As promised, I will explain the method of creating the aforementioned strain of flies. Just a disclaimer- the process is genetics-heavy; there’s not exactly a way to explain it thoroughly without delving into the technicalities, but I hope that my description is not too cluttered. Personally, I find the process itself highly fascinating; the techniques scientists have devised to manipulate flies and generate desired characteristics are so vast and so precise it’s insane.
One of the mechanisms of creating novel strains, which I mentioned earlier, is the transposable element. These elements are basically jumping sequences of DNA that move around within a genome, landing wherever they please. Certain transposable elements, such as “P” elements for example, have a high tendency to land near a “TATAA box” controlling a the expression of a certain gene. TATAA boxes are basically RNA polymerase binding sites; they are sequences of DNA that signal to RNA polymerases where to begin transcription. P elements tend to land right near these “TATAA” sequences, around the start of a specific gene. Say two genes are far apart from each other, P elements almost never land in the gene desert between them- they insert themselves close to the start of one of the genes. Although P elements can be predicted to land near TATAA boxes, there is no way to tell WHICH TATAA box they will land near. There are many genes on a single chromosome, and P elements have an equal chance on landing on any one.
Piggybac, on the other hand, has a truly random insertion pattern. At times, Piggybacs insert themselves in gene deserts, or at the ends of genes, in fact Piggybacs sometimes land in the middle of genes, disrupting the sequence of the gene and its expression.
The fly embryos into which I plan to insert my DNA plasmids in order to make transgenic flies automatically produce Piggybac transposases, (conveniently) the type of transposable element that I want to use. This makes my life much easier – I don’t have to manually mix the transposase plasmid with my DNA plasmid, because the Piggybac automatically produced by the embryo will bind with my DNA plasmid.
So, I’ll start with my first generation of flies. These flies have the following genotype:
Note: Fruit flies have four pairs of chromosomes- three autosomal pairs, and one X/Y pair. While reading Drosophila genotypes, the main thing to keep in mind is that the semicolons separate the different chromosomes. For example, the above genotype indicates that “w,” an eye color gene, is on the first chromosome, and that SP-1 and CurlyO are both on the second. The “plus” signs in the male genotype represent wild-type chromosomes, or chromosomes with normal genes, and the“W-“ (white mutant) produces white eye color (a.k.a. no eye color). Sp-1 and CurlyO are both homozygous lethal mutations – this means that any fly that has two copies of either gene will not survive. Any of the offspring of this parent generation of flies is that inherits a genotype of CurlyO/CurlyO, or Sp-1/Sp-1 will die in their larval stage. Using a Punnett Square, it can be determined that roughly ½ of the offspring will have either one of these genotypes, so only ½ of the total offspring will survive. The good news though, is that 100% of these offspring will have the CurlyO chromosome that produces Piggybac transposase, so all of our flies will express Piggybac transposase.
So I cross these flies (mate them), and wait for them to reproduce. Then, I will screen the progeny, or the offspring, of the flies I crossed, in order to select only the flies that have colored eyes (denoted by w+ genotype). These flies should also have the Piggybac transposase. Since the “w+” genes will be floating around in the “w-“ background, there will only be a fraction of the progeny that have the “w+” genes; only a subset of germ cells will actually take up the DNA we injected.
Now tubulin, whose promoter drives the expression of Piggybac, is a protein that is expressed in all cells, including eye and germ cells. Tubulin (along with the Piggybac transposase) will continue to move eye color genes around all over the fly, so the eye becomes mosaic colored, due to the constantly shifting “w+” gene.
Once these flies are chosen, I have to do a second cross. I cross above flies with males of the following genotype: (W-/Y;+/+;+/+)
Out of the progeny of this cross, I can identify which flies have CurlyO because those flies will also have mosaic colored eyes from the Piggybac transposase. (CurlyO produces tubulin which drives the expression of Piggybac, so the two will always be together). If I select the flies from the progeny that do not have CurlyO, then there is a chance that they will have solid eye color. This solid colored fraction of flies will have “w+” genes amidst the “w-“ background, and they will have no Piggybac without the CurlyO.
Now that I have these solid eye-colored flies, I will conduct more crosses using “balancer chromosomes” in order to find out which chromosome my Piggybac element actually landed on. Put simply, balancer chromosomes are genetic tools used to maintain heterozygous populations. For example, the Tm3 chromosome (a 3rd chromosome balancer that produces stubble hairs), is a balancer chromosome-any fly with two Tm3 chromosomes would not survive, since homozygous Tm3 is lethal. I could probably spend hours trying to explain more about how balancer chromosomes work and how useful they are; for anyone interested, I would suggest this article for a basic overview, and this book (starting on chapter 1 page 12) for a more detailed description. The reason I am using balancer chromosomes is to stabilize the fly populations I have created, so that I do not lose my injected Piggybac. Since my flies are heterozygous, it is possible that a portion of their offspring will be homozygous. Balancer chromosomes with homozygous lethal mutations allow me to eliminate flies with homozygous balancers, since they will die in their larval stages. Hence the only flies that survive from generation to generation are the heterozygous flies that contain my Piggybac element.
Now to piece all these steps back into the larger scheme of things- these now stable populations are what I, and the rest of my class at Exeter, will use to locate the Piggybac element we initially injected. Ideally, most flies will have this element in a different location due to the random hopping of the Piggybac element, and we want to cover as many locations as possible! Like I had mentioned, if we are able to map exactly where our Piggybac elements land, and publish these molecular locations on Flybase, our lines can be used to study the functions of genes that our Piggybac elements disrupted, or to study cells that express these genes!
I hope that all of this made sense, and if any part of it was messy, feel free to ask me about it via the “Questions?” tab at the top of the page!