Overview

Also Known As:N1IP::Cre^HI

The high-activity Notch1 activation-dependent reporter knock-in allele, N1IP::Cre^HI, has the endogenous Notch1 intracellular domain (NICD1) replaced with a nuclear-localized Cre recombinase. Compared to N1IP::Cre^LO, this N1IP::Cre^HI allele has significantly improved sensitivity with five- to ten-fold increase in Cre activity. This improved sensitivity allows users to label cells experiencing moderate-to-lower Notch activation thresholds; including most cells/tissues known to utilize Notch1 during their development and/or maintenance.

Donating Investigator

Raphael Kopan, Cincinnati Children's Hospital Medical Center

Genetic overview

Genetic Background	Generation

Notch1^tm4(cre)Rko

Allele Type	Gene Symbol	Gene Name
Targeted (Recombinase-expressing)	Notch1	notch 1

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Research Applications

Research Tools
Neurobiology Research
Cancer Research
Cell Biology Research
Developmental Biology Research

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Base Price

Starting at:

$2,854.50 Domestic price Cryo Recovery

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Details

Detailed Description

The Notch1 activity-trap knock-in allele, N1IP::Cre^HI, has the endogenous Notch1 intracellular domain (NICD1) replaced by a Cre recombinase with nuclear localization signal. Like endogenous Notch1, the Notch1-Cre fusion protein is expressed on the cell membrane and its extracellular domain binds Notch ligands (-Delta, Serrate and Lag-2), but proteolytic cleavage of the transmembrane domain tether results in release of nuclear-localized Cre rather than NICD1. Cre then enters the nucleus, where it results in the excision of floxed sequences. If a Cre-dependent reporter is present, Cre recombinase activity will 'trap' the cells and permanently mark its descendent lineage independently of any further Notch1 activation. Specific cell types are described below.
Heterozygous mice are viable and fertile with no gross phenotypic abnormalities. Homozygotes die at embryonic day 9.5 (E9.5).

The Notch1 activation-dependent reporter knock-in alleles, N1IP::Cre^LO (Stock No. 006953), N1IP::Cre^ERT2 (Stock No. 027235) and N1IP::Cre^HI (Stock No. 027234) are compared below.

N1IP::Cre^LO (and N1IP::Cre^ERT2 in the presence of tamoxifen) is relatively insensitive and seems to predominantly 'trap' cells in which moderate-to-high levels of sustained Notch activity or repeated activation cycles are known to occur (e.g., endothelium), while missing cells in many tissues known to rely on Notch1 activity (e.g., the hematopoietic system). The donating investigator speculates these differences could be attributed to the reduced transcript stability of the NICD (due to the insertion of 6xMyc-tags at the C-terminus of the nuclear-localized Cre recombinase). Also, in some tissues, sequences required for proper membrane trafficking of Notch may have been lost when NICD1 was replaced with Cre recombinase.

The N1IP::Cre^HI allele lacks the C-terminal 6xMyc-tag and has one more additional polyadenylation signal following the nuclear-localized Cre recombinase. These improvements restore the Cre recombinase C-terminus and increase transcript stability two-fold, respectively; resulting in a cumulative five- to ten-fold increase in Cre activity. The resulting Notch1-Cre fusion protein is cleaved as efficiently as endogenous Notch1, and has significantly improved sensitivity compared to N1IP::Cre^LO. This improved sensitivity allows users to 'trap' cells experiencing moderate-to-lower Notch activation thresholds; including most cells/tissues known to utilize Notch1 during their development and/or maintenance.

