A Novel Cre/lox71-Based System for Inducible Expression of Recombinant Proteins and Genome Editing

Abstract

In this study, we developed a novel Cre/lox71-based system for the controlled transient expression of target genes. We used the bacteriophage P1 Cre recombinase, which harbors a short, highly specific DNA-binding site and does not have endogenous binding sites within mouse or human genomes. Fusing the catalytically inactive form of Cre recombinase and the VP64 transactivation domain (VP16 tetramer), we constructed the artificial transcription factor Cre-VP64. This transcription factor binds to the lox71 sites within the promoter region of the target gene and, therefore, upregulates its expression. We tested the Cre-VP64/lox71 system for the controlled expression of several genes, including growth factors and the genome editor CRISPR/Cas9, and obtained superior efficiency in the regulation of transgene expression, achieving a high expression level upon induction together with low basal activity. This system or its modified forms can be suggested as a novel effective tool for the transitory controlled expression of target genes for functional genomic studies, as well as for gene therapy approaches.

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“…The same approach turned out to be ineffective in an ASC52telo mesenchymal stem line: a pair of gRNAs did not mediate excision of a miRNA-encoded DNA fragment, although sequencing results identified point indels within the cut sites of both gRNAs [ 72 ]. Presumably, this was due to a less effective expression of the Cas9 editor in ASC52telo cells compared to HEK293T, which may be compensated for via the use of ribonucleoprotein Cas9*gRNA complexes or by using a powerful inducible transgene expression system [ 73 , 74 , 75 ].…”