Reverse chunking, a simple and effective method for identifying unc-119(+) transgenics

Biolistic transformation is currently a popular method for making trangenic lines in C. elegans. Although this method is reliable, the fraction of successfully transformed animals that is obtained is extremely small, necessitating the use of a co-transformation marker. The most commonly used marker is the unc-119(+) gene which can dominantly rescue the severe Unc and dauer defective phenotypes of unc-119(ed3) mutants. Despite the utility of the unc-119(+) marker, screening for rare transgenic animals amongst the progeny of bombarded animals is time consuming and labor intensive. One approach is to allow the plates containing the bombarded animals to starve over the course of a few weeks. Each plate is then divided into chunks and the chunks moved to fresh plates containing food. These plates can then be screened a day or so later for animals with normal mobility. This approach is still time consuming and has the added drawback of amplifying the number of plates (and therefore the surface area) that has to be scanned. I have devised a simple alternative to this approach in which the worms do most of the work. One to two weeks after bombardment, just after the plates have starved, a fresh plate containing OP50 is cut up into little (1-2 cm²) chunks. Each of these chunks is placed on one of the plates containing the bombarded animals so that the side containing the bacteria is face up (away from the worms). The plates are left upright in the incubator overnight and the next day the bacterial lawn on top of the transferred chuck is checked for rescued worms. Because the animals have to climb up to the top of the chuck to reach the food, the rescued animals have a significant advantage. Using this approach, my group has on multiple occasions, been able to identify a single transformed worm from a 100 mm plate. This approach has the added advantage of being rapid; rather than scouring the entire plate for transgenic worms, only the small area on top of these "pedestals of rescue" has to be searched.