….and does it matter?
Yes, and no. On the one hand, if DMAP1 is essential, then that means important, right? Essential means you can’t live without it. On the other hand, if only our lethal potential DMAP1 mutants are really in DMAP1, implying the gene is essential, well then that’s a problem, because there is not much you can do with a dead fly. On the third hand, if the gene is lethal when completely knocked out, but has an interesting phenotype (like sterility) when partially knocked out, that might be the best of all possible worlds! When geneticists want to mutate a gene, they usually want A NUMBER of mutants, not just one. They want an ALLELIC SERIES – from weak hypomorphs (partial loss of function) to complete nulls (complete loss of function). And maybe a few weirdos like gains of function, dominant negatives or antimorphs.
But of course you have to want what you get, not get what you want. And we don’t know yet what we have with DMAP1. But there are some clues. We can use transgenic RNAi to tell us if DMAP1 might mutate to lethality. Huh?
Let’s start by taking a stab at the word transgenic. Most model organisms used for genetic analysis can be made transgenic these days. A text book definition usually describes a transgenic organism as one into which “foreign” genes have been inserted. And that is basically it – transgenic organisms are also widely used in agriculture. The specter of GMO frankenfoods includes transgenic soybean, corn, canola, cotton, rice etc. This is a huge, technical and emotional subject of course. I like this blog which is very eclectic and I have only really read the GMO part but it is very balanced.
Making a transgenic fly takes several months, and the genes that are inserted are not necessarily “foreign”, i.e., non-Drosophila genes. You can add additional copies of native Drosophila genes (endogenous genes). Or normal functioning copies of a mutated gene – gene therapy, if you like, for the fly (also called a rescue experiment). You can add a gene that includes all its own regulatory signals – a so-called genomic construct, so that if it lands in a conducive chromatin environment, it can be expressed just as it would in its native (endogenous) location. You can also target the gene you insert (let’s call it a transgene) – i.e., get it to integrate in a site in the genome of your choosing – using the same tools of homologous recombination the cell uses during prophase I of meiosis. There are lots of bells and whistles in this technique.
Here is a recipe for making a transgenic fly.
- Obtain a cDNA (DNA copy of a mRNA) of a gene of interest (let’s call it gene X).
- Cut and paste the gene X into a special DNA plasmid that carries signals that allow it to be integrated into a chromosome. This special plasmid is called a vector, because it will “carry” your gene of interest into the genome. The signals include a gene that expresses the red eye color as a reporter indicating successful integration (a wild type version of the white, or w+ gene), a promoter region upstream of gene X that can be induced in some way, and the inverted repeats of the P transposable element which in the presence of a transposase source, will make integration into DNA possible. The vector also contains bacterial sequences like antibiotic resistance markers and an origin of replication, because this part of the experiment, this cutting and pasting, is the cloning part, the recombinant DNA work, done largely in bacteria. The whole thing – vector plus insert – is often called a construct. It is a critical part of the experiment – you better sequence your construct multiple times to make sure there are no boo boos. Otherwise masses of time gets wasted down the line.
- Obtain a stock of white eyed flies (for example, w – mutant for the white gene on the X chromosome).
- Rear large numbers of young w females (mated with w males but get rid of them because they only take up space) and get them laying masses of eggs.
- Gently organize the eggs on double sided tape with their butt ends hanging over a bit.
- Mix your cloned construct with a “helper” plasmid (which produces transposase but cannot itself integrate) and load a very fine needle with the mix and inject the embryonic posteriors VERY CAREFULLY and VERY QUICKLY.
During the early development of Drosophila, the embryos are essentially a bag of nuclei (syncytium), no cell membranes. This means that the nuclei all share the same cytoplasm. There is some organization; the nuclei nearest to the posterior end of the embryo will become the germ line, so injecting the mix at this site increases the likelihood that the construct will integrate into DNA that will become the germline, and thus be stably inherited. The injection has to be relatively fast, however, because the embryos are alive of course, and will begin to cellularize, (the nuclei will become surrounded by cell membranes) after which integration of your construct is all but impossible.
