Rodent Genetic Models of Ah Receptor Signaling

Abstract

The aryl hydrocarbon receptor (AHR) is a ligand activated transcription factor that is a member of the PER-ARNT-SIM superfamily of environmental sensors. This receptor has been a molecule of interest for many years in the field of toxicology, as it was originally discovered to mediate the toxic effects of certain environmental pollutants like benzo(a)pyrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin. While all animals express this protein, there is naturally occurring variability in receptor size and responsiveness to ligand. This naturally occurring variation, particularly in mice, has been an essential tool in the discovery and early characterization of the AHR. Genetic models including congenic mice and induced mutations at the Ahr locus have proven invaluable in further understanding the role of the AHR in adaptive metabolism and TCDD-induced toxicity. The creation and examination of Ahr null mice revealed an important physiological role for the AHR in vascular and hepatic development and mediation of the immune system. In this review, we attempt to provide an overview to many of the AHR models that have aided in the understanding of AHR biology thus far. We describe the naturally occurring polymorphisms, congenic models, induced mutations at the Ahr locus and at the binding partner Ah Receptor Nuclear Translocator and chaperone, Ah receptor associated 9 loci in mice, with a brief description of naturally occurring and induced mutations in rats.

INTRODUCTION

The Aryl hydrocarbon receptor (AHR) is a central paradigm of modern toxicology (Bradshaw & Bell; Zhou, 2016). This ligand activated transcription factor is a member of the PER-ARNT-SIM (PAS) superfamily of environmental sensors and is essential for the adaptive metabolism of many xenobiotic compounds, including polycyclic aromatic hydrocarbons (PAHs) like benzo(a)pyrene (BaP). The AHR participates in this biology through the induction of phase I xenobiotic metabolizing enzymes including members of the cytochromes P450 family (e.g. Cyp1a1, Cyp1a2, and Cyp1b1) and phase II enzymes such as glucuronosyltransferase (Figure 1). In addition to adaptive metabolism, the AHR plays a central role in the toxicity of common polyhalogenated pollutants including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related polychlorinated and polybrominated biphenyls. The adaptive and toxic pathways often overlap, as AHR-mediated induction of detoxification enzymes like Cyp1a1 and Cyp1b1 metabolize ligands such as BaP to yield toxic diol epoxide intermediates (Gelboin, 1980). The mechanism of AHR-mediated toxicity of other ligands, such as TCDD, remains unclear. The dependence of many widespread environmental pollutants on AHR activation has made this signaling pathway the focus of many toxicological studies (Figure 1). A testament to this interest is the observation that a literature search cross referencing “AHR” and “toxicology” yields over 2,000 results (Web of Science, June 10, 2020).

Figure 1.

AHR signaling pathway. The AHR resides in the cytoplasm as part of a complex bound to two molecules of HSP90, one molecule of P23, and ARA9. Exogenous ligand readily diffuses into the cytoplasm of the cell, where it can bind to the cytosolic AHR complex. The ligand-AHR complex then translocates to the nucleus where the chaperones dissociate. In the nucleus, AHR binds to its obligate partner ARNT to form a heterodimer. The AHR-ARNT dimer recognizes and binds to AHREs located upstream of target genes in the DNA. Binding of AHR-ARNT to AHREs induces transcription of target genes including Cyp1a1, Cyp1a2, Cyp1b1, and Ahrr. The cytochromes P450 (CYP1A1, CYP1A2, and CYP1B1) negatively regulate the pathway by metabolizing the ligands that initiate AHR signaling. The AHRR represses the pathway through interactions with ARNT and binding of the AHRR-ARNT dimer to genomic AHREs.

