Recently, I am trying to make a pie-1 promoter-driven reporter. I have constructed a 2 kb pie-1 promoter + pie-1 first exon + first intron + GFP and made a stable transgene. I found that GFP localizes in the centrosome. I am wondering whether there are some wield sequences in pie-1’s first exon and intron. Thanks very much.
There are many plasmids available with PIE-1 promoters available that won’t have this potential problem already you could use. I’d be concerned given the following paper:
The PIE-1 protein and germline specification in C. elegans embryos
Craig C. Mello*†, Charlotte Schubert*‡, Bruce Draper*‡, Wei Zhang*, Robert Lobel* & James R. Priess*‡§
Department of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, 1124 Columbia Street, Washington 98104, USA
‡ Zoology Department University of Washington, Seattle, Box 351800, Washington 98195, USA
§ Howard Hughes Medical Institute at Department of Basic Sciences, Fred Hutchinson Cancer Research Center, 1124 Columbia Street Seattle, Washington 98104, USA † Present Address: University of Massachusetts Cancer Center, 373 Plantation Street, Worcester, Massachusetts 01605, USA.
TOTIPOTENT germline blastomeres in Caenorhabditis elegans contain, but do not respond to, factors that promote somatic differentiation in other embryonic cells1,2. Mutations in the maternal gene pie-1 result in the germline blastomeres adopting somatic cell fates3. Here we show that pie-1 encodes a nuclear protein, PIE-1, that is localized to the germline blastomeres throughout early development. During division of each germline blastomere, PIE-1 initially associates with both centrosomes of the mitotic spindle. However, PIE-1 rapidly disappears from the centrosome destined for the somatic daughter, and persists in the centrosome of the daughter that becomes the next germline blastomere. The PIE-1 protein contains potential zinc-finger motifs also found in the mammalian growth-factor response protein TIS-11/NUP475 (refs 4–7). The localization and genetic properties of pie-1provide an example of a repressor-based mechanism for preserving pluripotency within a stem cell lineage.
Does it localize to the centrosome with and without your protein of interest included?
My understanding is that many pie-1 vectors such as pAA64 and pFJ1 likely attach the 11 amino acid N-terminus of PIE-1 to the fusion protein. (MAQTKPIAEQM) Does anyone out there know if this accurate? At least one set of vectors uses a different version (pJK7, pJK3) without the 11 amino acid N-terminus of PIE-1. Are there any other versions of a cloning site at end of the pie-1 promoter in use? I would suggest building off of the work of others rather than designing a new version of the pie-1 promoter cassette.
All of the pie-1 vectors I can remember using also include the third intron of pie-1 (including an enhancer) cloned upstream of the promoter. Did you include this sequence?