AAV Recombineering with Single Strand Oligonucleotides
Figure 6
Oligo-Assisted AAV Genome Recombination of Circularization Dependent AAV.
A) The cartoon depicts single vector split gfp systems that are capable of GFP production after either intra- or inter-molecular dimerization. The relevant system features are depicted; the arrow represents the CMV promoter followed by the 5′ portion of the gfp coding sequence (cds) and an splice donor sequence. On the far left side is the hcg intron and the remaining gfp cds followed by a poly-adenylation sequence. The entire intron is highlighted in yellow. Both the single-strand (ss) and self-complementary (sc) versions are shown. B) AAV2 capsids packaged with either genome depicted in A were used to separately infect human embryonic kidney cells at the indicated viral genomes per cell. Then, either the H-Ib Fwd or a non-homologous (NH) oligo was used for transfection. Three days later, GFP+ cells were determined by flow cytometry. In all cases the single-strand construct demonstrated a significant (unpaired student t test) increase, yet less than 2-fold, with the H-Ib Fwd oligo compared to the NH control. No significant changes were note for the sc construct.