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Plasmid Constructions. pCAG/luc: A 1.7-kb fragment containing the luciferase coding sequence, released by digestion of pGL3-Basic vector (Promega) with BglII and XbaI, was ligated to a 3.4-kb BamHI + XbaI fragment derived from pEGFP-1 (Clontech) to create pLucf. The plasmid pCX-EGFP (a gift from J. Miyazaki, Kyoto University, Kyoto), was digested with SalI and EcoRI to release a 1.7-kb fragment. This fragment, containing the CAG sequence, was ligated to a XhoI + EcoRI digest of pLucf to create pCAG/luc.

RSV/FRT2PSA.FLP/EGFP. A 2-kb fragment containing Flp recombinase sequence, released by digestion of pOG-FLPe6 [a gift from A. Francis Stewart, European Molecular Biology Laboratory (EMBL), Heidelberg] with XbaI and SalI, was ligated to XbaI + XhoI-digested pMECA (1) to produce pMECA/FLP. This plasmid was then digested with XbaI and AgeI to release a 2-kb fragment that was ligated to NheI + NgoMI-digested pMECA to create pMECA/FLP (2). A 2.5-kb fragment containing the PSE-BC promoter sequence, released from the plasmid pPSE-BC (a gift from Lily Wu, University of California, Los Angeles) by digestion with XbaI and SalI), was ligated to XbaI + SalI-digested pMECA/FLPe (2) to create pMECA/PSA.FLP(dam-). A 4.5-kb fragment, released from pMECA/PSA.FLP by digestion with XbaI and AvrII, was ligated to NheI-digested pFRT2 (a gift from Susan Dymecki, Harvard University, Cambridge, MA) to generate pFRT2/PSA.FLP. Finally, this plasmid was digested with AgeI and XmnI to release a 4.5-kb fragment that was ligated to AgeI-digested pRSV/EGFP to produce RSV/FRT2PSA.FLP/EGFP.

pRSV/FRT2PSA.FLP/DT-A. The plasmid p22EDT1 (a gift from A. Francis Stewart, EMBL) was digested with BglII and NotI, releasing a 1.3-kb fragment containing DT-A sequence that was ligated to a 5.0-kb fragment released from the plasmid pIND by BamHI + NotI digestion to create pIND/DT-A. pIND/DT-A was digested with KpnI and XbaI, releasing a 1.3-kb fragment that was ligated to a KpnI + XbaI digest of pMECA to create pMECA/DT-A. This plasmid was digested with AgeI and XbaI, releasing a 1.3-kb fragment that was ligated to a 3.8-kb fragment deriving from an AgeI + NheI digest of pRSV/EGFP. The resulting plasmid, pRSV/DT-A, was digested with AgeI, and then ligated to a 4.5-kb fragment released from pFRT2/PSA.FLP by digestion with AgeI and XmnI to create pRSV/FRT2PSA.FLP/DT-A.

pRSV/EGFP. pEGFP-1 (Clontech) was digested with BamHI and AflII. The resulting 1-kb fragment was ligated into the BamHI and AflII sites of pIND (Invitrogen) to create pIND/EGFP. pIND/EGFP was then digested with SpeI and NheI. The resulting 1-kb fragment was ligated into the NheI site of pDC312/RSV (2) to create pRSV/EGFP.

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2. Peng, W., Verbitsky, A., Bao, Y. & Sawicki, J. A. (2002) Mol. Ther. 6, 537-545.