2.2 Plasmid construction
To construct a transposon-based GEV expression vector, the GEV gene was
amplified by PCR using pME-GAL4-ERT2-VP16 (#82588, Addgene, Watertown,
MA, USA)[28] as a template. A DNA fragment of the
Chinese hamster-derived EF1α promoter[8] was
chemically synthesized. A hygromycin-resistant gene expression unit
(selection marker) was obtained by PCR using pCEP4 (#V04450,
Invitrogen) as a template. These DNA fragments were inserted into a
PiggyBac transposon vector plasmid (#PB513B-1; System Biosciences, Palo
Alto, CA, USA) to generate PB/chEF1α/GEV_Hyg (Figure 1A).
Next, to generate the UAS/scFv-Fc expression vector, a DNA fragment of
scFv-Fc gene was obtained by cutting plasmid R2[8]with Eco RI and Spe I. A DNA fragment of the UAS response
element and minimal region of the adenovirus major late promoter were
chemically synthesized. A zeocin-resistant gene expression unit was
amplified by PCR using pcDNA4/GP[29] as a
template. By inserting these DNA fragments into the PiggyBac transposon
vector plasmid, PB/UAS/scFv-Fc_Zeo was constructed (Figure 1A). To
construct the UAS/GFP expression vector, a GFP gene was amplified
by PCR using pGreenFire1-RARE (#TR037PA-P, System Biosciences) as a
template. PB/UAS/GFP_Zeo was constructed by replacing the scFv-Fc gene
region in PB/UAS/scFv-Fc_Zeo with the resulting fragment (Figure 1A).
For PCR, KOD plus Neo DNA polymerase (#KOD-401; Toyobo, Tsuruga, Japan)
and the primers shown in Supplementary Table S1 were used for
amplification. All PCR products were subjected to gene sequence analysis
by Sanger sequencing to confirm correct sequences.