Figure 1.
Recombinase-based BV constructs and confirmation of minicircle formation. (A) Schematic illustration of BacΦC31o, BacCre, BacFLPo and BacALF containing the synthetic tandem recognition sites (attP/loxP/Frt and attB/loxP/Frt). After successful recombination, a ≈2.2 kb minicircle (pALF-CdE) was formed. A/L/F indicates the recombined tandem recognition sites. PCMV, cytomegalovirus immediate-early promoter, pA, polyadenylation signal. (B) Confirmation of recombination by d2EGFP expression. (C) Confirmation of recombination by PCR. HEK293 cells were co-transduced with BacALF (MOI 100), and the corresponding recombinase-encoding BV (MOI 100), or singly transduced with BacALF as negative control (NC). At 1 dpt, d2EGFP expression was observed (200×) and intracellular minicircle formation was verified by PCR assay using the primers spanning a 1.5 kb region in the recombined minicircle. The arrows indicate the primer binding sites. (D) Dependency of recombination efficiencies on MOI. HEK293 cells were co-transduced with BacALF and the recombinase-expressing BV at various MOI combinations and were subjected to flow cytometry at 1 dpt.