Overview

Also Known As:Calb1-2A-dgCre-D

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.

Donating Investigator

Hongkui Zeng, Allen Institute for Brain Science

Genetic overview

Genetic Background	Generation

Calb1^{tm1.1(folA/cre)Hze}

Allele Type	Gene Symbol	Gene Name
Targeted (Recombinase-expressing, Inducible)	Calb1	calbindin 1

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Research Applications

Research Tools
Cancer Research
Neurobiology Research

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Base Price

Starting at:

$2,854.50 Domestic price Cryo Recovery

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Details

Detailed Description

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 ecDHFR^R12Y/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 (Calb1-T2A-dgCre images).

The dgCre fusion gene (also called destabilized EGFP/Cre, DHFR-EGFP-Cre, hDHFR/EGFP/Cre or ecDHFR^R12Y/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 ecDHFR^R12Y/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).

Development

The Calb1-2A-dgCre-D targeted mutation (also called Calb1-T2A-dgCre-D, Calb1-2A-dgCre-Δ or Calb1-2A-dgCre-Δneo) has a viral 2A oligopeptide (T2A) that mediates ribosomal skipping and a destabilized EGFP/Cre fusion gene (dgCre) inserted immediately downstream of the calbindin 1 translational STOP codon. The specific details are below.

The targeting vector contained, from 5' to 3',
a partial Calb1 intron 6 through intron 10 sequence containing an frt3 site in intron 10,
a partial Calb1 exon 11 sequence up to (but not including) the endogenous stop codon,
a T2A sequence that is in-frame with the Calb1 coding sequence,
a dgCre fusion gene (described below) that is in-frame with the Calb1 coding sequence,
a bovine growth hormone polyA signal,
an AttB site,
a PGK-Neo-polyA cassette,
an frt5 site,
an mRNA splice acceptor,
the 3' portion of the hygromycin gene with SV40 polyA signal,
and an AttP site.

The dgCre fusion gene (also called destabilized EGFP/Cre, DHFR-EGFP-Cre, hDHFR/EGFP/Cre or ecDHFR^R12Y/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 targeting vector was electroporated into (129S6/SvEvTac x C57BL/6)F1-derived G4 embryonic stem (ES) cells. Correctly targeted ES cells were injected into recipient blastocysts. The resulting chimeric animals were bred to PhiC31-expressing mice (C57BL/6J congenic background; see Stock No. 007743) to delete the AttB/AttP-flanked sequences (PGK-neo-polyA::frt5::mRNA splice acceptor::3'hygro::SV40 polyA) and replace it with the recombined AttB/AttP site (AttL). The resulting Calb1-2A-dgCre-D mice were bred with C57BL/6J wildtype mice for additional generations (and the PhiC31 transgene was removed) prior to sending generation N4F1 males to The Jackson Laboratory Repository in 2013. Upon arrival, males were used to cryopreserve sperm. To establish the living Calb1-2A-dgCre-D mouse colony, an aliquot of the frozen sperm was used to fertilize oocytes from C57BL/6J inbred females (Stock No. 000664).

Expression Data

Expressed Gene	GFP, Green Fluorescent Protein,
Expressed Gene	cre/folA, cre/folA,
Site of Expression	Pattern correlates well with endogenous Calb1 expression: after trimethoprim induced Cre recombinase activity, Cre-inducible reporter allele expression is detected in scattered cells of the cortex, hippocampus, cerebellum and striatum, and restricted cell populations in thalamus and hypothalamus.

Control Suggestions

Wild-type from the colony
000664 C57BL/6J

Additional Information

Considerations for Choosing Controls

Selected References

Evans RC; Zhu M; Khaliq ZM. 2017. Dopamine Inhibition Differentially Controls Excitability of Substantia Nigra Dopamine Neuron Subpopulations through T-Type Calcium Channels. J Neurosci 37(13):3704-3720PubMed: 28264982 MGI: J:243762

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Genetics

Calb1^{tm1.1(folA/cre)Hze}

Allele Symbol: Calb1^{tm1.1(folA/cre)Hze}

Allele Name	targeted mutation 1.1, Hongkui Zeng
Allele Type	Targeted (Recombinase-expressing, Inducible)
Allele Synonym(s)	Calb1^(dgCre)Hze; Calb1-2A-dgCre-D; Calb1-2A-dgCre-Delta; Calb1-2A-dgCre-DeltaNeo; Calb1-2A-DHFR-EGFP-Cre-D; Calb1-2A-ecDHFR^R12Y/Y100I/EGFP/Cre-D; Calb1-T2A-dgCre-D
Gene Symbol and Name	Calb1, calbindin 1
Gene Synonym(s)
Expressed Gene	GFP, Green Fluorescent Protein,
Expressed Gene	cre/folA, cre/folA,
Site of Expression	Pattern correlates well with endogenous Calb1 expression: after trimethoprim induced Cre recombinase activity, Cre-inducible reporter allele expression is detected in scattered cells of the cortex, hippocampus, cerebellum and striatum, and restricted cell populations in thalamus and hypothalamus.
Strain of Origin	(129S6/SvEvTac x C57BL/6NCrl)F1
Chromosome	4
Molecular Note	The targeting vector inserted a viral 2A oligopeptide (T2A, mediating ribosomal skipping and a destabilized EGFP/Cre fusion gene (dgCre) inserted immediately downstream of thecalbindin 1 translational STOP codon. The dgCre fusion gene (also called destabilized EGFP/Cre,DHFR-EGFP-Cre, hDHFR/EGFP/Cre or ecDHFR/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) harboringthe G67S mutation and modified to also include the R12Y/Y100I destabilizing domain mutations. A PGK-neo-polyA cassette flanked by AttB and AttP sites was also part of the construct. Correctly targeted ES cells were injected into recipient blastocysts. The resulting chimeric animals were bred to PhiC31-expressing mice to delete the AttB/AttP-flanked sequences and replace it with the recombined AttB/AttP site (AttL). Trimethoprim-treatment is required to stabilize the cre protein. No EGFP is detected with or without induction.

