For this, oligos with HindIII and NotI overhangs were synthesised (Eurogentec, Belgium), annealed, and then ligated into HindIII- and NotI-opened pcDNA5/FRT/TO resulting in the modified vector pcDNAmod.
The cDNAs encoding mouse, the long (AAX12551) and short (AAX12552) isoforms of vezatin, were subcloned either in the pCMV expression vector, which enabled the production of the N-terminal Myc-tagged vezatin protein, or in the pCS2 modified vector (kindly provided by Dr FJ del Castillo, Ramon y Cajal Hospital, Madrid, Spain), which produced a C-terminal Myc-tagged vezatin protein.
For overexpression of Parkin, N-terminally FLAG-tagged full-length Parkin cDNA was cloned in pcDNA3.1 (Invitrogen's modified vector lacking V5/His tags).
The adduct in the CpGpT sequence was incorporated into a single-stranded vector, and following replication of the modified vector in E. coli strain AB1157, G→T transversions were observed at 100% frequency.
Moriya and co-workers examined the repair of the crotonaldehyde-derived N-dG :N-dG interstrand cross-links following replication of site specifically modified vector DNAs in various E. coli and mammalian cells (145).
The mutagenic properties of acrolein-induced and related model adducts have also been investigated following replication of site specifically modified vector DNAs in Escherichia coli and various mammalian cell lines (Table 1).
Consistent with this interpretation, (6 S,8 R -4-HNE-dGNE-dG 8 was less miscoding than (6 S,8 R -4-HNE-dGNE-dG 6 duR -4-HNE-dGation of site specifically modified vectoR -4-HNE-dGS-7 cells (82).
Fifty nanograms of the modified vector was transformed in BL21 (DE3) cells and plated on LB agar plates containing 70 μg/mL ampicillin and 50 μg/mL chloramphenicol.
The mutagenicities of (6 R,8 R)- and (6 S -crotonaldehydehyde adductS -crotonaldehyde1, row 18 adducts were ∼5 and ∼6%, respectively, when a single-stranded, site specifically modified vector was replicated in the COS-7 system (76).