- Transfer the injected embryos to vials with comfort food and wait. Those that survive this terrible insult will grow and hatch into adults – but if you are expecting to see red eyed flies indicating a successful germline transformation at this point, you will be disappointed. Some may have mottled red and white eyes, like Muller’s eversporting mutants, and that is a good thing, because it means that DNA has integrated into some nuclei, that have divided and multiplied and become incorporated into specific tissues, like the components of the compound eye. If you see this, you are observing a somatic mosaic, and you can then assume that if the soma (body) cells are mosaic for your construct, the germline probably is too.
- Mate your survivors to the original w stock and wait with great expectations for the next generation. Your survivors will produce gametes (sperm or eggs – usually easier to use males, so sperm) that may or may not have your construct – if a sperm from your survivor that HAS the construct fertilizes a w- egg, then hurrah! The progeny will be heterozygous for your construct in every cell!
- Use Ye Olde Fashionde Geneticse to map your construct to a particular chromosome and make the stock homozygous. You might have hit an essential gene, in which case the transgenic fly will carry a lethal – I usually chuck those.
That is the simple version. You can find the detailed protocol here – together with many beautiful images of different fly species…
So that is how you make a transgenic fly. It is the first step in a very commonly used scheme in the Drosophila world called the modularized miss-expression scheme – also known as the UAS-GAL4 method. I told you that the transgene is often induced to express in some way. It carries sequences that allow the experimenter to control when or where a particular transgene is expressed. Commonly, sequences from yeast are used – these ARE foreign DNA elements, tacked upstream of the gene of interest in the construct, called Upstream Activating Sequences – UAS – recognized specifically by the yeast GAL4 transcription factor. Yeast galactose metabolism is not happening as a rule natively in flies, thus if the experimenter can control when and where the GAL4 transcription factor is expressed, she can control when and where her transgene construct is expressed. See? The method is summarized in Figure 2A below.
The construct that responds to GAL4 because it has the UAS sequences is called, not surprisingly, the responder. When you want to express your gene X in a specific place or developmental stage, you would create the responder using the recipe described above. The construct that produces the GAL4 in some defined manner is called the driver. Yes, the driver is yet another transgenic fly that someone else has made. The main difference is that the construct was made by pasting a promoter region from a specific gene upstream of the yeast GAL4 transcription factor coding sequence. The promoter can come from anywhere. If you want to make an eye-specific driver – so you can drive and confine gene expression specifically in the eye (very useful, if you want to assess a mutant effect of an otherwise essential gene – flies can live with screwed up eyes) just tack on the eye-specific promoter region (taken from some gene you know is only expressed in the eye) to the GAL4 coding sequence.
There are hundreds of drivers available in stock centers nowadays. You don’t have to make your own. You just order them in. So if I want to drive expression of DMAP1 in, say, just the nervous system, I would make a UAS transgenic responder line that can be induced to express DMAP1, and then cross flies from that line to a nervous system-specific GAL4 driver line I would order from a stock center like Bloomington. Hey presto, the progeny from this cross would have both the driver AND responder, and would show…whatever the result would be. Here is an excellent review: PMID:12324939.
OK so we have covered transgenic flies, and how to control transgene expression. Now for RNAi. Oh heck let’s just let the Nobel prize winners earn their keep: http://www.nobelprize.org/nobel_prizes/medicine/laureates/2006/illpres/index.html
If you perused this nice slide show, you will now know that small double stranded RNA molecules are an evolutionarily conserved trigger for potent gene silencing. Who knew? The process of RNA interference, which is what RNAi stands for, is still revealing its elaborate secrets, and like anything that has evolved for millions of years, is…quirky. But the bottom line for experiments – that you can introduce into your model organism double stranded RNA molecules containing a sequence of a gene you wish to silence – has immense methodological power. Especially for organisms where classical genetics isn’t possible – no mutants, no way to access the DNA for mutagenesis. And there are implications for medicine too – silencing mutations that might be causing disease because of a GAIN of function mutation rather than a loss of function.
