SECOND GENERATION pie-1 VECTORS
Ingrid D’Agostino, Kim Reese and Geraldine Seydoux

These second-generation pie-1 vectors use GATEWAY sequences for directional cloning of virtually any ORF.

GATEWAY technology is licensed by Invitrogen.

Click here for Seydoux lab GATEWAY cloning protocols.

Some of the second-generation vectors also contain the unc-119 rescuing fragment, which can be used as a marker to identify transgenic worms during biolistic transformation (Praitis et al., 2001). Biolistic transformation usually yields low-copy integrants and is an efficient method to obtain stable expression of transgenes in the maternal germline (Praitis et al., 2001). In our experience transgenic lines obtained by bombardment are frequently more stable than lines obtained by injections.

Click here for Seydoux Lab BOMBARDMENT protocol.

For expressing ORFs in the germline:
pID2.02	unc-119 rescuing fragment/pie-1promoter-gateway destination cassette B- pie-1 3’
For expressing GFP-ORF fusions in the germline:
pKR2.40	pie-1promoter-GFP- gateway destination cassette B -pie-1 3’ This vector does not contain unc-119 sequences and therefore must be co-injected with a marker (typically we use pRF4 in complex array injections - Kelly et al., 1997)
pID3.01B	unc-119 rescuing fragment / pie-1promoter-GFP- gateway destination cassette B -pie-1 3’
(download sequence in MS Word) (download graphic map in PDF)