The myo-3p::FLAG::pab-1 contruct (pPRSK9) was assembled by ligating the myo3 promoter from a 2498 bp HindIII/Asp718 fragment from pPD95.86 (a gift from A. Fire1), a FLAG epitope encoded by oligonucleotides, and a PCR fragment containing genomic pab-1 (accession number U24123.1) into pBluescript II SK (Stratagene) cut with SacI/HindIII (see supplemental web site for more details). Previous work has shown that amino-terminal epitopes do not to interfere with the function of PABP2,3, and that the PABP-bound RNA best represents the mRNA made in a cell of several candidate mRNA-binding proteins 4.
A transgenic strain expressing myo-3p::FLAG::pab-1 was made by microinjecting5 wild-type animals with myo-3p::flag::pab-1 (25 ng/ul) and sur-5::gfp (pTG96; 100 ng/ul; a gift from Min Han)6, generating an extrachromosomal array, and then randomly integrating the extrachromosomal array into the genome using g-irradiation5. Transgenic worms containing the integrated array (gaIs146) were back-crossed to wild-type twice, resulting in strain SD1075.
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