Vector and inserts:
Genomic fragments obtained by PCR were cloned into the Timmons and Fire feeding vector (L4440), which is a modified version of Bluescript with a T7 promoter on each side of the MCS driving transcription of each DNA strand (Nature, 395, 854). Information about the L4440 vector (including sequence information) can be found at http://www.ciwemb.edu/. PCR fragments were obtained using Research Genetics GenePairs. The GenePairs primer sequences are available at http://cmgm.stanford.edu/~kimlab/primers.12-22-99.html and are displayed visually in WormBase (http://www.wormbase.org/).
Genomic fragments cloned into L4440 were transformed into HT115 (DE3), an RNase III-deficient E. coli strain with IPTG-inducible T7 polymerase activity (Gene, 263, 103-112). The strain is available from the Caenorhabditis Genetics Center (http://www.cbs.umn.edu/CGC/). The genotype is as follows: F-, mcrA, mcrB, IN(rrnD-rrnE)1, lambda -, rnc14::Tn10(DE3 lysogen: lavUV5 promoter -T7 polymerase) (IPTG-inducible T7 polymerase) (RNase III minus). This strain grows on LB or 2xYT plates (and is resistant to tetracycline, see below), and competent cells can be made using standard techniques.
Note on tetracycline:
The Tn10 transposon interrupting the rnc14 gene carries a tetracycline resistance gene. Therefore, bacteria should be subjected to tetracycline selection (12.5 µg/ml) to maintain the RNase deficiency. However, the transposon is quite stable, as we have not lost it in the absence of selection. Using our protocol (see below and Kamath et al. Genome Biology, 2, 1-10) inclusion of tetracycline during feeding significantly decreased the RNAi effect for several genes tested, so we recommend that it not be used in culture or in NGM plates during feeding using the method below. However, using the method of Timmons, et al. (Gene, 263, 103-112), an improvement in feeding results by including tetracycline was reported.
For feeding plates, use standard NGM agar plus the following ingredients:
Carbenicillin to 25 µg/ml final concentration
IPTG to 1 mM final concentration
Plates are poured fresh 1-3 days before use.
Feeding Protocol (from Kamath et al. (2000) Genome Biology, 2, 1-10):
1. Pick and grow bacteria 6 hours - overnight (but no longer than 18 hours) in LB + 50 µg/ml ampicillin, seed onto NGM agar plates including additives (above). (Do not add IPTG or tetracycline to the liquid culture, as this will reduce the RNAi effect.). Bacterial cultures grown shorter times (6 hours) sometimes give better results. The lawn quality is improved if the culture is dried quickly by leaving the lids off for about 20 minutes after seeding, but we don't know if this affects the RNAi effect. We also have anecdotal evidence that plates poured at least 1 week before use work better than freshly poured plates.
2. Let dry and induce overnight at room temperature.
3. The following day, transfer an L4-stage hermaphrodite onto first plate, minimizing the amount of OP50 bacteria transferred (we usually wash worms in M9 buffer and then aliquot them directly onto plates). Leave 72 hours at 15°C (or 36-40 hours at 22°C) for RNAi to take effect, then replica plate adult onto another plate seeded with the same bacteria. After 24 hours, remove the adult from the replica and score the progeny for phenotypes. Alternatively, aliquot embryos or larvae onto the feeding plates and score them later.
4. Note on temperature:
We have observed that some genes give different phenotypes at 15°C vs 22°C; thus, it may be worthwhile to test a given gene using both conditions.
If you have any questions, please contact Julie Ahringer (email@example.com)