RNAi Vector feeding protocol for distal tip cell (DTC) migration studies in C. Elegans
Lab/Group: Schwarzbauer Laboratory/CMC Discovery Initiative
Introduction
RNAi is an effective method for analyzing gene function in C. elegans that often phenocopies loss-of-function phenotypes 1. In RNAi, double stranded RNA (dsRNA) introduced into larvae or adults activates an enzymatic pathway that eliminates endogenous RNAs homologous to the dsRNA 2. Potent and persistent RNAi silencing in C. elegans results from secondary amplification of small amounts of the initial RNAi trigger by RNA dependent RNA polymerases 3, 4. RNAi can be induced in C. elegans using one of four methods: injecting in vitro synthesized dsRNA into the body cavity of the animal (injection RNAi), soaking in a solution of dsRNA (soaking RNAi), feeding animals bacteria engineered to express dsRNA (feeding RNAi), or through creation of transgenic animals that express dsRNA from DNA arrays maintained within cells (hairpin RNAi)5, 6. This protocol is slightly modified from 7, and can be used for candidate or broad RNAi screens in C. elegans 8, 9.
Materials
Reagents
NGM Plates: NaCl, peptone, agar, MgS04, CaCl2, cholesterol, K3PO4 (see recipe 10) + IPTG and carbinicillin
LB and ampicillin
Agarose and Glass slides
Lots of Petri Dishes
22×50mm glass coverslips
Equipment
4 to 6X (x10) dissecting scope, 20-40X (x10) light microscope with Nomarski optics. Autoclave, shaker, incubators (37°C, 23°C), microwave.
Procedure
Day 1.
Streak out culture from desired library well on LB/Amp plate (40 µg amp/ml) (optional).
Day 2.
1. Culture bacteria containing each RNAi clone in 1 ml LB medium containing 40 µg/ml ampicillin overnight (Table 1 shows vectors used in this particular study – link to RNAi primer info file).
2. Chunk a starved nematode plate and allow starved nematodes to recover on fresh E.coli OP50-seeded NGM plates for two days (until Day 4 of experiment).
Day 3.
In the 24 well format, 20 µl of each culture was spotted in a single well of a 24-well plate containing NGM agar, 6 mM IPTG and 25 µg/ml carbenicillin. In the 6 cm format, spread out 150µl of culture. I usually make duplicate plates, so I have a plate ready to score a second generation.
Day 4.
Release eggs from gravid hermaphrodites on the plates chunked on Day 2 using alkaline hypochlorite solution. Following washes in M9 buffer, transfer eggs to plates seeded with E. coli HT115(DE3) bacteria expressing double-stranded RNA (dsRNA) (Seeded Day 3) and incubated at 23°C. (For detailed protocol, see Hope (1999))
Day 5.
Take the day off.
Day 6.
1. Analyze animals with light microscopy.
Primary screen: Score the animals for clear patches using a dissecting microscopy. This is approximately 48h after hatching; animals should be young adults.
Secondary screen: Mount the animals on agar pads using 0.08M sodium azide as an anesthetic. View DTC migration using 40X Nomarski microscopy (see figure below - RNAi scheme).
2. Transfer 5 young adults to new plates seeded with RNAi bacteria.
Day 7.
Take the day off.
Day 8.
Analyze young adult progeny animals as described for Day 6. If the worms aren’t old enough (likely), analyze them on Day 9.
Troubleshooting
Critical Steps
Anticipated Results
References
1.Fire, A., et al., Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature, 1998. 391(6669): p. 806-811.PMID: 9486653
2.Grishok, A., RNAi mechanisms in Caenorhabditis elegans. FEBS Lett, 2005. 579(26): p. 5932-5939.PMID: 16162338
3.Pak, J. and A. Fire, Distinct populations of primary and secondary effectors during RNAi in C. elegans. Science, 2007. 315(5809): p. 241-244.PMID: 17124291
4.Sijen, T., et al., Secondary siRNAs result from unprimed RNA synthesis and form a distinct class. Science, 2007. 315(5809): p. 244-247.PMID: 17158288
5.Hull, D. and L. Timmons, Methods for delivery of double-stranded RNA into Caenorhabditis elegans. Methods Mol Biol, 2004. 265: p. 23-58.PMID: 15103067
6.Timmons, L., Delivery methods for RNA interference in C. elegans. Methods Mol Biol, 2006. 351: p. 119-125.PMID: 16988430
7.Kamath, R.S. and J. Ahringer, Genome-wide RNAi screening in Caenorhabditis elegans. Methods, 2003. 30(4): p. 313-321.PMID: 12828945
8.Cram, E.J., H. Shang, and J.E. Schwarzbauer, A systematic RNA interference screen reveals a cell migration gene network in C. elegans. J Cell Sci, 2006. 119(Pt 23): p. 4811-4818.PMID: 17090602
9.Cram, E.J., S.G. Clark, and J.E. Schwarzbauer, Talin loss-of-function uncovers roles in cell contractility and migration in C. elegans. J Cell Sci, 2003. 116(Pt 19): p. 3871-3878.PMID: 12915588
10.Hope, I.A., ed. C. elegans, A Practical Approach. The Practical Approach Series, ed. B.D. Hames. 1999, Oxford University Press: Oxford
Acknowledgements
