Figure 2. Construction of a novel dual expression lentiviral vector.

(A) Analysis of restriction enzyme digestion for the pLV-U6-EGFP-Puro plasmid that contains mutated XhoI and XbaI sites. PCR site-directed mutagenesis was performed using the pLV-U6-EGFP-Puro plasmid and the primers containing mutated XhoI and XbaI sites. Positive plasmids were screened by enzyme digestion and analyzed by agarose gel electrophoresis and then imaged as for Figure 1C. (B) The DNA fragment containing nine restriction sites was cloned into the pLV-U6-EGFP-Puro plasmid. A DNA fragment containing nine restriction sites was synthesized and cloned between BamHI and EcoRI sites in the pLV-U6-EGFP-Puro plasmid. Positive plasmids were screened by enzyme digestion; the gel image was obtained as for Figure 1C. (C) DNA sequencing result for the new MCS DNA fragment containing nine restriction enzymes. (D) The CMV promoter was cloned into the plasmid containing the new MCS. Positive plasmids were screened and analyzed as described in Figure 1C.

Abbrreviations: mutated pLV, pLV-U6-EGFP-Puro plasmid with mutated XhoI and XbaI sites; pLV-nMCS, pLV-U6-EGFP-Puro plasmid with a new MCS DNA fragment.