1 Biology 423L Oct 6 7 and Oct 13 14 and other days in those two weeks C elegans genetics and RNAi Report due Oct 27 28 Reading C elegans Hartwell Chapter 8 pp 255 256 Epigenetics Hartwell Chapter 18 pp 665 668 726 728 Verdel et al 2009 Int J Dev Biol 53 245 257 Eymery et al 2009 Int J Dev Biol 53 259 268 the papers above are available on course web site Objectives Become familiar with C elegans development Discriminate between wild type and mutant worms Learn how to interpret mutant analysis Perform a genetic cross with C elegans and identify dominant vs recessive mutations Use RNAi to phenocopy genetic mutations in C elegans Materials T A Prepare in advance 4 tubes containing 2ml of LB 50 g ml ampicillin 12 g ml tetracycline to grow cultures of RNAi bacteria strains described below Liquid cultures of OP50 bacteria used for regular worm culturing are kept at 4 C check with Ahmed lab to get bacterial strains and to get C elegans strains They also have a plate pourer machine to help with making NGM and RNAi plates Recipe for 1 litre of LB 10 g Tryptone 5 g Yeast extract 5g NaCl Add water to 1 liter and autoclave NGM plates Need 1 glue plate per group For mating 2 plates for parental cross 4 for F1s 8 for F2s all these will be seeded with OP50 bacteria RNAi plates 1 seeded with each type of bacteria for RNAi including a plate of vector only control Recipe for NGM plates 1 liter should make 200 35mm plates 17 g agar 2 5 g bacto peptone 3g NaCl 1 ml Cholesterol 5mg ml in EtOH Water to 1 L Autoclave cool down and add sterile CaCl2 to 1mM 2 MgSO4 to 1mM Potassium phosphate buffer pH 6 0 to 25mM For RNAi plates also add ampicillin to 50 g ml tetracycline to 12 g ml IPTG to 1mM make IPTG fresh from powder stored at 20 C Picks for everyone made from short Pasteur pipettes and platinum wire TA At least 1 week before first lab day Pour NGM and RNAi plates and let them solidify at room temp overnight You can use the Ahmed lab plate pourer machine Seed glue plates with OP50 bacteria the next day TA Two days before the lab day Streak RNAi bacteria as described below from frozen stocks on LB amp tet plates and grow overnight at 37 C Be sure C elegans strains will be ready TA Day before first lab Set up liquid cultures containing LB amp tet as described above from single colonies of RNAi bacterial strains and grow for 6 8 hours shaking at 37 C Spread the appropriate bacteria on RNAi plates and OP50 on NGM plates Use 50 100 l per plate The plates then need to dry for one day We will have a strain with the RNAi vector without an insert as a negative control and three strains to block gene expression 1 Dpy 2 Prz 3 Embryonic lethal pop1 4 Empty vector Each group needs 1 plate with each type of bacteria Each group needs at least 1 extra plate with no bacteria Each group needs a glue plate This is a NGM agar plate seeded with OP50 bacteria one week before For crosses Day 1 Need 2 plates per group seeded with OP50 bacteria Day 3 Need 4 plates per group seeded with OP50 bacteria Day 8 Need 8 plates per group seeded with OP50 bacteria C elegans strains Need wild type hermaphrodites staged to L4 for day 1 Get help from Ahmed lab Need double mutant dpy17 unc32 hermaphrodite and wild type males all staged at L4 for crosses on day 1 Note for 07 we can use very strong alleles of two unlinked mutations Ask Shawn Ahmed for help with the choice Need mutants of dpy prz and wild type for day 8 to compare phenotypes For RNAi experiments use a mutant C elegans in which the DNA dependent DNA polymerase is deficient This makes worms more sensitive to RNAi processing 3 Materials for class Wild type C elegans hermaphrodites dpy17 unc32 mutant hermaphrodites and wild types males staged to L4 If possible separate wild type males out for the students 10 per plate and one plate per group Be sure the correct genotype of worms is used for RNAi experiments Mutant strains to demonstrate phenotypes expected for loss of function of RNAi target genes RNAi plates with bacterial strains expressing dsRNAs for Dpy Prz and Embryonic lethal phenotypes 4 plates each per group and vector 4 plates per group Worm picks platinum wire and Pasteur pipets Bunsen burner needed to make picks Dissecting microscopes with base illumination Sterile tips 15 C incubator 20 C incubator alcohol lamps matches Method Students start here All manipulations have to be done sterily Sterilize your pick by burning the wire in a flame until it glows red Let it cool This should only take a few seconds All plates should be properly labeled with the worm genotype the date and your initials Day 1 2h RNAi 1 Collect worms Use the sterile metal pick to transfer worms from the plates provided to a fresh NGM plate OP50 bacteria Touch the worm pick to the glue plate and gently scrape it along the bacteria This will pick up some sticky bacteria Then touch the pick onto a worm gently The worm should stick to the pick Transfer the worm to the fresh plate Check to see if it is still moving 2 Transfer 2 worms P0 onto each fresh RNAi plate Choose animals in the last larval stage L4 L4 s can be recognized by a white semi circle near the center of the worm lengthwise which is the developing vulva The TAs will help you to recognize these worms 3 Incubate the plates at 15 C for three days We will need to use the incubator in Fordham Hall 4 Crosses 1 Take a plate seeded with OP50 bacteria on which the assistant has transferred 10 male wild type C elegans You may try to prepare this yourself if the assistant agrees Transfer 2 dpy17 unc32 hermaphrodites onto the plate Incubate at 20 C Day 4 30 min Friday for both groups if possible If necessary we will make a scheduled time on Saturday for the Wednesday lab RNAi The plates will be brought to the lab room at a time agreed on by the group Use the pick to remove the 2 adults you originally put on Burn them in your alcohol lamp flame to dispose of them Examine the small worms progeny for viability and movement Put the plates back at 15 C Crosses Pick one L4 hermaphrodite smaller than the adults with white patch from the F1 progeny on the original P0 crosses plate to a fresh NGM plate seeded with OP50 bacteria Prepare 3 or 4 plates with one F1 per plate C elegans hermaphrodites can fertilize themselves Incubate at 20 C Day 8 RNAi Examine the worms for phenotypes and compare them to the genetic mutants provided …