New variants of inducible Cre recombinase: a novel mutant of Cre-PR fusion protein exhibits enhanced sensitivity and an expanded range of inducibility

• Published in: Nucleic acids research 2001-05, Vol.29 (10), p.E47-47
• Main Authors: Wunderlich, F T, Wildner, H, Rajewsky, K, Edenhofer, F
• Format: Article
• Language: English
• Subjects: Amino Acid Substitution - genetics; Animals; Bacteriophage P1 - enzymology; Bacteriophage P1 - genetics; Base Sequence; Binding Sites; Cell Line; Cre recombinase; Enzyme Induction - drug effects; Gene Expression Regulation - drug effects; Gene Targeting - methods; Humans; Integrases - biosynthesis; Integrases - genetics; Integrases - metabolism; Ligands; Mifepristone - pharmacology; Mutation - genetics; NAR Methods Online; Protein Structure, Tertiary; Receptors, Progesterone - chemistry; Receptors, Progesterone - genetics; Receptors, Progesterone - metabolism; Recombinant Fusion Proteins - biosynthesis; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Recombination, Genetic - genetics; RNA Splice Sites - genetics; RNA Splicing - genetics; RNA, Messenger - genetics; RNA, Messenger - metabolism; RU486; Sensitivity and Specificity; Transfection; Viral Proteins
• ISSN: 1362-4962; 0305-1048; 1362-4962
• DOI: 10.1093/nar/29.10.e47

We have developed a novel inducible Cre mutant with enhanced recombinase activity to mediate genetic switching events. The protein, designated Cre*PR, is composed of a new Cre mutant at the N-terminus followed by the ligand-binding domain (LBD) of the progesterone receptor (PR). The response to low doses of inducer is significantly enhanced by elongating the C-terminus of the PR LBD from amino acid 891 to 914. The mutant Cre lacks the first 18 amino acids and contains a Val-->Ala substitution at position 336, thereby destroying a cryptic splice donor at the 3'-end of CRE: The latter mutation reduces unwanted background recombinase activity in the absence of the synthetic ligand RU486 by a factor of at least 10 to an almost undetectable level. Thus, the recombinase activity turns out to be inducible by a factor of >200. We expect Cre*PR to serve as a valuable tool for conditional expression of genes both in vitro and in vivo.