In addition to its role as the “dioxin receptor”, recent studies support the idea that the AHR also has a substantial physiological role independent of the adaptive metabolic response and toxicity (Bhaumik & Basu, 2017; Cella & Colonna, 2015; Esser et al., 2009; Gutiérrez-Vázquez & Quintana, 2018; Quintana et al., 2008; Stockinger et al., 2014; Veldhoen et al., 2008). Genetic tools such as the Ahr null mouse model have revealed an essential role of AHR in normal physiology (Fernandez-Salguero et al., 1995; Mimura et al., 1997; J. V. Schmidt et al., 1996). These observations have led to many hypotheses to explain its evolution, including the idea that the AHR is an environmental sensor that functions to survey local physiological chemistries, particularly at barrier tissues and during times of stress, and orchestrate appropriate biological responses. (Avilla et al., 2020; Esser & Rannug, 2015; Haas et al., 2016). Accordingly, many phenotypes of Ahr mutant models such as altered hepatovascular development and impaired immune function, do not appear to be directly related to TCDD toxicity or adaptive metabolism (Quintana et al., 2008; Stockinger et al., 2014; Veldhoen et al., 2008; Walisser et al., 2005) This idea is supported by the emerging observation that many physiological consequences of Ahr deletion are absent in many Cyp1 knockout animals (McKinnon & Nebert, 1998; Nukaya et al., 2009). While there remains much to be learned about AHR biology, we propose that mouse models have and will continue to serve as essential tools in deciphering this biology.

MOLECULAR CHARACTERIZATION

Molecular characterization of the AHR cDNA and its structural gene reveals a series of important characteristics. The structural gene is well conserved across species, comprised of 11 exons which map to human chromosome 7p16.9-17.3 or mouse chromosome 12:35.4-35.5 (Figure 2) (Poland et al., 1987; Jennifer V. Schmidt et al., 1993). Analysis of the mouse, human, and rat AHR cDNAs reveal a basic helix-loop-helix (bHLH) domain at the N-terminus (Burbach et al., 1992; Dolwick, Schmidt, et al., 1993; Ema et al., 1992). The bHLH domain has been shown to be responsible for nuclear localization, DNA binding, and dimerization with the obligate partner, the Aryl Hydrocarbon Nuclear Translocator (ARNT) (Burbach et al., 1992; Bert N. Fukunaga & Hankinson, 1996; Sanjay Jain et al., 1994). Of particular importance is the basic alpha helix of the bHLH domain, as this domain forms contacts with AHR-ARNT responsive elements (AHREs), genomic cis-enhancer elements also commonly referred to as dioxin response elements (DRE) or xenobiotic response elements (XRE), that are positioned proximal to target gene promoters. Adjacent to the bHLH domain is a consensus PAS domain that harbors two degenerate repeats, denoted PAS A and PAS B. The PAS A domain supports AHR-ARNT dimerization while the PAS B supports binding of ligand, chaperone proteins including heat shock protein 90 (HSP90), Ah Receptor Associated 9 (ARA9), and p23, as well AHR Repressor (AHRR) binding (Burbach et al., 1992; B. N. Fukunaga et al., 1995; Meyer & Perdew, 1999; Seok et al., 2017; Swanson et al., 1995; Swanson & Yang, 1996). The C-terminal region of the protein contains a transactivation domain (TAD) that appears to play roles in coactivator binding and activation of target promoters (Sanjay Jain et al., 1994; Q. Ma et al., 1995). More detailed descriptions of AHR signaling and receptor subdomains, as well as the role of the ARNT protein, can be found in a number of excellent reviews (Avilla et al., 2020; Michael S. Denison et al., 2002; Larigot et al., 2018; Daniel W. Nebert, 2017; J.V. Schmidt & Bradfield, 1996; Whitlock, 1990).

Figure 2.

The Ahrb1 (mouse) gene structure and protein map alignment. General domain names are listed in the top row (bHLH, PAS, and TAD), followed by known functions of each region. The bHLH encoding exons and domains are in blue (exons 1 and 2), PAS domains are in green (exons 3–9), and yellow domains represent the TAD (exons 10 and 11). Chromosomal location of the mouse, rat, and human Ahr are listed.