Disease/Phenotype

Disease Terms

Research Areas By Phenotype

This mouse can be used to support research in many areas including:

Research Tools
- Genetics Research
  - Mutagenesis and Transgenesis: Cre-lox System
  - Tissue/Cell Markers: Cre-lox System
  - Tissue/Cell Markers
  - Mutagenesis and Transgenesis
  - Tissue/Cell Markers: neurons
- Cre-lox System
  - Cre Recombinase Expression
  - Cre Recombinase Expression: Inducible
- Neurobiology Research
  - cell marker
Cancer Research
- Cre-lox System
  - Cre Recombinase expression: inducible
Neurobiology Research
- Cre-lox System
  - Cre Recombinase expression in neural tissue

Mammalian Phenotype Terms by Genotype

The following phenotype information is associated with a similar, but not exact match to this JAX^® Mice strain

Genotype: Calb1^{tm1.1(folA/cre)Hze}/Calb1⁺
involves: 129S6/SvEvTac * C57BL/6 * C57BL/6NCr

normal phenotype

no abnormal phenotype detected
- Heterozygous mice are viable and fertile with no gross physical or behavioral abnormalities.

References

Evans RC; Zhu M; Khaliq ZM. 2017. Dopamine Inhibition Differentially Controls Excitability of Substantia Nigra Dopamine Neuron Subpopulations through T-Type Calcium Channels. J Neurosci 37(13):3704-3720PubMed: 28264982 MGI: J:243762

Additional References

Gutierrez Herrera C; Girard F; Bilella A; Gent TC; Roccaro-Waldmeyer DM; Adamantidis A; Celio MR. 2019. Neurons in the Nucleus papilio contribute to the control of eye movements during REM sleep. Nat Commun 10(1):5225PubMed: 31745081 MGI: J:281478

Additional - Calb1^{tm1.1(folA/cre)Hze} related

Technical Support

CONTACT TECHNICAL SUPPORT

Genotyping Protocols
- Standard PCR:Calb1
- Genotyping resources and troubleshooting
Breeding Considerations

When maintaining a live colony, heterozygous mice may be bred to wildtype mice from the colony or to C57BL/6J inbred mice (Stock No. 000664). The phenotype of homozygous mice has not yet been determined (December 2013).
- Additional Breeding and Husbandry Support
Mating System
- Heterozygote x Wild-type
- Wild-type x Heterozygote
Citation
When using the Calb1-2A-dgCre-D mouse strain in a publication, please cite the originating article(s) and include JAX stock #023531 in your Materials and Methods section.

Animal Health Reports

Facility Barrier Level Descriptions

Production of mice from cryopreserved embryos or sperm occurs in a maximum barrier room, G200

Pricing & Availability

Cryo Recovery

Domestic
International

Live Mouse

Age

Genotype

Price

weeks

Cryorecovery - Not-For-Profit & Academic Pricing

Service/Product	Description	Price
	Heterozygous or wildtype for Calb1<tm1.1(folA/EGFP/cre)Hze>

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Frozen Mouse Embryo	B6.Cg-Calb1<tm1.1(folA/cre)Hze>/J Frozen Embryo	$3373.50
Frozen Mouse Embryo	B6.Cg-Calb1<tm1.1(folA/cre)Hze>/J Frozen Embryo	$3373.50

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Also Known As:Calb1-2A-dgCre-D

Donating Investigator

Genetic overview

Research Applications

Base Price

Detailed Description

Development

Expression Data

Control Suggestions

Additional Information

Selected References

Calb1tm1.1(folA/cre)Hze

Allele Symbol: Calb1tm1.1(folA/cre)Hze

Disease Terms

Research Areas By Phenotype

This mouse can be used to support research in many areas including:

Mammalian Phenotype Terms by Genotype

Genotype: Calb1tm1.1(folA/cre)Hze/Calb1+involves: 129S6/SvEvTac * C57BL/6 * C57BL/6NCr

normal phenotype

References

Additional References

Additional - Calb1tm1.1(folA/cre)Hze related

Genotyping Protocols

Breeding Considerations

Mating System

Citation

Animal Health Reports

Production of mice from cryopreserved embryos or sperm occurs in a maximum barrier room, G200

Live Mouse

Cryorecovery - Not-For-Profit & Academic Pricing

Related Products and Services

Payment Terms and Conditions

The Jackson Laboratory's Genotype Promise

Terms Of Use

Additional Use Restrictions Apply

Licensing Information

JAX® Mice, Products & Services Conditions of Use

No Warranty

Credit for PRODUCTS or SERVICES

Animal Care and Use for SERVICES

No Liability

Calb1^{tm1.1(folA/cre)Hze}

Allele Symbol: Calb1^{tm1.1(folA/cre)Hze}

Genotype: Calb1^{tm1.1(folA/cre)Hze}/Calb1⁺
involves: 129S6/SvEvTac * C57BL/6 * C57BL/6NCr

Additional - Calb1^{tm1.1(folA/cre)Hze} related