FIRST GENERATION pie-1 VECTORS
Melanie Dunn, Kim Reese and Geraldine Seydoux

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).

An 8kb genomic fragment from the pie-1 locus is sufficient to rescue a pie-1 mutation (Reese et al., 2000, Tenenhaus et al., 2001). We modified this fragment to allow expression of heterologous GFP fusions.

The resulting vector includes (from 5’ to 3’) the pie-1 large intron, the pie-1 promoter, GFP, a specific ORF (see below) , the pie-1 3’UTR, and additional 3’ sequences. The pie-1 vector is available in 4 different versions each with a different ORF inserted downstream of GFP.

1st generation pie-1 vectors:

pJH4.52	pie-1prom:GFP:histone H2B (F54E12.4) fusion.
pJH4.64	pie-1prom:GFP:act-1
pJH4.66	pie-1prom:GFP:beta-tubulin
pJH4.67	pie-1prom:GFP:gamma-tubulin
(download sequence in MS Word) (download graphic map in PDF)

Useful cloning sites:

SpeI sites flank each ORF, and can be used to replace these sequences with other ORFs to make new GFP fusions.
BamHI sites flank the GFP:ORF, and can be used to replace these fusions with others.

Complex array injections:

We use complex arrays (Kelly et al., 1997) to express these constructs in the germline.

Typical injection mix:

pJH4.52 linearized with SacII	1ug/ml
pRF4 linearized with EcoRI	1ug/ml
N2 genomic DNA digested with PvuII	60ug/ml

We routinely inject 50-60 worms and obtain 15-40 F1 rollers.

These are cloned to individual plates and checked for roller progeny (F2) - between 10 to 30% of F1 rollers will give F2 rollers.

Gravid F2 adult rollers are then examined for GFP expression in their germline and embryos. You should see some GFP expression in several lines, although expression levels can vary greatly from line to line.

In most lines, expression will diminish in the F3 and F4 generations, although a minority of lines may remain stable.

Keeping the lines at 25oC improves GFP expression although it may lead to sick worms.

What you should see:

All three constructs express in the adult germline (strongest in proximal germ cells), and in all blastomeres of early embryos.

pJH4.52: GFP on chromosomes
pJH4.64: GFP on cortical actin and cell division remnants.
pJH4.66: GFP on centrosomes, meiotic and (more weakly) mitotic spindles

With all constructs, you will also see significant expression in various somatic cells in adults; this expression may depend on N2 DNA sequences present in the complex array.