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Published in final edited form as: Methods Mol Biol. 2014;1187:47–61. doi: 10.1007/978-1-4939-1139-4_4

Overview of genetic tools and techniques to study Notch signaling in mice

Thomas Gridley 1, Andrew K Groves 2
PMCID: PMC4591040  NIHMSID: NIHMS725218  PMID: 25053480

Abstract

Aberrations of Notch signaling in humans cause both congenital and acquired defects and cancers. Genetically-engineered mice provide the most efficient and cost-effective models to study Notch signaling in a mammalian system. Here, we review the various types of genetic models, tools, and strategies to study Notch signaling in mice, and provide examples of their use. We also provide advice on breeding strategies for conditional mutant mice, and a protocol for tamoxifen administration to mouse strains expressing inducible Cre recombinase-estrogen receptor fusion proteins.

Keywords: Conditional mutations, Notch reporter lines, Notch receptor-Cre fusions, Fate mapping, Lineage analysis, Domain-swap mice, Tamoxifen administration

1. Introduction

The Notch signaling pathway is an evolutionarily ancient form of cell-cell communication. In vertebrates it plays a central role in the development and homeostasis of most major organ systems and has been implicated in many forms of hereditary and idiopathic diseases, including cancer. The many complexities of the Notch signaling pathway have been described extensively elsewhere (1–4). In this chapter, we describe how the laboratory mouse has been developed to study many aspects of Notch signaling in development and disease. In addition to the huge number of different genetically modified mouse lines that have been generated to cause loss or gain of function of different components of the Notch signaling pathway, recent years have seen the advent of new mouse lines to visualize Notch-responsive cells in the intact animal, to follow the fates of cells that have experienced Notch signaling, and to probe the structure-function relationships between different mammalian Notch receptors. In this chapter, we describe the current status of transgenic mouse technology that has addressed these questions, and provide some basic protocols for breeding and working with conditional mutants of the Notch pathway.

2. Loss and gain of function Notch pathway mutant mice

2.1. Constitutive loss of function mutant mice

The many different components of the Notch signaling pathway (2) has led to the generation of a huge number of genetically modified and mutagenesis-induced mouse lines that have loss of function in Notch signaling. As described in Section 6 below, there are comprehensive online databases that allow the investigator to search for a wide variety of different alleles of genes in the Notch pathway. Historically, these alleles were generated piecemeal in individual laboratories. More recently however, an international project has aimed to systematically produce targeted mutations in ES cells for every mouse gene (5, 6) (www.knockoutmouse.org/). Many of these ES cell lines have already been used to generate mouse lines, and an initial systematic analysis of these lines is ongoing (7). Individual investigators may order targeted ES cells from repositories derived from these projects, and in addition to generating mutant mice from such cell lines, it will be possible to re-engineer such lines for new purposes (for example, expression of fluorescent proteins, recombinases, optogenetic channels, calcium indicator proteins, or to add epitope tags) using technology such as recombination-mediated cassette exchange (8, 9).

2.2. Conditional loss of function mutant mice

The early embryonic lethality exhibited by many constitutive Notch pathway loss of function mutants, that is often a result of defects in formation of the embryonic vasculature (10), has emphasized the importance of generation of conditional and/or inducible Notch pathway mutants to facilitate study of Notch pathway function later in embryogenesis, and in the postnatal and adult mouse.

2.2.1: Generating conditional mutant mice with the Cre-Lox system

The ability of the P1 bacteriophage Cre recombinase to recombine palindromic LoxP sites at high efficiency (11) in mammalian cells (12), even when separated by large intervening DNA sequences, has made it the method of choice for creating spatially and temporally controlled gain and loss of function in mice (13–15). A huge armory of transgenic and knock-in lines expressing Cre recombinase under the control of different promoters and enhancers allows manipulation of genes in most tissues. Temporal control of Cre activity is also possible using fusions of the Cre protein with modified forms of the estrogen receptor, which allow nuclear localization and activation of Cre following administration of the estrogen analogue tamoxifen (16–20). Cre has also been fused with the progesterone receptor in a similar fashion to yield mice in which the recombinase activity can be activated by Mifepristone (RU486) (21), although this strategy is used far less than Cre-estrogen receptor fusions (22). Similarly, although the FLP-FRT recombination system has also been used to drive recombination in the mouse genome, its use has tended to be reserved for intersectional recombination strategies (23), or for the removal of selection cassettes following the construction of targeted alleles (24).

