Worm Breeder's Gazette 17(1): 32 (October 1, 2001)

These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

RNAi feeding to produce males

Darrell J. Killian, E. Jane Albert Hubbard

Department of Biology, New York University, 100 Washington Square East, 1009 Main Building, New York, NY 10003

Heat-shock has traditionally been used as a means to obtain males, but particular worm strains will not tolerate this process. This problem may be overcome through genetic means by introducing a him mutation but this involves additional steps and may complicate later experiments. In an effort to obtain males from strains that are not amenable to heat-shock, we turned to RNA mediated interference as an approach.

 

We made and tested a him-14 feeding construct (many thanks to Yuji Kohara for the him-14 cDNA and Lisa Timmons and Andy Fire for the L4440 vector) to see if males could be generated. Hermaphrodites that consume HT115(DE3) bacteria producing him-14 dsRNA consistently produce a low but significant number of males. Interestingly, the males appear among the progeny at the end of the brood. We typically see 5-7% males in the last 100 progeny from three pooled hermaphrodites at 20ºC. Our best results are obtained by feeding L4 hermaphrodites for about 50-55 hours and then transferring these worms to a fresh RNAi plate and inspecting the progeny from the end of the brood for males. We did not observe an increase in the percentage of males if the RNAi feeding was carried over additional generations. To test if the effect persists once worms are removed from the dsRNA-producing bacteria, hermaphrodite siblings of RNAi-induced males were transferred to OP50 bacterial lawns. These animals threw fewer than 1% males (from a pooled total of 12 bulk-inspected hermaphrodite broods) in the first generation and none in the subsequent generation. We have also confirmed that males produced by him-14 RNAi can sire cross progeny. Finally, we did not see evidence of embryonic or larval lethality.

 

We stress that feeding him-14 dsRNA to produce males is effective and reversible, but not efficient. We have not successfully obtained sufficient numbers of males from strains that are somewhat unhealthy or that have a reduced brood size. Since males appear at the end of a normal brood, low brood size may hinder the production of males by this approach. Furthermore, we found that the bacteria used to produce males must be freshly grown from a healthy plate colony and used immediately. Ultimately, other genes may be more useful for this purpose. Indeed, large scale RNAi screens have identified many genes that give Him phenotypes when targeted by RNAi (e.g., Fraser et al., 2000).

 

Theresa Stiernagle kindly agreed to distribute GC363, the HT115 (DE3) bacterial strain carrying pGC8 [him-14 partial cDNA in the Fire L4440 vector]. Many thanks to David Greenstein for discussions that led to this experiment. These results were also described in a March, 2001 note to the C. elegans newsgroup.