The FR-MAR has enhancer-blocking activity. (A) Restriction map of the pGEM42 plasmid used for FR-MAR cloning. (E) EcoRI; (Hd) HindIII; (Hc) HincII; (K) KpnI restriction sites. (B) For transfection into HeLa cells, the various plasmid constructs were linearized. Transformants were grown in the presence of 1.2 mg/mL G418 and viable colonies counted at day 10. (C) The number of inserted copies of pNeo1–5 constructs in stably transfected cell lines. DNA was purified from transfected HeLa cells, dot-blotted, and hybridized either with a probe for the Neor gene or the control GAPDH gene. The quantification was carried out using the standard concentrations of plasmid DNA mixed with nontransfected HeLa DNA as described in Methods. (D) Analysis of expression of Neor gene in stably transfected cell lines. Total RNA was purified from transfected HeLa cells, separated by gel electrophoresis, blotted, and hybridized either with a probe for the Neor gene or the control GAPDH gene. The gene expression was quantified using the ImageQuant software (Fuji).
