STOCK Notch1^tm4(cre)Rko/J

Stock number: 027234
Product name: N1IP::Cre^HI
Research areas: Cancer Research, Cell Biology Research, Developmental Biology Research, Neurobiology Research, Research Tools

Description

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.

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).

Allele

Symbol: Notch1^tm4(cre)Rko
Name: targeted mutation 4, Raphael Kopan
MGI accession ID: MGI:5009236
Generation method: Targeted
Attributes: Recombinase-expressing
Marker symbol: Notch1
Marker name: notch 1
Chromosome: 2
Strain of origin: 129X1/SvJ
Expressed genes: cre,cre recombinase,bacteriophage P1
Site of expression: Endothelial cells.
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.