Transfecting two plasmids into immortalized keratinocytes can be achieved using various methods. Here are some of the techniques supported by recent studies:

1. Co-transfection with HPV E6 and E7: Co-transfection of plasmids containing the E6 and E7 oncogenes from human papillomavirus type 16 (HPV16) has been shown to effectively immortalize human foreskin keratinocytes. This method demonstrates that the simultaneous introduction of both plasmids can lead to indefinite growth potential in the cells, as both proteins cooperate in the immortalization process.

2. Fibrin-mediated transfection: A matrix-mediated transfection system using a self-hardening fibrin matrix has been developed for delivering plasmids to human keratinocytes. This method has shown successful transfection rates, and it can be adapted for co-transfection by inoculating the matrix with both plasmids before resuspension with keratinocytes.

3. Poly-L-ornithine and DMSO shock: This method involves using poly-L-ornithine to enhance the uptake of plasmid DNA, followed by a dimethylsulfoxide (DMSO) shock. The optimal conditions include incubating the cells with poly-L-ornithine and plasmid DNA, followed by a brief DMSO treatment. This approach has been shown to yield higher transfection rates and can be adapted for co-transfection of two plasmids.

4. Polybrene-mediated transfection: Polybrene can be used in conjunction with DMSO to enhance the transfection efficiency of plasmids in keratinocytes. This method has been successfully adapted for co-transfection, allowing for the introduction of multiple plasmids into the cells.

5. Use of PDMAEMA-based polymers: Poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA)-based polymers have been tested for their ability to transfect immortalized keratinocytes. These polymers can be engineered to facilitate the delivery of multiple plasmids simultaneously, providing a potential method for co-transfection.

These methods highlight the versatility of transfection techniques available for immortalized keratinocytes, allowing for the introduction of multiple plasmids to study gene function or for therapeutic applications.