Many conditional alleles have been generated for components of the Notch signaling pathway. Conditional alleles are available for the Notch1, Notch2, and Notch3 receptors, for the Dll1, Dll4, Jag1 and Jag2 ligands, as well as several non-canonical ligands such as DNER, Contactin1 and MAGP1 (25). Conditional alleles have also been generated for modifying and processing enzymes of Notch and its ligands, for nuclear co-activators and for direct downstream targets of Notch signaling. Section 6 gives advice for identifying and locating mouse mutants for various Notch signaling pathway components. More recently, the advent of large scale mouse targeting projects, such as the Knockout Mouse Project (KOMP) and the European Conditional Mouse Mutagenesis Program (EUCOMM), has generated a large number of new targeted mutations of genes in the Notch pathway. Many of these alleles have used the “knockout first” targeting strategy, in which a targeted null allele can be converted to a conditional allele by mating with ubiquitously-expressing FLP lines (26).

In addition to conditional “floxed” alleles, some mouse lines have been developed to produce dominant-negative mutants of Notch pathway components. For example, ROSA26 dnMAML1-GFP mice express a Cre-inducible fusion protein of a truncated Mastermind-like1 (MAML1) and GFP knocked into the ROSA26 locus (27, 28). This fusion protein is capable of binding Rbpj/CSL, but cannot recruit transcriptional co-activators to this complex and thus acts as a dominant-negative protein. The use of a dominant loss-of-function mutation can make breeding strategies easier; however, it is possible that two mutant alleles may be required to give a strong loss of Notch signaling in some tissues.

2.2.2: Selection and verification of Cre driver lines

Several databases listing Cre driver lines have been developed (29). The Mouse Genome Informatics database maintains and curates an extensive and rapidly growing list of published Cre driver lines (www.informatics.jax.org/recombinase.shtml) that target many different tissues, cell populations and stages of development. The activity and recombination pattern of these lines can be monitored by crossing them with a variety of Cre reporter lines, in which the expression of a fluorescent or enzyme marker is activated after Cre recombination. Many of these lines are available from the Jackson Laboratory (cre.jax.org/crereporters.html), which also maintains a Cre driver line characterization pipeline (30). Additional Cre driver databases are maintained in Canada and Europe (31, 32).

It is strongly recommended that each investigator independently verifies the activity of a Cre driver line imported into their mouse colony before starting a conditional knockout experiment. It is common for Cre lines to produce patterns of recombination that are partially different from their first published descriptions. This can be due to differences in genetic background, or to differing sensitivities of the Cre reporter lines used. This advice is doubly true for studies using tamoxifen-inducible versions of Cre recombinase. Here, the degree and efficiency of recombination is exquisitely sensitive to the dose and route of administration of tamoxifen, and careful monitoring of recombination efficiency is essential for correct interpretation of results using such mice. If possible, it is recommended to include a Cre reporter allele alongside a conditional allele, so that recombination can be observed directly in the conditional mutant strain. A protocol for tamoxifen administration is given below (Section 2.2.4).