The fact is, RNAi is widely used in medicine and basic research, even while we are discovering how complex it is. Not all portions of a given coding sequence are equally potent at eliciting the silencing response. The knock down is not necessarily complete, which could be useful, if it were possible to predict accurately (which it often is not). There are other pathways in the cell that produce double stranded RNAs for developmental purposes (the micro RNA pathway). In mammals, long double stranded RNA molecules can bugger up the cell cycle and trigger cell death – a disappointing result if you are using a cell culture model. And so on.
But that shouldn’t stop us. How to make RNAi transgenic. If you have read this far, the solution should be easy. Make a transgenic responder line of flies that instead of expressing a gene of interest when induced by a driver, expresses instead a double stranded RNA molecule targeting a gene of interest (Figure 2B). There are a few ways of doing this: sewing a coding sequence in an inverted repeat manner, or putting promoters on either side of it…but it is quite possible. There are two main transgenic RNAi projects – one in Vienna, Austria, and the other in Harvard, USA. The Viennese project (I hear waltzes) does not target their RNAi responders, i.e., does not control where their RNAi responder transgenes integrate. This means any given responder might show position effects – that is, may variably upregulate genes in the vicinity of the insertion site (the double stranded RNA is being induced by GAL4 after all) or have landed in an inhospitable chromatin environment (making access to the UAS by GAL4 difficult). On the other hand, the Viennese project has A LOT of RNAi responders, so if you get the same results with multiple responders for the same gene, that is promising.
The other project – the Transgenic RNAi Project or TrIP in Harvard uses targeting technology to control for position effects. But the chosen sites themselves may have position effects, so it is all a compromise really.
Anyway, how is this relevant to DMAP1? Because we have no mutants for DMAP1, RNAi is the only way at present to assess whether or not the gene might mutate to lethality, therefore indicating that it is essential. In the preamble to the DMAP1 project, I described the results of a paper in which the biological effect of DMAP1 was assessed using RNAi. Also, in Flybase, data from a number of sources indicates that RNAi targeting DMAP1 causes lethality. But the P element we are using to make our DMAP1 mutants is really in the upstream gene, CG33785…for which there is very little information. It looks like an RNA polymerase subunit, specifically RNA pol III, which transcribes things like tRNAs, some rRNA and many of the burgeoning small RNA molecules complicating the story of RNA interference among other things (PMID: 21540878). There are other peculiarities about this gene – it is bicistronic (huh? For now, a weirdo gene structure which I seem condemned to study) and no one seems interested in it so who knows what it is actually doing.
I obtained RNAi responders for DMAP1 and CG33785/CG33786 (two gene number designations because of the bicistronic structure). They came from the Vienna center, because Harvard had none for these genes. Hard to believe it, but the point of this blog entry is right here: when using a ubiquitous driver (ACT5 GAL4) DMAP1 is lethal, and CG33785/6 is not. I only had one DMAP1 RNAi responder, but had THREE for CG33785/6. If I wanted to be sure, I would do QPCR (quantitative PCR) on pools of mRNA from all these experiments, to confirm gene knock down etc. Possibly, the three CG33785/6 RNAi stocks are non-functional (meaning the RNAi doesn’t work) and the one DMAP1 stock is lethal because of something in the background. But I am inclined to get what I want here. I am going to assume that DMAP1 can be mutated to lethality, whereas CG33785 – probably not. A summary of the results is shown below: the driver is balanced (marked with the dominant mutation CyO – more about balancers here) so if a particular driver/responder combination results in lethality (presumably because knock down of the gene in question is lethal) no NON balanced (CyO+) progeny will survive.
Which brings me back to my first point. Does essential mean important? DMAP1 – whatever it does – I am betting is not as ubiquitous a function as RNA polymerase III. And yet, loss of function in DMAP1 is probably lethal, while the cell can probably survive losing CG33785/6. Perhaps a better way to think of this is to take Mother Nature’s point of view. If something is important, REALLY important, required to get to the next generation, are you going to have only ONE copy of it?