C. elegans has been used to the teach fundamentals of genetic mapping for the last three years in the Neurobiology summer course at the Marine Biological Laboratories (MBL). The nine week course is primarily aimed at graduate students and postdoctoral fellows. The molecular section of the course I help teach lasts three weeks including lectures in the morning and lab work in the afternoons and evenings. Two students work with each of the six faculty members in the molecular section of the course. The two students assigned to me spend about 120 hours each working in the lab spread out over a three week period. Consistently, the students have had no experience in C. elegans and, often, little or no prior experience in molecular biology. Given the short duration the molecular section, and the desire to accomplish something of substance, the project has to be chosen with care. The short life cycle of C. elegans makes it an ideal organism to teach the fundamentals of genetic mapping in a limited amount of time. The project chosen last year was recombinant mapping of an interesting mutation,
rt70 , using single nucleotide polymorphisms (SNPs) from the Hawaiian strain, CB4856.
rt70 causes ASH neuron degeneration and is described in an abstract/poster by Emily Bates, et al. Prior to the start of the course last year, a
lon-2(
e678)
rt70 egl-15(
n484) chromosome was generated. All mutations are recessive. Heterozygous
lon-2 rt70 egl-15 / CB4856 animals were generated and picked one per plate. These heterozygous animals were transported to MBL along with hundreds of seeded plates. The students practiced picking C. elegans , then singled animals carrying recombinant chromosomes/
lon-2 rt70 egl-15 for the first few days (putative recombinant lines). We attempted to generate homozygous recombinant lines by picking single 9 animals in the next generation and were successful about 80%of the time; the remaining 20% were repicked for analysis at a later date. The homozygous recombinant lines were 1) frozen in a 96 well format using Michael Koelle's protocol and 2) allowed to starve and lysed for PCR analysis using previously defined SNPs identified by Stephen Wicks. To facilitate analysis, only previously characterized SNPs creating a restriction site polymorphism were utilized. Screening more than 100 lines by PCR was not a problem and we were able to refine the
rt70 map position. The biggest difficulty we had was scoring
egl-15(
e484) . In retrospect, I should have chosen marker mutations with very obvious phenotypes so they could be scored unambiguously and easily. PCR reactions were optimized while we were waiting for the recombinant strains to grow. The MBL students also had time to learn how to generate transgenic C. elegans by microinjection. PCR machines for the course were generously loaned by MJ Research, Taq was kindly provided by Fisher and NEB generously donated restriction enzymes. Thanks to all who donated their time, reagents and help- especially MBL students Michael Long and Ryohei Yasuda for their unflagging enthusiasm!