Tools of the trade

Imagine trying to get to the lab first thing on a Monday morning without your alarm clock and hot shower. It is all too easy to take the technical achievements around us for granted, although we would quickly be stopped in our tracks without them. The same is true in research: perhaps only those who have been in the field long enough can truly appreciate the importance of methods that overcame technical hurdles. In developmental biology, one of the most significant examples has been the ability to manipulate gene expression in the whole animal.

By the early 1980s, it was possible to alter expression of specific genes in mammals by microinjecting DNA into eggs to create transgenic mice. Rubin and Spradling, who were working on the fruitfly Drosophila melanogaster, decided to take a Trojan-horse approach to the same problem. They took a P element — a natural transposon vector that is known to jump in and out of the fly genome at will — and asked whether it could be used as a vehicle to deliver genes of interest into the genome. In 1982, they showed that this approach did indeed work: a P element containing the rosy eye colour gene could permanently restore normal eye colour in embryos lacking this gene. The main advantage over previous microinjection techniques was that integration of the P element occurred efficiently and was stably inherited by progeny. This basic idea of using P elements as a gateway into the Drosophila genome formed the foundation of most gene manipulation techniques that have since been developed in the fly: this includes the development in the 1990s of the FLP-FRT system of mosaic clone analysis by Golic, and Xu and Rubin, and the Gal4 system by Brand and Perrimon — two key techniques for disrupting gene expression in a small subset of cells, and for expressing genes ectopically in your cells of choice.

	It was the single discovery that … made Drosophila an organism that could take advantage of advances in recombinant DNA technology and apply them to classical genetics and embryology   recalls Matthew Freeman

Meanwhile, in the mouse, Capecchi and Smithies were both busy trying to tackle the challenge of engineering specific mutations into any gene. The approach they took was to take advantage of the homologous recombination machinery in cells and to make vectors to target a gene of interest. In 1986, Smithies rescued a mutation in the human beta-globin locus, and in 1987, Capecchi succeeded in showing that the Hprt (hypoxanthine phosphoribosyl transferase) gene could be knocked out in embryonic stem (ES) cells. This revolutionary technique had the potential to be used to introduce any type of mutation in every gene and for the mutant cells to then be stably propagated. Mutant ES cells could, in turn, be used to generate germ-line chimaeras, thereby allowing targeted knockouts or insertions, which could alter the expression of particular genes in the whole animal.

One challenge in making mouse knockouts even today is the time they can take to produce. It was a method initially observed in plants and then developed in the worm — RNA interference — that now provides the most rapid means to inhibit gene expression in mammals. In 1998, Fire and Mello were investigating the potency of different antisense RNAs to suppress gene expression, and inadvertently discovered that double-stranded RNAs were far more effective. Reminscent of dsRNA viruses that suppress gene expression in plants, this technique required only a few molecules of dsRNA and was inherited non-stoichastically. At the time, they speculated that the underlying mechanism might entail a catalytic amplification process.

Over the past 6 years, we have learnt that RNA interference works by hijacking a molecular pathway present from flies to mammals. Its success has now spread to mammals, following the realization that chopping the RNAs into smaller fragments (small interfering RNAs or siRNAs) largely prevents an unwanted interferon response. Although it remains imperative that care be taken to demonstrate specificity in siRNA experiments, it is proving to be the most rapid and universal technique for inhibiting gene expression. In addition, it is now becoming clear that another regulatory mechanism that also involves small RNAs — the microRNA pathway — affords an additional level of control over gene expression used by the organism itself, although the breadth of its functions during development is only now being realized.