AAV Recombineering with Single Strand Oligonucleotides
Figure 3
Characterization of Oligo-Assisted AAV Genome Concatemerization.
A) Human embryonic kidney (HEK) cells were co-infected by AAV2 single strand split gfp vectors at a multiplicity of infection (MOI) of 200 for each vector. Then, either H-Ib Fwd or the non-homologous (NH) oligos were used for transfection (80 nM). GFP positive cells were quantitated by flow cytometry at the indicated times. B) HEK cells were co-transduced with the indicated MOI of each split gfp vector and subsequently transfected with the H-Ib Fwd oligo or a NH control (80 nM). Three days later the number of GFP+ cells was determined by flow cytometry. C) HEK cells were co-transduced with the AAV2 split gfp vectors at a MOI of 200 and the indicated concentrations of H-Ib Fwd was used for transfection. GFP+ cells were determined on day 3. The presented results were not normalized to the efficiencies of co-infection and oligo transfection. D) Oligo size analysis for stimulation of OAGR. Cells were co-infected with the split vector system as above and then either the H-Ib Fwd oligo or derivatives of that sequence were then used for transfection. %GFP positive cells were determined on day 3 by flow cytomtery. nt = nucleotide.