2.2.3: Breeding strategies for generation of conditional mouse mutants

A typical breeding scheme to generate conditional loss-of-function mouse mutants using the Cre-LoxP system is shown in Figure 1. In brief, a Cre driver mouse is crossed with a mouse from a second line that is heterozygous for a null allele of the gene of interest. The offspring from this cross are screened for mice carrying both the Cre and null alleles. These mice are then crossed with mice carrying two alleles of the gene of interest that has all or part of its coding region flanked by loxP sites (“floxed” or “flox” alleles). Approximately 25% of the resulting offspring will inherit the Cre allele, a null allele and a floxed allele of the gene of interest. These mice will then display a loss of function of the gene of interest in cells in which the Cre recombinase is active. If a tamoxifen-inducible version of Cre recombinase is used, recombination will not occur until the mice are dosed with this drug.

Fig. 1.

Fig. 1

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A sample conditional breeding strategy to generate conditional loss of function mutations in Notch pathway genes. GOI = Gene of interest, for which a null allele (GOInull) conditional, “floxed” allele (GOIflox) are available. The Cre driver allele is referred to as DriverCre. This mating scheme will produce approximately 25% offspring that carry a conditional loss-of-function mutation.

Notes

  1. Anecdotal evidence suggests that recombination is more efficient if a null allele is crossed on to the Cre driver line as opposed to a floxed allele. This is presumably due to the presence of fewer loxP sites in the target genome. A recent published study has verified these anecdotal findings (33). A null allele can easily be generated from a floxed allele by mating with a ubiquitously-expressed Cre line.

  2. Since the maintenance of Cre driver; null allele mouse lines requires genotyping at each generation and does not yield 100% of the desired genotype, it is common to use male studs carrying these alleles, as a single stud can be mated repeatedly to multiple females. Since the propagation of a homozygous floxed line is far more efficient, homozygous floxed females are typically used in this mating scheme.

  3. Before commencing a conditional breeding strategy, it is always advisable to check the chromosomal location of the Cre driver allele and the gene of interest to be used in the project, as well as any reporter alleles that may be included in the breeding scheme. Although the probability of two given loci occurring on the same chromosome in mice is low, a quick check may save months of wasted time.

2.2.4: Protocol: Administration of tamoxifen to mice by oral gavage

1. Introduction

As described above, it is strongly recommended that investigators perform preliminary experiments to verify the fidelity of their Cre lines, and to determine the speed and efficiency of Cre-mediated recombination after administration of tamoxifen. It is possible to observe the first evidence of recombination within 6–8 hours of tamoxifen administration (34), although this will obviously depend on the strength of the regulatory sequences in each Cre driver line. It should also be noted that recombination may continue for days, or in some cases weeks, after cessation of tamoxifen administration (35), so studies that make use of pulses of tamoxifen should be interpreted with appropriate caution.

2: Materials

  1. Warm 5–6 ml corn oil (Sigma, C8267) in a 15 ml tube at 42°C for 30 minutes. Corn oil can be stored at room temperature, but replace every 3–6 months to prevent the oil from becoming rancid.

  2. Weigh out 100 mg tamoxifen (Sigma T5648) into a second 15 ml tube. Tamoxifen is light-sensitive, and should be stored in the dark at 4°C with a desiccant. Allow the tamoxifen to equilibrate to room temperature before weighing.

  3. Add 5 ml of the warm corn oil to the tamoxifen, wrap the tube in foil to protect from light and incubate in a 37°C water bath to dissolve. The solution should be vortexed frequently to aid solubilization, which usually takes several hours. There should be no precipitate left in the solution.

  4. The tamoxifen solution can be stored in the dark at 4°C wrapped in foil. Discard the solution after one month, or sooner if a precipitate starts to develop.

3: Methods

  1. Weigh each mouse on a balance before administering tamoxifen. Accurate recording of weight is important to obtain consistent dosing.

  2. Curved reusable metal gavage needles with a ball tip are available from a number of suppliers (for example, Fine Science Tools 18060-20) or from institutional veterinary staff. A 20 gauge needle with a tip of between 1–2 mm is suitable for adult mice. Alternatively, disposable needles are also available (for example, Fine Science Tools 18061-20).