Considerable variation in the size and primary amino acid sequence of the AHR is common across and within animal species, with receptor molecular weights ranging from 95 kDa (C57BL/6 or B6 mouse) to 124 kDa (hamster) (Pohjanvirta et al., 1999; Poland et al., 1991). In some cases, receptor structural variation within a species corresponds to differing affinities for ligand, sensitivity to ligand-induced responses, or receptor stability (Andreasen et al., 2002; Henck et al., 1981; Sanjay Jain et al., 1994; Poland et al., 1994; Schwetz et al., 1973). Laboratory mice have served as a popular example of this phenomenon, where differences in ligand binding affinity, sensitivity to ligand activation, and thermostability vary considerably across inbred strains (Table 1, Figure 3)(Poland et al., 1994). For example, strains such as B6 display a higher affinity for ligand binding and exhibit a more robust ligand-induced response than do strains such as the DBA/2 (D2) or 129Sv (129) strains (Daniel W. Nebert et al., 1975; Poland & Glover, 1975). Similarly, the protein product encoded in the B6 mouse is more thermodynamically stable than that encoded in the D2 or C3H mouse strains. These differences in receptor size, affinity, sensitivity, and stability have been used to support the idea that the AHR is an evolutionarily malleable molecule that adapts to different chemical environments (M. S. Denison & Nagy, 2003; M. E. Hahn, 2002; Mark E. Hahn et al., 2017).

Table 1. Naturally occuring Ahr alleles in Mice.

Descriptions of the Ahr alleles found in common laboratory mouse strains. A brief overview of each allele highlights responsiveness to AHR xenobiotic ligands and receptor size.

Allele	Strain	Responsiveness	Size (kD)	Length (aa)
Ahrb1	C57BL/6, C58, MA/My	High	95	805
Ahrb2	BALB/cBy, A, C3H	High	104	848
Ahrb3	MOLF/Ei, Mus caroli, Mus spretus	High	105	883
Ahrd	AKR, DBA/2, 129, SWR, RF, NZB	Low	104	848

Figure 3.

Protein maps of mouse AHR naturally occurring alleles and alignments with mouse ARNT. There are four characterized naturally occurring alleles in mice, mAhrb1, mAhrb2, mAhrb3, and mAhrd. The bHLH domain is marked by blue, the PAS domains are shown in green, and the Q rich region of the TAD is shown in yellow. Polymorphisms are shown by the single letter amino acid code on the protein maps. Alignment with the binding partner ARNT is shown to overlap between the bHLH and PAS A domains.

MOUSE MODELS

Many of the signaling pathways and consequences of AHR activation are organ system dependent and cell specific, making this biology difficult to recapitulate in vitro. This complicated biology has thus driven the widespread use of mouse and rat models and the development of induced genetic models. The natural variation at the Ahr locus, particularly in rodents, has been an essential tool in the study of AHR biology but complicates the genetics of widely used models. One goal of this review is to describe the natural variation and how this variation affects many widely used Ahr genetic models such as the conditional null mouse, a model built on a low affinity allele. Another goal for this review is to compile available resources with the benefit of hindsight. In the example of the Ahr null models, the three models originally reported conflicting phenotypes, many of which have been resolved over the decades since the initial report. Given the reliance on rodent models and the profound impact that genetic variation has on receptor biology, it seemed imperative to compile a resource describing Ahr model genetics for the community. To this end, we reviewed many of the natural and induced mutations at the Ahr locus in an effort to outline available rodent models with the associated structural maps, phenotypic and genotypic information, and a limited commentary on their utility (Figure 4, Table 1, Table 2, Table 3). To streamline this process, we have set our focus only on the alleles generated homologously at the locus of interest, in this case Ahr, and two additional loci known to influence AHR signaling, Arnt and Ara9. Many transgenic models are not reviewed here with the hopes that such a task can be accomplished at a later date. (Bhaumik & Basu, 2017; Cella & Colonna, 2015; Esser et al., 2009; Gutiérrez-Vázquez & Quintana, 2018; Quintana et al., 2008; Stockinger et al., 2014; Veldhoen et al., 2008)(J. V. Schmidt et al., 1996)(Avilla et al., 2020; Esser & Rannug, 2015; Haas et al., 2016)(McKinnon & Nebert, 1998; Nukaya et al., 2009)The generation of three independent Ahr null mouse models (Ahr^Δ1, Ahr^Δ2, and Ahr^LacZ) described in more detail below, has revealed a significant role for the AHR in hepatic and vascular development as well as for the maintenance of barrier tissues (e.g., intestine, skin, and lung) and the immune system (Fernandez-Salguero et al., 1995; Mimura et al., 1997; J. V. Schmidt et al., 1996). Early characterization of these null mice from our own laboratory uncovered significant vascular phenotypes, including the patent ductus venosus (DV) in the liver and a hyaloid artery in the eye, both of which are normal fetal structures that typically resolve shortly after birth (Lahvis et al., 2000; Lahvis et al., 2005; J. V. Schmidt et al., 1996). Hepatic phenotypes, including reduced liver size, hepatic portal fibrosis, neonatal hepatic steatosis, and reduced hepatocyte size, were also observed (Fernandez-Salguero et al., 1995; Harstad et al., 2006). Early studies from other laboratories revealed numerous developmental defects, including impaired seeding, homing, function, and lifespan of certain lymphocyte populations (Fernandez-Salguero et al., 1995). Recent studies have expanded upon these early characterizations to show that AHR influences the maintenance of certain resident immune cells, such as innate lymphoid cells in the gut, while also aiding in the orchestration of immune responses such as cytokine production (Cella & Colonna, 2015; Eberl et al., 2015; M. Kim & Kim, 2016; Zhou, 2016). The AHR is now commonly thought of as being highly active in the mounting and coordination of immune responses in barrier organs like the skin, gut, and lung in addition to the previously defined roles of xenobiotic metabolism and toxicity (Díaz-Díaz et al., 2018; Esser & Rannug, 2015).

