The Calb1-2A-dgCre-D (or Calb1-T2A-dgCre-D) targeted mutation has a viral 2A oligopeptide (T2A) that mediates ribosomal skipping and a destabilized EGFP/Cre fusion gene (dgCre) inserted downstream of the calbindin 1 translational STOP codon. This is designed to have both endogenous gene and dgCre expression directed to Calb1-expressing cells by the endogenous promoter/enhancer elements. 

The ecDHFRR12Y/Y100I domain of dgCre leads to proteosomal degradation of the entire EGFP/Cre fusion protein, resulting in reduced overall Cre recombinase activity. Administration of the DHFR inhibitor, trimethoprim (TMP), prevents degradation of the dgCre fusion gene and results in increased Cre recombinase activity. The EGFP sequences included in the DHFR-Cre cassette contribute to the destabilization of the entire dgCre fusion protein in the absence of TMP.

When Calb1-2A-dgCre-D mice are bred with mice containing loxP-flanked sequences, TMP-enhanced Cre recombination will result in deletion of floxed sequences in the Calb1-expressing cells of the double mutant offspring. 

Specifically, the donating investigator reports that Calb1-2A-dgCre-D mice have trimethoprim-inducible increases in Cre recombinase activity (reporter gene expression) detected at high levels in scattered cells of the cortex, hippocampus, cerebellum and striatum, and restricted cell populations in thalamus and hypothalamus, in a pattern that correlates well with endogenous Calb1 expression. In the absence of trimethoprim, these same regions exhibit small-to-moderate reductions in Cre recombinase activity that is variable between brain regions. The donating investigator reports no EGFP fluorescence with or without TMP. Immunohistochemical detection of EGFP levels, if any, have not yet been evaluated in the presence or absence of TMP (December 2013).

Heterozygous mice are viable and fertile with no gross physical or behavioral abnormalities. The donating investigator did not examine dgCre activity in tissues other than brain, and did not attempt to generate homozygous mice to date (December 2013).

For characterization information, see images at the Allen Institute for Brain Science website (<a href=http://connectivity.brain-map.org/transgenic/imageseries/list/1.html?gene_term=Calb1-T2A-dgCre>Calb1-T2A-dgCre images</a>). 

The dgCre fusion gene (also called destabilized EGFP/Cre, DHFR-EGFP-Cre, hDHFR/EGFP/Cre or ecDHFRR12Y/Y100I/EGFP/Cre) is an enhanced green fluorescent protein/Cre recombinase fusion gene with an N terminal fusion of 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 dgCre leads to proteosomal degradation of the entire fusion protein, resulting in reduced 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 dgCre fusion gene, resulting in increased Cre recombinase activity. The EGFP sequences included in the DHFR-Cre cassette contribute to the destabilization of the entire dgCre fusion protein in the absence of TMP. The donating investigator reports no EGFP fluorescence with or without TMP. Immunohistochemical detection of EGFP levels, if any, have not yet been evaluated in the presence or absence of TMP (December 2013).

Calb1-2A-dgCre-D (or Calb1-T2A-dgCre-D) mice express a trimethoprim-inducible EGFP/Cre fusion gene directed by endogenous calbindin 1 promoter/enhancer elements. When induced, small-to-moderately increased Cre recombinase activity is directed at high levels to scattered cells of the cortex, hippocampus, cerebellum and striatum, and restricted cell populations in thalamus and hypothalamus.

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B6.Cg-Calb1<tm1.1(folA/cre)Hze>/J Frozen Embryo