New variants of inducible Cre recombinase: a novel mutant of Cre-PR fusion protein exhibits enhanced sensitivity and an expanded range of inducibility.
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.
Wunderlich
F T
FT
Institute for Genetics, University of Cologne, Weyertal 121, D-50931 Cologne, Germany.
Wildner
H
H
Rajewsky
K
K
Edenhofer
F
F
D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D017101:Bacteriophage P1 / Q000201:enzymology / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D004790:Enzyme Induction / Q000187:drug effects; D005786:Gene Expression Regulation / Q000187:drug effects; D018390:Gene Targeting / Q000379:methods; D006801:Humans; D019426:Integrases / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D008024:Ligands; D015735:Mifepristone / Q000494:pharmacology; D009154:Mutation / Q000235:genetics*; D017434:Protein Structure, Tertiary; D022821:RNA Splice Sites / Q000235:genetics; D012326:RNA Splicing / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011980:Receptors, Progesterone / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic / Q000235:genetics*; D012680:Sensitivity and Specificity; D014162:Transfection; D014764:Viral Proteins
D008024:Ligands; D022821:RNA Splice Sites; D012333:RNA, Messenger; D011980:Receptors, Progesterone; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D015735:Mifepristone; C045073:Cre recombinase; D019426:Integrases