Posted By Caulimovirus on January 17, 2007
â€œMaybe We Could Just Shoot It?â€
.: Suppose youâ€™re a geneticist, and you discover a gene responsible for a few nifty traits. Naturally, your inherent disrespect for the purity of nature will compel you to try to incorporate that gene into as many different organisms as possible. For single-celled organisms like bacteria, this can be done pretty easily with something called a plasmid, which is a small circle of DNA that is capable of replicating itself. But for the so-called higher organisms, like plants and animals, plasmids by themselves arenâ€™t enough; youâ€™ll need something else too.
.: But first, letâ€™s learn more about the other ways of inserting foreign DNA into cells. Bacteria sometimes have plasmids, and these plasmids contain only a few genes at most and are separate from the bacteriumâ€™s genomic DNA. An interesting feature of some bacteria is their ability to temporarily combine (conjugation) to another bacterium and exchange plasmids. This occurs naturally in some species, but scientists can also induce artificial transference in others.
.: There are two popular methods for introducing plasmids into cells incapable of conjugation: heat-shock and electroporation. In heat-shock, the cells are cooled in the presence of CaCl2 and then heated briefly. The plasmids are then able to enter the cells. With electroporation, the cells are subjected to an incredibly brief but exceptionally powerful electric shock, which create holes in the cellsâ€™ membranes, allowing the plasmid DNA to pass through.
.: Those methods are fine and good for bacterial cells, but what about plants and animals? Plant cells, like bacterial cells but unlike animal cells, have cell walls, and they can be bastards to work around. Whereas bacterial cell walls are composed of peptidoglycan, plant cell walls are usually made out of cellulose mixed with other polysaccharides and various proteins. Cellulose is tough. Although electroporation is still possible with intact cell walls, scientists usually remove them to create a, and then go from there. Itâ€™s difficult for scientists to grow a whole new plant from a single protoplast, so they developed another method.
.: Enter biolistics.
.: I can imagine a scientist going over a mental checklist: â€œDid we try shocking it? Check. Did we try heating it? Check. Have we tried poking it? Yes. Say, why havenâ€™t we tried shooting it? We can try coating tiny little bullets with bits of DNA and shooting them into plant cells, and then the bits of DNA will recombine with the plant DNA, and weâ€™ll have all the transgenic affronts to nature weâ€™ll ever need!â€ It almost sounds like an Urban Legend, but thatâ€™s essentially what the process entails.
.: Basically, scientists coat microprojectiles (in this case, microscopic balls of tungsten) with plasmid DNA. They then place the microprojectiles on a sled, which is situated on a monorail-like track. Behind the sled is an explosive chamber, much like a blank bullet, which shoots the sled down the track and into a stopping plate. The stopping plate, owning up to its name, stops the sled, which then sends the microprojectiles hurling towards a plate of plant tissue. (Think of a real sled and that one time you didnâ€™t see that huge log straight in front of you, and how far you flew after the collision.) The microprojectiles penetrate the walls of the cells, and regular cellular mechanisms are able to mend the holes created in the process. From there the plasmid DNA falls off the microprojectiles and recombines into the chromosomes of the plant cells, giving the scientists all the new nifty traits they could possibly conjure.
.: Of course, there are several interesting details that need mentioning. Consider this simple experiment: go outside and pick up a good-sized rock, about the size of your palm, and throw it as far as you can. Then pick up a tiny pebble and, using the same amount of force, throw it as far as you can. The pebble wonâ€™t travel nearly as far as the rock because of air resistance. Now imagine shrinking that pebble even further to the size of 1.2 Î¼m, or one millionth of a meter, in diameter. Thatâ€™s how large the microprojectiles are and why air resistance can be a formidable obstacle to researchers. To solve that problem, they simply place the dish of cells in a chamber and decrease the air pressure to about 1/10 of the atmosphere. Less air, less resistance.
.: Another problem that applies equally well to other methods of DNA transfer is the following conundrum: how do scientists identify the cells in which transference was successful? How can they look inside the cells to see if the plasmid really got in there? The answer is simple: they donâ€™t have to. One way to solve this problem is to include a gene for antibiotic resistance in the plasmids and expose the tissue culture to antibiotics. This process weeds out the cells that didnâ€™t undergo transference, and all thatâ€™s left are the newly transgenic cells.
.: But how is the plasmid DNA incorporated into the chromosomal DNA once itâ€™s in the cells? Plants and animals donâ€™t have free-floating plasmids like bacteria do, so what happens? The answer is homologous recombination, but I think Iâ€™ll save that for another post.
.: Lastly, Iâ€™ve tried to explain the process to the best of my abilities, but sometimes I find that words simply arenâ€™t effective. Therefore, Iâ€™ve created a simple schematic that summarizes the entire process:
.: I think that about clears up any potential misconceptions.