Development

The high-activity Notch intramembrane proteolysis-Cre knock-in allele (Notch1::Cre^HI, N1::Cre^HI, or N1IP::Cre^HI) was designed by Dr. Raphael Kopan (while at Washington University School of Medicine, St. Louis). First, the targeting construct for Notch1::Cre^LO (Stock No. 006953) was modified to remove the C-terminal 6xMyc epitope tag (restoring the original C-terminus), to add a third SV40 polyadenylation signal following the Cre recombinase sequence (to match the number of polyadenylation signals in wildtype Notch1 and to improve mRNA stability), and to introduce a single nucleotide variation in exon 28 to facilitate genotyping (C5178A; corresponding to a silent mutation at the third position for L1726). The final construct was designed to replace Notch1 exons 29-34 (encoding the intracellular domain) with a Cre recombinase sequence and nuclear localization signal, followed immediately downstream by a 3xSV40 polyadenylation sequence, and frt-flanked Pgk1/EM7 promoter-driven neomycin selection cassette. The construct was electroporated into 129X1/SvJ-derived SCC10 embryonic stem (ES) cells. Correctly targeted ES cells were injected into blastocysts to generate chimeric mice. Animals that transmitted the N1IP::Cre^HI allele through the germ line were obtained.

Mice were bred with C57BL/6N-congenic Flp-deleter mice (Stock No. 019100) to remove the selection cassette. After this, N1IP::Cre^HI mice were bred to remove the Flp-deleter transgene.

At some point, N1IP::Cre^HI mice were also bred with C57BL/6-congenic R26-LSL-lacZ reporter mice (Stock No. 003474) and
C57BL/6J-congenic Ai3(RCL-EYFP) reporter mice (Stock No. 007903). The R26-LSL-lacZ and Ai3(RCL-EYFP) alleles were later bred away.

N1IP::Cre^HI mice on a mixed C57BL/6;129SV;CD1 genetic background were sent to The Jackson Laboratory in 2015. Upon arrival, males were used to cryopreserve sperm. To establish the living N1IP::Cre^HI 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	cre, cre recombinase, bacteriophage P1
Site of Expression	Endothelial cells.

Control Suggestions

Wild-type from the colony

Additional Information

Considerations for Choosing Controls

Selected References

Liu Z; Turkoz A; Jackson EN; Corbo JC; Engelbach JA; Garbow JR; Piwnica-Worms DR; Kopan R. 2011. Notch1 loss of heterozygosity causes vascular tumors and lethal hemorrhage in mice. J Clin Invest 121(2):800-8PubMed: 21266774 MGI: J:171829

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Genetics

Notch1^tm4(cre)Rko

Allele Symbol: Notch1^tm4(cre)Rko

Allele Name	targeted mutation 4, Raphael Kopan
Allele Type	Targeted (Recombinase-expressing)
Allele Synonym(s)	N1::Cre^hi; NIP::Cre^HI; Notch1::cre^Hi
Gene Symbol and Name	Notch1, notch 1
Gene Synonym(s)
Expressed Gene	cre, cre recombinase, bacteriophage P1
Site of Expression	Endothelial cells.
Strain of Origin	129X1/SvJ
Chromosome	2
Molecular Note	The Notch1 intracellular domain (IC) is replaced by the cre recombinase sequence and includes the SV40 3' UTR polyA signal while introducing a single nucleotide variation (SNV) at C5178A to facilitate genotyping. This allele does not have the 6xMyc tag present in the Notch1^tm3(cre)Rko allele, and displays enhanced activity relative to that allele.

Disease/Phenotype

Disease Terms

Research Areas By Phenotype

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

Research Tools
- Cancer Research
  - Cre-lox System
- Cardiovascular Research
  - Cre-lox System
- Cre-lox System
  - Cre Recombinase Expression
- Developmental Biology Research
  - Cre-lox System
- Genetics Research
  - Mutagenesis and Transgenesis: Cre-lox System
  - Tissue/Cell Markers: Cre-lox System
  - Mutagenesis and Transgenesis
  - Tissue/Cell Markers
  - Tissue/Cell Markers: multiple
- Reproductive Biology Research
  - Cre-lox System
Neurobiology Research
- Cre-lox System
  - Cre Recombinase expression in neural tissue
Cancer Research
- Genes Regulating Growth and Proliferation
- Cre-lox System
Cell Biology Research
- Genes Regulating Growth and Proliferation
- Signal Transduction
Developmental Biology Research
- Embryonic Lethality (Homozygous)