Figure 4.

Gene maps for Ahr allelic series. Gene maps of the naturally occurring and induced mutations at the Ahr locus map to a general protein map. Exons encoding for the bHLH are blue, PAS-encoding exons are green, and the TAD is shown in yellow. Exons are numbered and introns are lettered. The light gray region at the 5’ end of exon-1 and the 3’ of exon-11 are the untranslated regions of the exons. Exons that are untranslated as a result of induced mutations and premature stop codons are shown in gray. Alleles are organized by naturally occurring alleles and induced mutations, which is subdivided by the method by which mutations were introduced, either homologous recombination or CRISPR-mediated gene editing. The red arrows represent loxP sites.

Table 2. Phenotypes reported in Ahr null models.

Summary of Ahr null phenotypes originally reported in the three models.

Model	Formal Name	Construction	Phenotypes
AhrΔ1/Δ1	Ahrtm1Gonz	Exon-1 replaced with NeoPGK	Normal 1:2:1 Mendalian genetics
			Null pups died or were cannibalized 1–4 days after birth
			Lymphocyte infiltration into organs (gut, lung)
			Slow growth rate within first 2–4 weeks of life
			Abolished Cyp1a1 and Cyp1a2 induction following agonist treatment
			Decreased constitutive amounts of Cyp1a2
			Reduced liver weight: body weight ratio by 50%
			Mild to moderate inflammatory changes in bile ducts
			Eosinophilia toxicity in hepatocytes
			Centrilobular hypercellularity
			Glycogen depletion
			Smaller periarterial lymphatic sheaths
			Fewer splenic lymphocytes in adolescent (80% reduction) and geriatric mice
			Delay/alteration in seeding, homing, emigration, or lifespan of peripheral T and B lymphocytes
			Normal numbers of B and T cells
			Reduced fertility (46% of null moms successfully raised pups to weening)
AhrΔ2/Δ2	Ahrtm1Bra	Exon-2 removed	Normal 1:2:1 Mendalian genetics
			Slow growth rate within first 3 weeks of life
			Abolished Cyp1a1 activity following agonist treatment
			Decreased constitutive amounts of Cyp1a2
			Reduced liver weight: body weight ratio by 25%
			Microvesicular fatty metamorphosis of hepatocytes and prolonged extramedullary hematopoiesis in livers of 1 week old pups
			Mild to moderate portal hypercellularity with thickening and fibrosis by 2 weeks of age
			Congestive splenomegaly and enlarges spleen in 50% of pups
			Normal numbers of B and T cells
			Patent ductus venosus into adulthood
			Altered liver lobe weights (left lobe is smaller)
AhrLacZ	Ahrtm1Yfk	LacZ inserted after exon-1	Normal 1:2:1 Mendalian genetics
			Slightly slower growth rate
			Abolished Cyp1a1 and 1a2 induction following agonist treatment
			Low levels of constitutive CYP1A2 remain intact
			Hepatic portal fibrosis (less so)
			Females are sub-fertile due to impaired follicle development and ovulation

Table 3. Summary of induced mutations at the Ahr locus in mice.

