feeding for males

Jane Hubbard jane.hubbard at nyu.edu
Tue Mar 13 18:36:25 EST 2001


Dear colleagues,
In an effort to obtain males from strains that are not amenable to 
heat-shock, we fed him-14dsRNA to N2 hermaphrodites (many thanks to 
Yuji Kohara for the him-14 cDNA). We thought this reagent might be 
useful to others in the community, so here is a summary of our 
results. We will put more details of our studies in the next WBG 
(fall, 2001). The short answer is that males are produced by this 
method, though not abundantly. Interestingly, males appear 
consistently at the end of an hermaphrodite brood. At 20 degrees, we 
see 5-7% males among the last 100 or so total progeny from 3 pooled 
hermaphrodites. Therefore, to feed for males, we suggest feeding L4 
hermaphrodites for about 50-55 hours, then transferring them to a 
fresh RNAi plate and inspecting the progeny from the end of the brood 
for males.

We performed several additional tests with these animals. To see if 
the effect can be enhanced by another generation of feeding, 
hermaphrodites were taken from male-producing plates and fed 
him-14dsRNA for another generation. We got the same results: low but 
significant numbers of males appearing at the end of the brood. To 
test if the effect persists once worms are removed from the 
him-14dsRNA, hermaphrodites from plates that produced males were 
transferred to OP50. These animals threw males at less than 1% (total 
of 4 males from bulk-inspected progeny of 12 individual 
hermaphrodites). Hermaphrodites from the OP50 plates that produced 
these 4 males were followed another generation on OP50 and produced 
no males (progeny of 12 hermphrodites inspected). Males produced by 
him-14dsRNA can sire cross progeny as tested by crosses to 
unc-13(e51) hermaphrodites. Finally, we did not see evidence of 
embryonic or larval lethality.

Theresa Stiernagle has kindly agreed to distribute the HT115(DE3) 
bacterial strain carrying pGC8 [him-14 partial cDNA in the Timmons 
and Fire L4440 "double T7" vector]. Also, many thanks to David 
Greenstein for discussions that led to this experiment.

Darrell Killian (djk232 at nyu.edu)
Jane Hubbard
--
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
E. Jane Albert Hubbard
Department of Biology
New York University
1009 Main Building
100 Washington Square East
New York, NY 10003-6688
office: 212-998-8293
lab: 212-998-8274
fax: 212-995-4015
e-mail: jane.hubbard at nyu.edu
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