Mammalian Phenotype Terms by Genotype

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

Genotype: Notch1^tm2Rko/Notch1^tm4(cre)Rko
involves: 129X1/SvJ * C57BL/6

cardiovascular system phenotype

abnormal vitelline vasculature morphology
- defective angiogenic remodeling of the yolk sac vasculature is observed

embryo phenotype

abnormal vitelline vasculature morphology
- defective angiogenic remodeling of the yolk sac vasculature is observed

mortality/aging

embryonic lethality during organogenesis, complete penetrance
- death at E10.5 is observed, likely resulting from higher loss of heterozygosity (LOH) frequency compared to that in Notch1^tm2Rko/Notch1^tm3(cre)Rko embryos; higher LOH occurs because of improved cre expression

References

Liu Z; Turkoz A; Jackson EN; Corbo JC; Engelbach JA; Garbow JR; Piwnica-Worms DR; Kopan R. 2011. Notch1 loss of heterozygosity causes vascular tumors and lethal hemorrhage in mice. J Clin Invest 121(2):800-8PubMed: 21266774 MGI: J:171829

Additional - Notch1^tm4(cre)Rko related

Technical Support

CONTACT TECHNICAL SUPPORT

Genotyping Protocols
- Standard PCR:Notch1
- Genotyping resources and troubleshooting
Breeding Considerations

When maintaining a live colony, heterozygous mice are bred to wildtype mice from the colony. Homozygotes die in utero.
- Additional Breeding and Husbandry Support
Citation
When using the N1IP::Cre^HI mouse strain in a publication, please cite the originating article(s) and include JAX stock #027234 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 - Pricing

Service/Product	Description	Price
	Heterozygous or wildtype for Notch1<tm4(cre)Rko>

Related Products and Services

Frozen Mouse Embryo	STOCK Notch1<tm4(cre)Rko>/J	$2595.00
Frozen Mouse Embryo	STOCK Notch1<tm4(cre)Rko>/J	$2595.00
Frozen Mouse Embryo	STOCK Notch1<tm4(cre)Rko>/J	$3373.50
Frozen Mouse Embryo	STOCK Notch1<tm4(cre)Rko>/J	$3373.50

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The Jackson Laboratory has rigorous genetic quality control and mutant gene genotyping programs to ensure the genetic background of JAX® Mice strains as well as the genotypes of strains with identified molecular mutations. JAX® Mice strains are only made available to researchers after meeting our standards. However, the phenotype of each strain may not be fully characterized and/or captured in the strain data sheets. Therefore, we cannot guarantee a strain's phenotype will meet all expectations. To ensure that JAX® Mice will meet the needs of individual research projects or when requesting a strain that is new to your research, we suggest ordering and performing tests on a small number of mice to determine suitability for your particular project. We do not guarantee breeding performance and therefore suggest that investigators order more than one breeding pair to avoid delays in their research.

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Also Known As:N1IP::CreHI

Donating Investigator

Genetic overview

Research Applications

Base Price

Detailed Description

Development

Expression Data

Control Suggestions

Additional Information

Selected References

Notch1tm4(cre)Rko

Allele Symbol: Notch1tm4(cre)Rko

Disease Terms

Research Areas By Phenotype

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

Mammalian Phenotype Terms by Genotype

Genotype: Notch1tm2Rko/Notch1tm4(cre)Rkoinvolves: 129X1/SvJ * C57BL/6

cardiovascular system phenotype

embryo phenotype

mortality/aging

References

Additional - Notch1tm4(cre)Rko related

Genotyping Protocols

Breeding Considerations

Citation

Animal Health Reports

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

Live Mouse

Cryorecovery - 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

Also Known As:N1IP::Cre^HI

Notch1^tm4(cre)Rko

Allele Symbol: Notch1^tm4(cre)Rko

Genotype: Notch1^tm2Rko/Notch1^tm4(cre)Rko
involves: 129X1/SvJ * C57BL/6

Additional - Notch1^tm4(cre)Rko related