In addition the large mRNAs from HIV have inherent RNA processing problems and are not efficiently exported from the nucleus in the absence of a helper protein (REV).
It'll take some touch to get it right, so it helps to have a couple of helpers to rev the engine, check the number, and twist the distributor.
FugW3-GFP as a vector control and FugW3-SIRT1-WT, -TFEB-WTY, -TFEB-WT, and -TFEB-K116R constructs were transfected into 293T-packaging cells with three other helper plasmids (RRE, REV, and VsVg) respectively, using Lipofectamine LTX.
To overcome the dramatic reduction in the titer of anti-HIV siRNA lentivirus vectors, we developed a modified rev helper plasmid that allows for high titer lentivirus preparations containing anti-HIV siRNA transgenes.
Each NRP1WT, NRP1ΔSEA, and NRP1ΔCyto in the pHAGE lentiviral backbone vector was co-transfected with the helper plasmids (tat, rev, gag-pol and VSV-G) to HEK293 cells as described previously (Shimizu et al., 2008).
Lentiviral vectors were prepared by co-transfecting 293T cells (Invitrogen) with helper plasmid pRsv-REV, pMDlg-pRRE and pMD2G (Invabio) using the calcium phosphate technique.
Briefly, HEK293T cells were cotransfected with FUGW or FUmChW vectors (with or without LIMK1shRNA) plus VSV-G, pMDLg and RSV-Rev helper plasmids.
To produce live virus for gene transfer, we ligated Arnt and Gdi2 cDNAs into the NotI-XbaI site of pHIV-ZsGreen and transferred the recombinant plasmids to HEK293T packaging cells by transfection in combination with helper plasmids pMDLgRRE, pRSV-Rev, and pCMV-VSV-G.
The lentiviral expression module was transfected into 2×105 293T cells, along with the helper plasmids pLP1 (gag-pol), pLP2 (Rev) and pLP/VSV-g (VSV g) (all obtained from Invitrogen).
