize the glowing protoplasts! It takes 2 days of work, but it is a really cool result! And when we are talking about genetic modification, 2 days is barely a blink of an eye compared to stable transformations. This method will let me test a lot of different genes in a relatively quick fashion to see what works and what does not.

Not to mention, I'm making GLOWING CELLS.. how cool is that?!?! :-D

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