Question

Explain the recombinase mediated cassette exchange (RCME) technique in detail

Answer 1

                     RECOMBINASE-MEDIATED CASSETTE EXCHANGE(RCME)

                              RCME is a procedure in reverse genetics allowing the systematic, repeated modification of higher eukaryotic genomes by targeted integration, based on the features of site-specific recombination processes. For RCME, this is achieved by the clean exchange of a pre-existing gene cassete for an analogous cassete carrying the "gene of interest"(GOI)

                         

                                The genetic modification of mammalian cells is a standard procedure for the production of correctly modified proteins with pharmaceutical relevance. To be successful, the transfer and expression of the transgene has to be highly efficient and should have a largerly predictable outcome. Current developments in the field of gene therapy are based on the same principles. Traditional procedures used for transfer of gene of interest(GOI), are not sufficiently reliable, mostly because the relevant epigenetic influences have not been sufficiently explored; transgenes intergrate into chromosomes with low efficiency and at loci that provide only sub-optimal conditions for their expression. As a consequence the newly introduced information may not be realized (expressed), the gene's may be lost and (or) re-insert and they may render the target cells in unstable state. It is exactly this point where Recombinase-mediated casseste exchange enters the field. The whole procedure was introduced in 1994 and it uses the tools yeasts and bacteriophages have evolved for the efficient replication of important genetic information.

                               The applications of Recombinase - mediated cassete exchange is in

• Generation of transgenic animals      :   Generation of transgenic knock out/in mice and their genetic modification by RCME.
• Tagging and cassete exchange in DG44 cells in suspension culture               :    Insertion of a target cassete in a mammalian host cell line and exchange with an ER stress reporter construct via targeted integration.

                            In short Recombinase mediated combinase exchange provids a versatile platform for gene targetting.