Worm Breeder's Gazette 15(5): 18 (February 1, 1999)
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.
Dept. of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, 725 N. Wolfe St./515 PCTB, Baltimore, MD 21205.
We have used sequences from the pie-1 gene to construct a vector designed to express GFP fusions in the adult germline and in early embryos. The pie-1 gene encodes a maternal RNA transcribed in the adult germline and inherited maternally in embryos (Mello et al., 1992, 1996; Tenenhaus et al., 1998). We have shown previously that an 8kb genomic fragment from the pie-1 locus is sufficient to tag PIE-1 with GFP and rescue a pie-1 mutation (Fig. 1; Dunn et al., Reese et al., 1998 East Coast Worm Meeting Abstracts).
We now have modified this construct to allow expression of heterologous GFP fusions. The resulting vector (Fig. 2) contains the pie-1 promoter, the pie-1 3’UTR, and the pie-1 large intron, all of which appear to be important for robust expression in the adult germline and in early embryos. We have tested this new vector in 4 configurations: as is (GFP alone), and with one of three different open-reading frames inserted at the SpeI site after the GFP: his-11 (histone H2B), C36E8.5 (beta tubulin), and act-1 (actin). These constructs were injected into N2 hermaphrodites using the Fire lab’s complex array injection method which permits germline expression of transgenes (Kelly et al., 1997, Genetics 146: 227-238). For all constructs, we obtained a number of lines (10% to 50% of all roller lines) with GFP expression in the germline and in early embryos. In most lines, expression was restricted to the F2 generation after injection. In all cases, the subcellular localization observed matched that expected for each fusion: throughout cells for GFP alone, on DNA for GFP:histone H2B, on centrosomes and meiotic (and more weakly mitotic) spindles for GFP:beta-tubulin, and on cell cortices and cell division remnants for GFP:actin. (In one GFP:actin line, we also saw nuclear GFP). In addition to germline expression, all lines also expressed GFP in a number of somatic cells.
We are hopeful that this vector could be used by others to express additional GFP fusions in the germline and in early embryos. Anyone interested can obtain the pie-1 vector from Melanie Dunn at firstname.lastname@example.org.