Molecular biology has many applications where the introduction of large (>100 kb) DNA molecules is required. Current methods of large DNA transfection using conventional transfection reagents are very inefficient. The Targefect-BAC reagent can efficiently deliver BAC DNA into a variety of mammalian cells.
Increased transfection efficiency results from the inclusion of unique enhancers which enhance gene delivery and increase transgene expression. Two enhancers are provided iwth the kit and two protocols are suggested- one protocol uses Virofect, an adenovirus-derived enhancer formulation, which complexes with plasmid DNA via Targefect, an efficient cationic transfection reagent. Virofect enhances gene transfer by using adenoviral receptors on the cell surface to enhance intracellular delivery of transfection complexes. Following internalization, Virofect helps escape of the transfection complexes from degradation in the lysosome, and increases the duration of transgene expression. The Targefect-BAC reagent (Targefect-F2) alone can efficeintly deliver BAC DNA into aome common cell types (60% efficiency of BAC DNA In HEK-293 cells), but the Virofect enhancer greatly enhances the efficiency of transfection. Cell types tested include HEK-293 cells, CV-1 cells, Vero cells, Hela cells,primary human umbikilical vein endothelial cells, ovarian cancer cell lines.
In an alternate protocol, a combination of the Targefect-BAC transfection reagent with the Peptide Enhancer from Targeting Systems (also included in the Targefect-BAC kit as an optional reagent) is used for transfection of BAC DNA. into several cell types. The advantage of the peptide enhancer protocol is that it uses significantly less BAC DNA amd works far more efficiently than the virofect protocol in certain cell types. It is therefore suggested that both protocols be tested