FIG. 2.
Schematic representation of vector constructs and their corresponding titers. The starting control vector (pZN) is a derivative of the pHIT111 (32), which in turn is derived from pLXSN (22). The lacZ reporter marker gene is expressed from the LTR and an internal SV40 promoter promotes Neor gene expression. Both expression cassettes use the same 3′ LTR R/U5 polyadenylation signal. This vector already contains the MLV wt-SD, which suboptimally pairs with a cryptic SA contained elsewhere within the packaging signal (22). The sequential cloning steps to make pICUT-2-ZN from this starting vector are represented by the intermediate vectors shown. First, the st-SD and, next, the c-SA were added (to make the vectors pZN-SD and pICUT-ZN, accordingly); subsequently, the wt-SD only or this as well as the cr-SD were disabled to make the vectors pICUT-1–ZN and pICUT-2–ZN, respectively. Finally, a pICUT-2 vector in which the lacZ marker is replaced with a multiple cloning site is also shown. The corresponding titers are the average LFU per milliliter recorded for each vector from three separate assays (with <5% deviation).