08 / 09 - Evangelische Kirchen in Erfurt

1284 R. M. Alcazar, R. Lin and A. Z. FireFigure 9.—Explicit test of male transmission ofsilencing. (A) Males during copulation transmitboth sperm and seminal fluid. The male-derivedsilencing efficacy can be explained by at least twomodels: (1) The silencing factor is inside thesperm and (2) the silencing factor is transmittedthrough the male in the seminal fluid. (B) Schematicof crosses designed to test the male transmissionof silencing through the sperm or theseminal fluid. It was critical for this experimentthat we identify self-progeny animals that hadbeen fertilized after their parent hermaphroditehad received male sperm and seminal fluid. Toensure this, we transferred the parent hermaphroditeseach day and scored only self-progeny thatderive from mothers that had previously producedcross-progeny. Operationally, this was carriedout by mating individual F 1 male silencingcarriers with five naive hermaphrodites for 6–12hr, transferring the hermaphrodites to individualfresh plates to allow egg laying for 1 day (firstbrood), and transferring hermaphrodite mothersto a second plate for an additional day (secondbrood). Of 50 mated hermaphrodites, 6 metthe criterion that they had some cross-progenyon the first day of transfer and some self-progenyon the second day of transfer. The self-progenybroods on these six plates from the second transferconsist of self- and cross-progeny that were fertilizedsubsequent to the transfer of sperm andseminal fluid from males to the mother hermaphrodite.We then compare silencing transmissionto self-progeny and cross-progeny from thesebroods. The boxes summarize the viability of F 3and F 4 cross- and self-progeny from first and secondtransfers at 25°. The data show that carriermales transfer the silencing trait to cross-progenyand not to self-progeny. This is consistent with asignal intrinsic to sperm and not one carried inthe seminal fluid. The asterisk indicates that a singleviable F 3 larva was produced from 1 of the 36F 2 animals in this experiment; this animal yieldedno F 4 progeny and may have represented a rare‘‘spontaneous rescue’’ affecting 1in10 4 progenyof oma-1(zu405) mothers.DISCUSSIONWe found that a single dose of dsRNA targeting thegermline-active gene oma-1 can lead to silencing thatlasts multiple generations. Heritable silencing wasinitiated by the RNAi response, with a biphasic timecourse involving nearly complete but temporary suppressionof the target locus lasting three to fourgenerations, followed by a much lower frequency oflong-term silencing. Heritable silencing frequency andpersistence was dependent on the dose of inductiontrigger. When the trigger was limited, the silencingefficacy was preferentially distributed in early bornprogeny. Both sperm and oocytes were capable of transmittingthe silencing signal to descendant populations.Surprisingly, the frequency of silencing after spermtransmission appeared to be higher than for oocytetransmission. The silencing achieved by sperm transmissioninvolved a signal intrinsic to sperm.Multigenerational silencing after RNAi treatment:Earlier analysis of heritable silencing in C. elegans hadbeen carried out with a number of loci. Followingmicroinjection of pos-1 dsRNA, one study (Grishok