Description of the names used to refer to each mutation as well as the responsiveness of the allele to AHR xenobiotic ligands.

Mutant	Name	Formal Name	Parental Strain	Allele (Responsiveness)
Ahr null	AhrΔ1/Δ1	Ahrtm1Gonz	129 ES cells backcrossed onto B6	Ahrd (none; null allele)
Ahr null	AhrΔ2/Δ2	Ahrtm1Bra	129 ES cells backcrossed onto B6	Ahrd (none; null allele)
Ahr null	AhrLacZ	Ahrtm1Yfk	129 ES cells backcrossed onto B6	Ahrd (none; null allele)
Congenic Ahrd	Ahrd-B6	B6.D2N-Ahrd/J	Ahr from D2 backcrossed onto B6	Ahrd (low)
Congenic Ahrb1	Ahrb1-DBA	D2.B6-Ahrb-1/J	Ahr from B6 backcrossed onto D2	Ahrb1 (high)
Ahr conditional	Ahrfx	Ahrtm3.1Bra	Ahrfxneo	Ahrd (low)
Ahr hypomorph	Ahrfxneo	Ahrtm3Bra	129 ES cells backcrossed onto B6	Ahrd (low)
Nuclear localization	Ahrnls	Ahrtm2Bra	Ahrd	Ahrd (low)
DNA-binding	Ahrdbd	Ahrtm4Bra	Ahrd	Ahrd (low)
V375A	AhrV375A	Ahrem1Bra	Ahrfx	Ahrd (high affinity ligand binding domain)
NG367R	AhrNG367R	Ahrem2Bra	Ahrfx	Mutated Ahrd
Ter383	AhrT383	Ahrem3Bra	Ahrfx	Mutated Ahrd
Humanized Ahr	AhrHum	Ahrtm1(AHR)Mym	129 ES cells backcrossed onto B6	Human Ahr (lower than Ahrd)
Constitutively Active	AhrCAIR	Ahrtm1Lgzh	B6 ES cells	Conditional constitutively active AHR fused to IRES-GFP

The importance of genetic background

To begin this discussion, we must first highlight some considerations that are essential in understanding the models described herein. First, while this will be an excessively Ahr-centric review, we must point out the importance of all other loci in the mammalian genome, something we define here as “genetic background”. In this regard, one of the great powers of mouse genetics is the availability of numerous genetic backgrounds that exist through the sharing and archiving of mouse strains. While considering the Ahr locus itself is important, the influence of genetic background on AHR signaling and response cannot be underestimated. It is well known that genetic background can contribute considerably to certain phenotypic endpoints, as illustrated by the observation that topical application of TCDD causes hyperplasia and hyperkeratosis in hairless mice (HRS/J) while this endpoint is absent in B6 mice (Knutson & Poland, 1982). This observation exists despite B6 mice being sensitive to TCDD-induced hepatotoxicity and induction of P450s (D. W. Nebert & Gelboin, 1969). This exemplifies the idea that other genetic elements influence AHR biology and must be taken into account when comparing strains.

To nullify the contribution of other genetic loci on a phenotype, alleles can be introduced into a desired genetic background through a series of backcrosses to a particular parental strain. After 10 backcrosses, this process yields mice that are congenic, or only differ at the selected locus and proximate genomic sequences. True congenic mice are generated through a series at least 10 backcrosses, at which point mice are expected to contain 99.9% of their genetic information from the backcrossed background (Snell, 1948). Generating congenic mice has also proven to be an important consideration during the era of induced mutations generated through homologous recombination. The technology available to generate many of the targeted AHR mutant mice in the 1990s and early 2000s was limited to only a handful of pluripotent embryonic stem cell lines (ESCs), many of which were derived from 129 mice. The ESCs themselves were often heterogenous and could vary depending on origin. The remnants of these limitations must be considered today, as many of ESC-derived models remain widely used (Gardner & Brook, 1997). The homologous recombination technique used to generate the Ahr null and conditional nulls models described here required the use ESCs derived from 129 mice. Additionally, 129 mice harbor the low affinity Ahr^d allele (Poland et al., 1994). That is, mutations targeted to disrupt or alter AHR were altering the isoform of AHR that binds ligand with low affinity. This became a limitation when studies using the conditional allele were insensitive to AHR ligands (Walisser et al., 2005). To address this issue, backcrossing to a common background such as B6 is an essential step in ridding residual genomic heterogeneity resulting from the early incorporation of the 129 genome, as well as differences between ESCs themselves. This step is often overlooked and has likely caused much of the ambiguity and dissidence between mouse models over the years.