  3. Connect the needle to a syringe and fill with tamoxifen solution. Grasp the mouse by the scruff of the neck, and secure the tail in the manner one would use for an intraperitoneal injection. It is important that the mouse be securely restrained but relatively relaxed.

  4. Introduce the needle tip into the side of the mouse’s mouth. Pass the needle tip to the rear of the mouth along the roof. Push the mouse’s head back and pass the needle fully down the esophagus into the stomach to deliver the tamoxifen dose. A minimum of force is required and the entire procedure should be performed as gently as possible to avoid distress. If the animal attempts to struggle, it is possible that the windpipe is obstructed – this should be avoided.

4: Notes

  1. Some studies have administered tamoxifen by intraperitoneal injection. While this may require less training to administer, many anecdotal reports suggest pregnant females show signs of discomfort from the added burden of corn oil in their abdomens.

  2. Adult mice can tolerate doses of tamoxifen as high as 0.3 mg/g body weight. Pregnant females and their litters are more sensitive to tamoxifen, so dosage should be determined empirically for each mouse strain. Females past the 10th day of pregnancy can typically tolerate as much as 0.12 – 0.13 mg/g body weight administered twice a day. Administration of tamoxifen at earlier stages can cause developmental problems or resorption of embryos, and the dose may need to be reduced appropriately (36).

  3. As tamoxifen is an estrogen analogue, it can cause complications with pregnancy, especially in the final few days before delivery. To prevent late fetal abortion, progesterone (Sigma P3972) can be administered with the tamoxifen, typically at 50% of the dose of tamoxifen.

  4. It is recommended that pregnant dams be disturbed as little as possible by laboratory and animal care staff for several days before and after delivery.

  5. Tamoxifen can be administered to neonatal pups in several ways. Direct intraperitoneal injection of tamoxifen works effectively, although care must be exercised when handling the pups. Alternatively, tamoxifen is known to be transmitted in maternal milk (37), so continued administration of tamoxifen to the mother will provide dosing to neonates, albeit in a more variable fashion.

  6. Tamoxifen is a carcinogen and appropriate caution should be taken when handling the powder and solution. Dispose of materials that have come into contact with tamoxifen in a manner consistent with local environmental safety regulations.

  7. All handling and use of animals requires approval by local monitoring agencies and should adhere to local and national animal care guidelines. It is strongly recommended that users learning the gavage procedure be trained by a veterinarian or veterinary technician, ideally by first practicing on an animal that has been euthanized.

2.3. Conditional gain of function mice

Transgenic mis-expression of Notch pathway components can have very strong biological effects, such that constitutive gain of function transgenic or knock-in alleles are often lethal, or difficult to maintain. This has led a number of investigators to generate conditional gain of function mice, whose expression is activated by the Cre-Lox system. These alleles contain a transcriptional stop sequence, flanked on either side by a loxP site, located between the promoter and the coding sequences for the Notch pathway gene that will be expressed. These alleles have been generated either as transgenes or as knock-in alleles into the widely-expressed ROSA26 locus. Commonly, the gene being mis-expressed is the transcriptionally-active Notch intracellular domain (NICD). For example, mice that conditionally express NICD1 (38) or NICD2 (39) have been reported; many additional publications using these or similar mice exist. Aifantis and colleagues recently reported generation of four knock-in strains, one for each NICD (1 through 4) (40). Each line used the same allele design, in which coding sequences for the NICD were inserted into the ROSA26 locus behind a loxP-stop-loxP cassette. NICD coding sequence was followed by an internal ribosome entry site-yellow fluorescent protein (IRES-YFP) cassette to facilitate monitoring of expression. Interestingly, when activated by a human ubiquitin C promoter, only the NICD1 line was capable of causing T cell leukemia, despite comparable levels of expression of all four different NICDs, demonstrating sequence-specific differences among the four NICDs in vivo (40), similar to those previously described in vitro (41).

