Figure 4.

Schematic representation of the DNA vector-mediated homologous recombination in mammalian cells. The homologous recombination donor plasmid DNA vector carries four multiple cloning sites (MCS) to facilitate: (1) sub-cloning and insertion of a therapeutic gene (sub-cloned at MCS3) and it's promoter (sub-cloned at MCS2) to a desired site of the host genome via two homology arms (sequences homolog to the insertion site are sub-cloned at the MCS1 and MCS4 flanking the therapeutic gene expression cargo). (2) Non-viral delivery of the HR donor DNA vector into the anticipated cell provides translocation of this vector into the nuclei. (3) Homology arms facilitate recognition of homolog sequence on the target cell's genome. (4) HR occurs at the homology sites and the therapeutic transgene expression cargo would integrate into the host chromosome through HR. Adding the LoxP sites (red triangles) to the HR donor DNA vector would facilitate removal of homology arms, or other unnecessary sequences, after insertion of the therapeutic cassette into the host genome via expression of the Cre recombinase and its excision activity on the loxP sites through “Cre/LoxP recombination systems.”