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Technology

This technology takes advantage of the Cre-lox system. The enzyme Cre recombinase can catalyze the site-specific recombination of DNA strands encoding corresponding LoxP sequences. Recombinations can lead to either the deletion or the flipping of the DNA sequence between the two LoxP sites. In this invention, two LoxP pairs along a DNA insert allow for Cre-induced recombination to occur, which simultaneously deletes a strand of DNA encoding an antibiotic resistance gene and the Thy1.1 cell surface marker protein and flips another antisense strand of DNA into the sense orientation. This strand of DNA contains a selectable marker, such as a gene for a fluorescent marker, as well as a 3' UTR (untranslated region) that encodes a micro-RNA which can knock down a gene of interest. The sequence is engineered with self-cleavage at the C-terminal of the fluorescent marker protein upon protein translation. The 3' UTR, which encodes the miRNA sequence, is engineered for high levels of transcription. This elegant system can be customized with different selectable markers, different antibiotic resistance genes, different miRNA knockdown sequences, and different promoters for gene expression. Moreover, this method leaves the doxycycline/tetracycline channel open, introducing the possibility of creating a  gene knockdown system that is both cell-type-specific and time-dependent.