3. Notch signaling reporter lines

Signaling pathway-specific transgenic reporter lines are very useful in analyzing pathway activation in both wildtype and mutant genetic backgrounds. These lines generally contain a gene encoding a reporter protein, such as β-galactosidase or a fluorescent protein, under the control of a promoter that is transcriptionally regulated by the specific pathway being analyzed (e.g., the Notch pathway). The two most commonly used transgenic Notch reporters are the Transgenic Notch Reporter (TNR) EGFP reporter line (42), and the Notch Activity Sensor (NAS) nuclear lacZ reporter (43). Neither of these Notch reporter lines, however, has gained the extremely wide utilization of the Wnt pathway transgenic reporter lines TopGal (44) and BatGal (45). The TNR line utilizes a synthetic promoter consisting of four Rbpj binding sites linked to the basal SV40 promoter to drive EGFP expression, while the NAS reporter uses 12 multimerized Rbpj binding sites upstream of a minimal promoter from the beta globin gene. Other Notch reporters use endogenous Notch target genes containing Rbpj binding sites to drive reporter gene expression. Transgenic or knock-in reporters utilizing the Hes1 (46) and Hes5 (46–48) promoters to drive β-galactosidase or EGFP expression have been described.

Novel transgenic Notch pathway reporters continue to be developed. Two groups have developed new reporters that utilize improved, brighter fluorescent proteins in order to better enable in vivo or ex vivo imaging at the single cell level, as well as flow cytometry analyses. The Hes1-emGFP line is a knock-in allele that places the emerald variant of the GFP protein under the transcriptional control of the endogenous Hes1 promoter (49). Hes1-emGFP mice have been used in the analysis of Notch signaling in intestinal stem cells (49), during mammary gland development (including the sorting by flow cytometry of emGFP-expressing cells from the mammary gland) (50), and in the analysis of Notch pathway activation in specific hematopoietic cell lineages during both normal and stress hematopoiesis (40). The CBF:H2B-Venus transgenic line (Cbf1 is an alternative symbol for the Rbpj gene) expresses a fusion protein comprising human histone H2B (to provide nuclear localization) sequences linked to the yellow fluorescent protein Venus (51), and uses the same synthetic Notch target promoter as the TNR reporter line (42). The sensitivity of the CBF:H2B-Venus reporter facilitated identification of new sites of Notch signaling in embryos, particularly within the epiblast of the early postimplantation embryo, and could be used for live cell imaging in ES cells, primary mouse embryonic fibroblasts, and in cultured postimplantation embryos (51).

4. Notch receptor-Cre fusions for fate mapping and functional analyses

One criticism of Notch reporter lines such as those described in the preceding section, which utilize promoters containing synthetic arrangements of Rbpj binding sites or natural Notch target promoters containing Rbpj binding sites, is that they do not distinguish signal reception mediated by the different Notch receptor proteins. Several groups have generated knock-in or transgenic lines expressing fusions of Cre recombinase or the transcriptional activator with Gal4VP16 with various Notch receptors. These lines can be used for both fate mapping of Notch receptor-expressing cell lineages, or for functional analyses of these lineages.

Two groups have developed bi-transgenic Notch1 reporter systems that are dependent on gamma secretase-mediated proteolysis of a modified Notch1 gene to identify cells that have undergone active (i.e., proteolysis-mediated) Notch1 signaling. Kopan and colleagues used homologous recombination to generate a Notch1 knock-in allele in which sequence encoding the Notch1 (N1) intracellular domain downstream of the gamma secretase cleavage site were replaced with sequence encoding Cre recombinase (52). The line, currently named N1::CreLO (but referred to in the original publication as N1IP-Cre, for Notch1 Intramembrane Proteolysis-Cre), when crossed with a Cre reporter such as the ROSA26R strain (53), permits in vivo mapping of Notch1 activation by gamma secretase-mediated proteolytic cleavage. While not a Notch receptor-Cre fusion, Radtke and colleagues have developed a conceptually similar bi-transgenic Notch1 reporter system (54). They constructed a bacterial artificial chromosome (BAC) transgenic line (N1-Gal4VP16) containing a modified Notch1 cDNA, in which the intracellular domain of the Notch1 receptor was replaced with sequence encoding the Gal4VP16 transcriptional activator. When crossed to a transgenic Gal4 reporter line, such as UAS-lacZ mice (55), the N1-Gal4VP16 line permits the identification in vivo of cells undergoing active Notch1 signaling (54).

