Question

RNAi is used to functionally inactivate genes in cells and whole organisms like C. elegans. Describe the basics of how you would knock down the expression of a gene required for muscle formation in C. elegans and what method could you use to confirm that your results were specifically attributed to the RNAi?

Answer 1

RNA interference (RNAi) is a commonly used technique for reverse genetic approaches in Caenorhabditis elegans. Feeding RNAi is the most convenient and inexpensive method for performing genome-wide RNAi screens. In Caenorhabditis elegans, many of the critical proteins involved in this process have been uncovered through mutational screens focusing on uncoordinated movement and embryonic arrest phenotypes. Caenorhabditis elegans is a valuable model organism for the study of muscle due to the similarity of worm body wall muscle to vertebrate muscle, along with its semi-transparent cuticle that allows for visualization of muscle structures in vivo. The basic protein components within a C. elegans sarcomere have vertebrate counterparts, with only minor differences in protein composition and organization

MATERIALS AND METHODS

Acquiring C. elegans strains

Wild-type C. elegans strains

Preparing E. coli OP50 food source

C. elegans utilizes E. coli OP50 as a food source when the E. coli is spread as a lawn on culture plates. A starter E. coli OP50 culture can also be obtained through CGC. A starter culture is prepared by aseptically transferring a single colony from the streak plate into 250ml sterile Luria Broth (lOg Bacto-tryptone, 5 g Bacto-yeast, 5g NaCl, H2O to 1- liter, pH to 7.0 using IM NaOH). The innoculated cultures grow overnight at 37°C after which the bacteria can be used to seed NGM plates. The liquid culture is stored at 4“C and is stable for several months

Preparing NGM petri plates

Standard Falcon 60mm petri plates are used to maintain C. elegans. Nematode Growth Medium (NGM) agar is prepared by mixing 3g NaCl, 17g agar, 2.5g peptone and 975ml H2O in a 2-liter flask which is autoclaved for 50 minutes. After the flask cools to 55°C in a water bath, 1ml IM CaCl2 ,1ml 5mg/ml cholesterol dissolved in ethanol, 1ml IM MgS0 4 and 25ml IM KP0 4 are added and mixed. Using sterile technique, warm NGM mixture is poured into the 60mm petri plates until they are 2/3 full. The plates sit for 2-3 days at room temperature before they are seeded to allow moisture to evaporate and to detect contamination

Seeding NGM Plates

A volume of 50 pi of OP50 bacterial culture is aseptically transferred to the center of an NMG plate on a flat surface. The bacterial lawn will grow overnight at room temperature. The seeded plates are stored on a countertop in a sealed air-tight container for 2-3 weeks

Maintaining Worm Cultures

The worms are maintained by transferring a chunk of NGM agar every three days to a new seeded bacterial plate. This is performed using a sterilized spatula to cut a 0.5cm x 0.5cm piece of agar with worms from an old plate and flipping the chunk over and placing it near the bacterial lawn of a fresh seeded plate. The worms will crawl out from under the chupk and feed on the new lawn

Preparing RNAi bacteria

The RNAi bacterial cultures are prepared in 5ml LB with 50ng/ml ampicillin or lOpg/ml tetracycline and 25pg/ml carbenicillin depending on the antibiotic resistance genes present in the plasmid (plasmid maps are provided by the donating lab). The cultures are grown overnight in a shaking incubator at 37°C.

Preparing RNAi/NGM plates

Standard NGM plates are prepared as described above with the addition of 25pg/ml carbenicillin and ImM IPTG to the NGM agar mix prior to pouring plates. The plates must be poured 4 days before being seeding with RNAi bacteria to allow time for them to dry, but covers should remain on during this time. If the plates are wet, the RNAi phenotype will not be as strong

Preparing C. elegans transfer tool

The transfer tools are prepared using Fisher brand 5 V* inch glass plpets. Platinum wire (99.95%), 0.05% iridium, 0.01-inch diameter, 30G can be purchased at Tritech Research (PT-9901

Transferring worms to RNAi plate

Using the transfer tool two or three L2 to L3 stage worms are moved to an unseeded NGM plate and given 30 minutes to allow the worms to wiggle off excess OP50 bacteria C. elegans prefer to eat OP50 bacteria to RNAi bacteria and excess OP50 will cause a weaker RNAi phenotype. Using the transfer tool, the RNAi bacteria are scooped up from an RNAi seeded plate. Using the transfer tool with the RNAi bacteria stuck to it, the worms are picked up from the unseeded plate. The worms are transferred onto the RNAi seeded plate. The plates should be kept in the dark on a countertop. The phenotypes can be scored 3 days later. The worms are staged using a standard staging reference

RESULTS

Genes that were knocked down in this experiment included bli-4, unc-22, rol-6, lon-2, dpy-5 and ama-1.