Reconstitution of the Myeloid and Lymphoid Compartments after the Transplantation of Autologous and Genetically Modified CD34 Bone Marrow Cells, Following Gamma Irradiation in Cynomolgus Macaques - Genetic Engineering: Recent Developments in Applications - page 127

Lentiviral Vector
Two SIV-derived vectors were produced, one for in vitro studies and the other for
in vivo studies: 1) pRMES8 is a minimal packaging-competent SIVmac251-based
vector[34]. It contains the enhanced green fluorescent protein (eGFP) marker gene
under control of the mouse phosphoglycerate kinase (PGK) promoter, placed be-
tween the SIVmac251 LTRs and leader sequences. It carries the SIVmac251 RRE
region and minimal sequences of the gag and pol genes encompassing central
polypurine tract/central termination sequence (cPPT/CTS) regions (figure 1A).
pRMES8 was used for in vitro assays investigating the susceptibility of CD34 +
cells from primate bone marrow to transduction with SIVmac251-derived vec-
tors. 2) For in vivo assays, we used pGASE; this plasmid is an optimised version
of pRMES8, with a 3'-SIN-LTR for safety and insertion of an exon splicing en-
hancer (ESE) upstream the PGK promoter to increase titer [41]
Figure 1. Schematic representation of SIV-derived SIN vector, helper construct and VSV-g encoding plasmid.
An SIVmac251-derived vector was produced by cotransfecting 293T cells with three plasmids: A. a plasmid
pGASE containing the eGFP gene under control of the PGK promoter; B. a plasmid pSIV3 + containing viral
genes; C. a plasmid pGREV containing the VSV envelope gene. Cis genetic elements are symbolized with white
boxes, whereas promoters and genes are depicted by shadowed boxes. pCMV, early cytomegalovirus promoter;
pPGK, mouse phosphoglycerate kinase-1 promoter; RRE, REV-responsible element; SA, SIV Rev/Tat splice
acceptor; cPPT and PPT, central and 3' polypurine tracks, respectively; GFP, the gene encoding the enhanced
green fluorescent protein; LTRsin, partially U3 deleted 3'LTR; LG, leader and a 5' GAG region.
pSIV3+ is the packaging plasmid derived from the BK28 molecular clone of
SIVmac251, as described elsewhere[33]. Briefly, the pSIV3+ gag/pol expression
plasmid was obtained by replacing the 5' LTR of SIVmac251 (nucletotides 1
to 506) by the human cytomegalovirus (CMV) early-immediate promoter and