Supplementarymaterial(doc112K)-Nature.doc

Supplemental Online Material A. Schambach et al. Equal potency of gammaretroviral and lentiviral SIN vectors for expression of O6-methylguanine-DNA-methyltransferase in hematopoietic cells (Note: Reference numbering differs from published paper) Vector construction The MGMT-P140K cDNA was subcloned into pBluescript SK+ (Stratagene, La Jolla, CA) and its sequence was confirmed. From there it was transferred into the retroviral vector pMP71 [1] as an NcoI / EcoRI fragment. Subsequently, the posttranscriptional regulatory element (PRE) of Woodchuck Hepatitis Virus (WHV) was inserted via BamHI / HindIII to obtain pMP71.MGMTp. This PRE version has several possible ATGs mutated and ends before the X protein ORF [2]. To obtain SF91.MGMTp, the MGMTp cassette was introduced into the retroviral vector pSF91 [1]. To construct a retroviral self-inactivating (SIN) retroviral vector, the 3’ SIN LTR was excised from pSinSF91P [3] with HindIII/XhoI and cloned into pSF11 [1]. Into the resulting vector pSin11.GFP, we inserted the SFFVp U3 promoter (including the enhancer; -342 to +18, relative to the transcriptional start site, GenBank acc. No. AJ224005) and the PRE (see above), thus obtaining pSin11.SF.GFP.p. To clone pSin11SF.MGMTp, the GFP was excised and substituted by MGMT-P140K cDNA. The lentiviral construct pRRL.PPT.PGK.GFPpre was kindly provided by L. Naldini (Milano, Italy). For pRRL.PPT.PGK.MGMTp the MGMT/P140K cDNA was inserted via AgeI and SalI. For the corresponding retroviral vector, pSin.PGK.MGMTp, pRRL.PPT.PGK.GFPpre was cut with EcoRV and SalI to excise the PGK promoter and the GFP. In a second step, the GFP cDNA was replaced by MGMT-P140K. To design pRRL.PPT.SF.MGMTp and pRRL.PPT.SF91.MGMTpre, the SFFVp promoter (see above) alone or the SFFVp promoter followed by a retroviral intron was excised from pSSF91P [3] and cloned into the lentiviral constructs. For pRRL.PPT.EF1?.MGMTp the EF1? (intron-containing) promoter was excised from pHR’ EF1? EGFP (kindly pr