Genetically engineered mouse models are useful tools for studying gene functionsin vivo.The discovery of Cre recombinase from bacteriophage P1 to induce DNA sequence-specific recombination in mammalian cells has achieved big advances in mouse models.

Applications involving Cre recombination have included conditional mutagenesis, gene replacement and chromosome engineering in mice, and conditional gene expression. However, process of generating Cre-mediated genetically engineered mouse models is expensive and time consuming. Also, the use of site-specific recombination in genetic studies is often hampered by difficulties expressing the recombinase enzyme in specific-type of cells and the desired developmental stage. Moreover, even conditional mutants induced by tissue-specific Cre expression may interfere with tissue development, thus, precluding later studies in terminally differentiated cells.

To overcome the limitation, the delivery of proteins with cell-penetrating peptides (CPP) into cells across impermeable cell membranes has been proposed as an attractive strategy to deal with genetically engineered mouse models to define gene functions. The directly introduced cell-permeable recombinant protein is likely to be much easier, quicker, and less cost than other methods, such as using Cre mouse or viral delivery.

For this purpose, we havenewly invented improved cell-permeable (CP) Cre recombinant protein fused to novel hydrophobic CPP, aMTD, to greatly increase their solubility, yield, and efficiency of membrane penetrating ability. iCP-Cre recombinant proteins induced high levels of recombination in a variety of cultured cells and all tissues examined in various genetically engineered mice (ROSA26-LSL-LacZ, ROSA26-LSL-EYFP, ROSA26nT-nG, SOCS3f/f etc.) following intravenous (IV) or local administration for whole-body or organ-specific (brain, liver, kidney etc.) recombination in vivo.

The use of protein transduction using cell-permeable Cre recombinase for genome engineering in animalsprovides a rapid and efficient tools to define gene function in mouse models.