DNA sequence encoding the scFv recognizing transferrin receptor (TfR) was generated by PCR amplification, with scFv-expressing vector clones [26 (link)] as the template, using the following primers: 5′-GCG GCC CAG CCG GCC ATG G-3′ and 5′-CTT GCG GCC GCA CCT AGG ACG GTC AGC TT-3′. The SfiI/NotI-digested PCR products were subcloned into pTNH6-Haals [27 (link)] at the corresponding restriction sites, resulting in pTNH6-HaalsαTfR-#1-8, respectively (Additional file 1 Table S1). A BsiWI-fragment of pCAG-T7F [19 (link)] was inserted into the BsiWI site of pVITRO1-neo-mcs (InvivoGen), resulting in pVF#9. A BsrGI-fragment of pTNH6-HaalsαTfR-#5 was inserted into the BsrGI site of pVF#9, resulting in pVF#9-TfR.
Plasmids were linearized by restriction digestion with PvuI (NEB) before transfection. HAC donor CHO cells (8 × 104/well in 24-well plates (Nunc)) were co-transfected with 0.3 μg each of pTNH6-H and pCAG-T7-F, and 0.25 μg of pDsRed-Monomer-N1 (Clontech) using Lipofectamine 2000 (Invitrogen). At 24 h after transfection, the cells were re-plated at low density and selected for 14 days with 800 μg/ml of G418 (Nacalai). Drug-resistant cells were recovered as a mixed population. HT1080 cells (2 × 106/6 cm dish) were co-transfected with 0.4 μg each of pTNH6-H and pCAG-T7-F. After culture for 6 h, syncytium formation was tested under the microscope.
Plasmids were linearized by restriction digestion with PvuI (NEB) before transfection. HAC donor CHO cells (8 × 104/well in 24-well plates (Nunc)) were co-transfected with 0.3 μg each of pTNH6-H and pCAG-T7-F, and 0.25 μg of pDsRed-Monomer-N1 (Clontech) using Lipofectamine 2000 (Invitrogen). At 24 h after transfection, the cells were re-plated at low density and selected for 14 days with 800 μg/ml of G418 (Nacalai). Drug-resistant cells were recovered as a mixed population. HT1080 cells (2 × 106/6 cm dish) were co-transfected with 0.4 μg each of pTNH6-H and pCAG-T7-F. After culture for 6 h, syncytium formation was tested under the microscope.
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