CaSpeR5, a family of Drosophila transgenesis and shuttle vectors with improved multiple cloning sites.

The pCaSpeR (1) and pUAST vectors (2) are two of the most commonly used Drosophila transformation vectors. However, although they have great utility in their current form, their multiple cloning sequences (MCSs) have a limited number of unique restriction sites (Figure 1). This is particularly true for the pUAST vector, whose MCS has only five unique sites. Further, neither of the MCSs in pCaSpeR or pUAST are present in small shuttle or cloning vectors, which is problematic, because the large size (>8 kb) of the transgenesis vectors requires sequence manipulations such as site-directed mutagenesis or deletion dropouts to be done in small plasmid vectors and the modified DNA to be moved to the transgenesis vectors. The lack of matching shuttle vectors further constrains the usable cloning sites and can make moving large genomic fragments between a cloning vector and a transgenesis vector problematic. To overcome the above limitations, we engineered a new MCS based on the pCaSpeR4 MCS that adds five new 6-base cutter sites, but most importantly flanks the entire MCS by two 8base cutters on each side (Figure 1). We call this improved vector pCaSpeR5, since the new vector retains all the restriction sites of the pCaSpeR4 MCS in their original order. We also created pUAS-C5 by replacing the MCS of pUAST with a modified version of the pCaSpeR5 MCS (C5 MCS) that lacked the ATG-containing SphI site (Figure 1). Although the pUAST has many common 6-cutter sites in its backbone, the C5 MCS nonetheless adds five new 6-cutter and the flanking four 8-cutter sites to the pUAS expression vector. The pCasper5 and pUAS-C5 vectors were shown to be functional by cloning a GMR-nvYFP transgene (3) into pCaSper5 and nvYFP (3) into pUAS-C5 (Figure 2). Injection of these constructs into embryos yielded typical numbers of transgenic offspring (>33 and >46 transgenic lines per 200 injected embryos for pCaSpeR5 and pUAS-C5, respectively) that expressed yellow fluorescent protein (YFP) strongly in eye discs (pCaSpeR5:: GMR-nvYFP) or in the tracheal system [pUAS-C5:: nvYFP lines crossed to the tracheal btl-gal4 driver (4)] at levels at least comparable or stronger than to similar constructs in other transgenesis vectors (Figure 2). To facilitate clone manipulation, we created a small ampicillin (Amp)resistant vector named pBS-C5 by replacing the MCS of pBlueScript® (Stratagene, La Jolla, CA, USA) with the C5 MCS. BbvCI and PmeI sites flanking the C5 MCS were also added CaSpeR5, a family of Drosophila transgenesis and shuttle vectors with improved multiple cloning sites