Conclusion
We provide a new high-efficiency cloning system for Rosa26 knock-in constructs to express either cDNA or miRNA fragments. Our system will enable high-throughput approaches for controlled expression of cDNA or miRNA constructs, with the latter providing a potential high-speed alternative for conditional knock-out models.
Results
We have optimized the cloning procedure to generate targeting vectors for Cre-regulated expression of constructs within several days with minimal hands-on time, thereby enabling high-throughput approaches. We demonstrate that transient expression of Cre still results in expression of the construct, as shown by the expression level and via functional assays. In addition to its well-established possibilities in expressing cDNA constructs, we show that the Rosa26 locus can be used to drive expression of functional miRNA constructs from its endogenous promoter.