Kopan and colleagues have generated several additional Notch receptor-Cre fusion knock-in lines, including N1::CreHI (which, not surprisingly, expresses higher levels of Cre recombinase than the N1::CreLO line) (56), N1::CreERT2 (57), and N2::CreLO (57, 58). Like the N1::CreLO line, all of these lines have Cre coding sequences replacing the Notch receptor intracellular domain, and must be activated by gamma secretase-mediated proteolytic cleavage in order to generate Cre recombinase activity. In addition to fate mapping studies (52, 58–60), some of these lines have been used in functional assays. Since the Cre coding sequences replace the Notch receptor intracellular domain in these alleles, they are functional null alleles that must be analyzed as heterozygotes. In a clever utilization of this system, trans-heterozygous N1::CreLO/N1flox mice were generated. The low efficiency of the N1::CreLO allele created sporadic clones of cells with Notch1 loss of heterozygosity (LOH) in these mice, which subsequently experienced widespread vascular tumors and lethality secondary to massive hemorrhage (56).

Notch receptor fusion lines such as N1::CreLO and N1-Gal4VP16 identify cell lineages that have undergone proteolysis-mediated Notch1 signaling. Artavanis-Tsakonas and colleagues have developed four CreERT2-expressing lines that, when mated to Cre reporter lines, identify cell lineages expressing the Notch1 through Notch4 genes without requiring their activation by proteolytic cleavage (49). These lines have been used recently to identify hematopoietic cell lineages that express each of the Notch receptors during hematopoietic differentiation (40). By crossing each of the four Notch-CreERT2 lines to the ROSA26lslRFP reporter line (61), administering tamoxifen and waiting for various chase periods, the authors found that only Notch1-CreERT2 and Notch2-CreERT2 were expressed in bone marrow. Notch1-CreERT2 primarily labeled cells with lymphoid potential, while Notch2-CreERT2-labeled cells were found mostly in nonlymphoid progenitors (40). The Notch2-CreERT2 line also was used to identify, by mating to conditional β-galactosidase and Tomato red fluorescent protein Cre reporter lines, two previously unrecognized mammary epithelial cell lineages that are distinct from classical luminal and myoepithelial cells (50). The Notch2-CreERT2 line was used to functionally assess the role of these lineages during mammary gland development in virgin and lactating females, by mating the line to a conditional diphtheria toxin receptor line (62) to perform cell ablation of Notch2-CreERT2-expressing cell lineages. These experiments demonstrated that the Notch2-CreERT2-expressing cell lineages were critical for the formation of tertiary branches of mammary gland ductal trees (50).

5. Domain-swap mice

In vivo structure-function studies are a powerful tool to dissect protein function in mice. Kraman and McCright generated a knock-in allele (Notch2N1in) in which sequences encoding the carboxy-terminal 426 amino acids of the Notch2 protein were replaced with the equivalent region of the Notch1 protein (63). Despite the fact that the amino acid sequences of the replaced region are only 37% identical, no aberrant phenotypes were detected in mice homozygous for the Notch2N1in allele, indicating that the carboxy-terminal region of the Notch1 intracellular domain is able to functionally replace that of the Notch2 protein in vivo.

