Breaking flies

By Peter Dearden 08/10/2010 2

SM Morgan

I am at the point in my PhD studies where I am ordering pre-made mutant lines of flies from the US to test out some of the results I found early in my research. It is amazing that we can do this — and for so remarkably little expense. There are vast warehouses in the States and elsewhere that hold rows upon rows and shelves galore of different fly mutant lines — populations of flies carrying a mutation in a single gene. This makes things easy for us in that we can completely skip the mutant generation steps of the experiment and simply order online the mutants we require.

This is all well and good — but I am very much a ‘learn by doing’ type of person and my undergraduate studies appear further away every day.

So — a recap. Drosophila mutant generation and identification 101.

The website we order from provides a search box where you can simply type in the name or symbol of the gene in which you want a mutant line, and a page pops up with every line available with that gene affected. The mutants are all listed with delightful codes and links, and appear at first glance to be some sort of mystic language.

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Figure 1. Every option for mutant lines in this particular gene. Different ‘shops’, order numbers, and genome description. Figure adapted from FlyBase.

The particular mutant I have ordered for this gene has an entry of its own which shows us its geneotype — with each element a link which directs us to further information about that part.

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Figure 2. Partial Stock Centre Record for mutant Drosophila line 18166. Adapted from FlyBase.

The line ‘FlyBase Geneotype’ tells us all we need to know about that line. The first code, ‘w1118’ tells us that the entire line carries the white eyed marker — this is so that you can tell the flies that are carrying the specific gene mutation (with pink to red eyes) from those that are wildtype, or have no extra mutation in their genome (white eyes). The white eyes are caused by a naturally occurring mutation in the white gene, which is responsible for production and distribution of normal pigment in the eye. In mutant flies like this one, the transposon carries a mini-white gene which partially rescues eye colour – only in flies with that transposon inserted into the genome. Fully wild type flies have red eyes and the white eyed gene mutant was one of the first identified when people started using the fruit fly in genetic studies.

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Figure 3. Variation in eye colour — the eye at the bottom is a complete knockout of the white gene. By ArrogantScientist .

So: completely red eyed flies (wild type) should not be in my pots at all, since the entire mutant line is in the white eyed background. White eyed flies could be present but will not carry my special gene mutation. Pink/redish eyed flies will have my gene of interest knocked out. Easy! I can see their eye colour without aid of a microscope.

The second code, ‘PBac{RB}Ect4 e03540’, means firstly that the mutant was created with a transposable element ‘PBac[RB]’. A transposable element, or transposon, is a small section of DNA which is capable of moving around the genome — copying itself out of the sequence and then inserting itself in again at another region. PBac{RB} is a synthetic transposon made up from piggyBac (a transposon originally found in moths), a small piece of the white gene, a FRT site, and a splice acceptor site.

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Figure 4. piggyBac transposon. Figure adapted from Thibault et al 2004.

The only part of this that we need to understand right now is that the small piece of white gene (mini-white) is enough to restore partial function of the protein and cause pigmentation of the eyes — from white to yellow or even pale pink/red. As discussed above this eye colour is an easy marker for picking out flies containing the gene mutation. The {RB} in the code is simply an identifier for that version of synthetic piggyBac transposon. It does not matter where in the genome the piggyBac jumps in — the mini-white gene will work to restore eye pigmentation no matter what.

As an aside, the FRT site is used to make deletion mutants — if two transposons are inserted in the genome relatively close to one another the two FRT sites can loop together and the fragment of genome in between is cut out. It was initially a segment of a yeast plasmid, and combined with Flp recombinase (an enzyme which facilitates this looping process) is capable of causing deletion mutants. Deletions mutants are when the gene is cut out — not just interrupted.

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Figure 5. Diagram of FRT site recombination adapted from Parks et al, 2004.

The next segment of the code, Ect4e03540, tells us where in the genome the transposon (the piggyBac) has inserted itself. The original screen for mutants in Drosophila involved shooting this wee piece of DNA throughout the genome and then growing up fly lines in which one transposon was present in one gene — so you end up with a library of flies, each with a different gene knocked out. When I say ‘knocked out’ I mean normal function of the gene is stopped completely, or inhibited. A big piece of nonsense in the middle of a gene is usually enough to halt its normal function.

The code, e03540, links us to an entry in the online databases, and shows us exactly where this transposon is.

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Figure 6. Section of the genome, showing the insertion location of piggyBac in this particular example highlighted yellow. Figure adapted from FlyBase.

The numbers under the hash-crossed line at the top tell us where on the chromosome this is — in number of bases. The ‘3L’ above tells us this is on Chromosome 3, on the L arm. You can see the chromosomes in Drosophila in Figure 7, and locate 3L. As you can see in Figure 6, there are many insertions in the gene Ect4, each represented by a blue triangle. The gene itself is represented by the longer blue bar under the chromosome location line at the top.

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Figure 7. By Steven J. Baskauf.

The second-to-last section of the code, /TM6B, tells us that this mutant line is over the balancer chromosome TM6B. Balancer chromosomes are used when two copies of the mutant gene are lethal — the balancer chromosome keeps the mutant copy in the line of flies, and keeps it single copy only. A balancer chromosome is the same as the chromosome it ‘balances’ but has had big chunks of itself turned over or deleted. This stops recombination; a mixing and swapping of genes that occurs when gametes are being made — so that the new generation have a different set of genes and a greater chance at survival should a threat emerge that favours one genetic code over another.

You can see the available balancers in Figure 8, the F, S and T in the codes stand for chromosome the First, Second and Third. (Original right?!) The phenotypic markers in the third column are the physical characteristics we can look out for to identify the flies carrying the balancer. There is no balancer chromosome for the fourth chromosome since it is so small and considered to undergo no recombination anyway.

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Figure 8. Drosophila balancer chromosomes. From HoxfulMonsters.

So no mixing and swapping keeps the mutant gene mutant, and then a marker on the balancer keeps it in the line. This involves a bit of effort — you pick out the flies carrying that marker so that they can then breed — you kill any flies that have dropped/lost the balancer chromosome. This ‘marker’ is similar to the white eye/red eye situation — an easily visible phenotype, and is represented in the last section of the code — in this case, Tb1.

The marker on a balancer is always dominant — only one copy is needed to be able to see it manifest as a phenotype. In this particular line, the marker is Tubby — the larvae carrying the balancer chromosome (and thus, the marker) are shorter and fatter than their balancer-less compatriots. Thus you can easily pick out the larvae you want to work with. The balancer chromosomes also contain recessive mutations, which are lethal if there are two copies of them. So you can only have one balancer present in a genome, and you can tell if it is there by looking at the shape of the larvae.

In the end: you have a mutant line of flies that which you can tell carry the balancer by their shape, and which carry the mutation by the colour of their eyes.

Brilliant. Now you can start some experiments!

2 Responses to “Breaking flies”

  • Hey Morgan,

    what a wonderful explanation, you have given over here. may i have your email id please to have some technical suggestion like when we got knockout, how can we confirm the gene deletion has been done and thats for sure and secondly, how much part of the target sequence has been deleted.

    thanking in anticipation for your cooperation

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