ACTIVE MOTIVE Chariot™

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Chariot™ is a revolutionary new transfection reagent capable of efficiently delivering proteins,peptides and antibodies into cultured mammalian cells in less than two hours.Current transfection techniques include microinjection1, calcium phosphate coprecipitation2,cationic liposomes3, viral vectors4and electroporation5. These methods are capable of transport-ing DNA into cells, but the techniques can be cumbersome and even cytotoxic. Once transfec-tion has been completed, the researcher must wait 12-80 hours post-transfection to detectexpression of the gene of interest.Recently, novel methods to deliver proteins have been reported and commercialized, i.e.HIV-1TAT, DrosophilaAntennapedia homeotic transcription factor and herpes simplex virus-1 DNAbinding protein VP22 (for a recent review, see ref. 6). Transduction via TAT-fusion proteins resultsin inactivation and denaturation of the protein. To deliver an active protein, correct renaturationis required upon internalization. In addition, TAT must be covalently linked to the compound ormacromolecule to be delivered by a chemical reaction.Penetratin 1 is a 16 amino acid peptide corresponding to the third helix of the homeodomain ofAntennapedia protein. Activated Penetratin has an N-terminal pyridyl disulfide that is used tocovalently couple it to the macromolecule to be delivered. However, chemical coupling can becumbersome and requires the macromolecule being delivered to carry a free thiol group.Another delivery system utilizes the translocation properties of the 38 kDa herpes simplex virus-1DNA binding protein VP22. VP22 must be fused to the peptide/protein to be delivered and,therefore, requires the construction of a suitable expression vector.Unlike these current transfection techniques, Chariot forms a non-covalent complex with theprotein, peptide or antibody of interest. This completely bypasses the transcription-translationprocess associated with gene expression, reducing the time until the cells can be assayed fromdays to under two hours. The Chariot-macromolecule complex stabilizes the macromolecule andhelps to protect it from degradation during the transfection process7, 8. Upon internalization, thecomplex dissociates and the macromolecule is free to proceed to its target organelle. Moreover,efficient delivery is observed at 4ºC, suggesting that the delivery mechanism is independent ofthe endosomal pathway8. Therefore, the macromolecule is not subjected to the conditions ofthat pathway, which can modify the structure of the macromolecule during internalization.Chariot is non-cytotoxic and serum-independent8. It is ideal for in vivostudies because fixing isnot required. Chariot has been used to efficiently transfect a variety of cell lines, including:

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