In a genetic engineering tour de force, Kopan and colleagues recently reported much more extensive, and reciprocal, intracellular domain swaps of the Notch1 and Notch2 proteins (58). They described construction of two knock-in lines in which the genomic regions encoding the intracellular domains (ICD) of the Notch1 and Notch2 proteins were interchanged. The N12 allele encodes the extracellular domain (ECD) of Notch1 with the ICD of Notch2, while the N21 allele encodes the ECD of Notch2 with the ICD of Notch1. The swapped regions extended from exon 28, encoding the transmembrane domain, to the stop codon in exon 34. In order to retain transcript-specific regulation of mRNA stability and translation (by microRNAs, for example), the 3′ untranslated regions of the two genes were not interchanged. Mice homozygous for either allele (N12/N12 or N21/N21) were viable. The initial publication was confined to an analysis of kidney development in these mice (58). The authors found that just a single copy of Notch1-ICD could fully rescue the loss of Notch2-ICD, if it was expressed from the Notch2 locus. These results demonstrated that, at least for kidney development, the Notch1-ICD and Notch2-ICD were fully interchangeable. They further demonstrated that the Notch2-ECD increased Notch receptor localization to the cell surface during kidney development, and was cleaved more efficiently than the Notch1-ECD upon ligand binding (58).

6. Locating mouse genetic resources for in vivo analyses of Notch signaling

One of the greatest factors contributing to how widely a mouse line is utilized by the research community is whether the line is available from a public strain repository (64). Many of the lines summarized in this overview are available from such repositories. Researchers can search the web site of the International Mouse Strain Resource (IMSR; www.findmice.org) to locate mouse lines available from strain repositories around the world. The search options on the IMSR site permit one to filter the results in various ways, and it can be useful to exclude the ES cell category (which can contain hundreds of gene trap alleles that have never been made into mice) from the search (Fig. 2). Information on different alleles of particular genes of interest is also available on the Mouse Genome Informatics site (www.informatics.jax.org). Table 1 provides examples of Notch signaling reporter and Notch receptor-Cre lines available (or soon to be available) from public mouse strain repositories. We have not attempted to summarize individual Notch pathway mutants deposited in mouse repositories, as the number of these strains is too numerous, but encourage investigators to utilize the search procedures described here, and elsewhere (5, 6, 29, 64).

Fig. 2.

Fig. 2

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Results of a search on the IMSR web site (www.findmice.org) for Notch1-related targeted mutations. Not all of the returned results are shown. The results show examples of live mice and cryopreserved sperm or embryos, available from the Jackson Laboratory (JAX), Taconic (TAC), and the RIKEN BioResource Center (RBRC).

Table 1.

Notch reporter and Notch-Cre fusion lines available from strain repositories

Namea Reporter/Cre Repository Stock Number Reference
TNR EGFP JAX 18322 (41)
NAS β-galactosidase EMMA 2412 (42)
CBF:H2B-Venus Venus-YFP JAX 20942 (50)
N1::CreLO Cre JAX 6953 (51)
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a

This is the name used in the author’s publications, and in this review. The repositories may have these strains listed under different names. We recommend searching repository stock lists with the stock number. JAX: Jackson Laboratory; EMMA: European Mouse Mutant Archive.

Strains not deposited in repositories may be available directly from the investigator who produced them. Recovering cryopreserved strains from repositories generally costs several thousand dollars, or its equivalent in other currencies. It is not unusual to see requests from investigators on email list servers such as MGI-list (hosted by the Mouse Genome Informatics site), inquiring whether anyone willing to share breeder stock has a certain strain live on the shelf. It may also be useful to contact laboratories that have published recently using the desired strains.

7. Concluding remarks

The number and variety of genetic reagents and tools to study Notch signaling in mice has increased dramatically in recent years, and is sure to continue. The creation of large scale international programs to generate and distribute mouse genetic resources (5, 6), the ability to re-engineer these alleles (8, 9), and the development of novel techniques for genome engineering and generation of genetically-engineered mice (65, 66), all promise to keep mouse biologists interested in studying the Notch pathway busy for many years to come.

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