A gene-trap cassette incorporating the Mhc intron 18 SA site51 (link) was PCR amplified from pP-GC (gift of Xavier Morin and William Chia)15 (link) with primers SA-XbaI-F and SA-PstI-R. The resulting PCR fragment was cut with XbaI and PstI and subcloned into pBS-KS-attB1-2, cut with XbaI and PstI, resulting in the mini gene-trap plasmid, pBS-KS-attB1-2-GT-SA. The SA is followed by a multiple cloning site (PstI, EcoRI, XhoI, BamHI and HindIII).
A mutagenic GAL4 gene-trap cassette, encompassing the GAL4 coding sequence and Hsp70 polyadenylation signal, was obtained from plasmid pChs-GAL4 (Drosophila Genomics Resource Center)55 (link), and PCR amplified with primers GAL4-Hsp70-EcoRI-F and GAL4-Hsp70-BamHI-R. A mutagenic QF gene-trap cassette, encompassing the QF coding sequence and Hsp70 polyadenylation signal, was obtained from plasmid pattB-QF-Hsp70 (Addgene)30 (link), and PCR amplified with primers QF-SV40-EcoRI-F and QF-SV40-BamHI-R. A mutagenic Flp fate mapping gene-trap cassette, encompassing the FLPo56 (link) coding sequence and SV40 polyadenylation signal, was obtained from plasmid pQUAS-DSCP-Flpo (Addgene)30 (link), and PCR amplified with primers Flpo-SV40-EcoRI-F and Flpo-SV40-BamHI-R. The resulting PCR fragments were cut with EcoRI and BamHI and subcloned into pBS-KS-attB1-2-GT-SA, cut with EcoRI and BamHI, resulting in the plasmids pBS-KS-attB1-2-GT-SA-GAL4-Hsp70, pBS-KS-attB1-2-GT-SA-Flp-SV40, and pBS-KS-attB1-2-GT-SA-QF-Hsp70 respectively.
A mutagenic GAL4 gene-trap cassette, encompassing the GAL4 coding sequence and Hsp70 polyadenylation signal, was obtained from plasmid pChs-GAL4 (Drosophila Genomics Resource Center)55 (link), and PCR amplified with primers GAL4-Hsp70-EcoRI-F and GAL4-Hsp70-BamHI-R. A mutagenic QF gene-trap cassette, encompassing the QF coding sequence and Hsp70 polyadenylation signal, was obtained from plasmid pattB-QF-Hsp70 (Addgene)30 (link), and PCR amplified with primers QF-SV40-EcoRI-F and QF-SV40-BamHI-R. A mutagenic Flp fate mapping gene-trap cassette, encompassing the FLPo56 (link) coding sequence and SV40 polyadenylation signal, was obtained from plasmid pQUAS-DSCP-Flpo (Addgene)30 (link), and PCR amplified with primers Flpo-SV40-EcoRI-F and Flpo-SV40-BamHI-R. The resulting PCR fragments were cut with EcoRI and BamHI and subcloned into pBS-KS-attB1-2-GT-SA, cut with EcoRI and BamHI, resulting in the plasmids pBS-KS-attB1-2-GT-SA-GAL4-Hsp70, pBS-KS-attB1-2-GT-SA-Flp-SV40, and pBS-KS-attB1-2-GT-SA-QF-Hsp70 respectively.
