The Fezf1-2A-dCre-D targeted mutation (also called Fezf1-2A-dCre-Δhygro) has a viral 2A oligopeptide sequence (T2A) that mediates ribosomal skipping and a destabilized Cre fusion gene (dCre) inserted downstream of the Fez family zinc finger 1 coding region. This is designed to have both endogenous gene and dCre expression directed to Fezf1-expressing cells by the endogenous promoter/enhancer elements.
The ecDHFRR12Y/Y100I domain of dCre leads to proteosomal degradation of the entire fusion protein, resulting in reduced overall Cre recombinase activity. Administration of the DHFR inhibitor, trimethoprim (TMP), prevents degradation of the dCre fusion gene and results in small increases in Cre recombinase activity.
When Fezf1-2A-dCre-D mice are bred with mice containing loxP-flanked sequences, TMP-enhanced Cre recombination will result in deletion of floxed sequences in the Fezf1-expressing cells of the double mutant offspring. Specifically, the donating investigator reports that Fezf1-2A-dCre-D mice have trimethoprim-inducible increases in Cre recombinase activity (reporter gene expression) detected in restricted populations within striatum and hypothalamus, as well as scattered positive cells in amygdala and cortex, in a pattern that correlates well with endogenous Fezf1 expression. In the absence of trimethoprim, small reductions in Cre recombinase activity are observed in these brain regions. Heterozygous mice are viable and fertile with no gross physical or behavioral abnormalities. The donating investigator did not examine dCre activity in tissues other than brain, and did not attempt to generate homozygous mice to date (September 2014).
For characterization information, see images at the Allen Institute for Brain Science website (Fezf1-2A-dCre images).
The dCre fusion gene (also called destabilized Cre, hDHFR/Cre or ecDHFRR12Y/Y100I/Cre) is Cre recombinase fused at its N terminus to the first 159 amino acids of the Escherichia coli K-12 strain chromosomal dihydrofolate reductase gene (DHFR or folA) harboring the G67S mutation and modified to also include the R12Y/Y100I destabilizing domain mutations. The ecDHFRR12Y/Y100I domain of dCre leads to proteosomal degradation of the entire fusion protein, resulting in little or no Cre recombinase activity. The donating investigator reports that administration of the high affinity DHFR inhibitor trimethoprim (TMP; at a concentration of 0.25-0.30 mg/g body weight) prevents degradation of the dCre fusion gene, resulting in Cre recombinase activity.