Naturally Occurring Polymorphisms in the Mouse Aryl Hydrocarbon Receptor

Differences in amino acid sequence between these naturally occurring alleles has been useful in determining the impact of certain amino acids on receptor behavior. There are four naturally occurring alleles characterized in common strains of laboratory and feral mice (Poland et al., 1994). These alleles are classified based on their molecular weight as well as their responsiveness to AHR ligands. High responders are designated Ahr^b (for B6, the first strain identified as responsive) and the low responders are named Ahr^d (for D2, the first low responsive strain identified). Subsequent examination of additional laboratory (C3H, MOIF/Ei, BALB/cBY) and feral (Mus caroli, Mus spretus) mouse strains revealed the existence of two additional alleles, both of which were high responders and therefore designated Ahr^b. To differentiate the high-responding alleles, the Ahr^b group was subdivided based on molecular weight. The B6 mouse was reclassified as Ahr^b1 (96kD), while the two larger receptors were designated Ahr^b2 (104kD) and Ahr^b3 (105kD) (Table 1, Figure 3). Importantly, the differences in length are due to alternate stop codons and elongated C-termini rather than differences in sequence across other domains.

Characterization of the naturally occurring polymorphisms in AHR have served as a prototype for studying structure-function relationships. Molecular analysis of the three Ahr^b alleles reveals an Alanine at residue 375 whereas the Ahr^d allele harbors a Valine at this position (Poland, Palen, and Glover 1994) (Table 1, Figure 3). Comparisons between the Ahr^b2 and Ahr^d were particularly useful because these receptors differ at three residues (348, 375, 758), yet have a 10-fold difference in sensitivity to radiolabeled TCDD (Figure 3)(Poland et al., 1994). Early mutagenesis experiments using these two alleles in vitro showed that residue 375 is largely, though not completely, responsible for the differential binding affinities between the Ahr^b alleles and Ahr^d (Ema et al., 1994; Poland et al., 1994). Mapping experiments revealed that residue 375 mapped to the ligand binding domain, supporting the identity of this residue as essential for ligand affinity (Burbach et al., 1992; Ema et al., 1994; Xing et al., 2012). Another prominent polymorphism that is thought to affect receptor behavior is a point mutation in exon-11 of the Ahr^b2, Ahr^b3, and Ahr^d alleles that extends the open reading frame, resulting in a longer translation product with decreased thermostability. The protein product from Ahr^b1 is more stable and binds HSP90 more readily than the Ahr^b2 and Ahr^d counterparts, which might also influence responsiveness (Poland et al., 1994). Despite these limitations, naturally occurring variation in mice and derivative congenic models were essential tools in providing a foundational understanding of AHR domains and the importance of residue 375 and the elongated C-terminus.

Congenic Models

B6.D2N-Ahr^d:

The congenic Ahr^d mouse harbors the D2- derived Ahr^d allele at the Ahr locus on the B6 background. This mouse was generated by intercrossing the D2 mouse and the B6 mouse, selecting for the Ahr^d allele. Subsequent backcrossing and intercrossing proceeded for over 17 generations, at which point this line was considered congenic for the B6 background, with the incorporation of the Ahr^d at the Ahr locus (Poland & Glover, 1980, 1990; Poland et al., 1994). These mice are useful in examining the consequences of Ahr^d versus Ahr^b on the same background, therefore eliminating the possibility of non-Ahr genomic elements on endpoints such as susceptibility to BaP carcinogenesis and aryl hydrocarbon hydroxylase activity (Boobis & Nebert, 1976; Poland et al., 1976). This mouse continues to be widely used as a nonresponsive control for studies examining the AHR-mediated effects of ligands like TCDD and BaP on common end points such as carcinogenesis and inflammation (Kennedy et al., 2014; Wong et al., 2018).

D2.B6-Ahr^b1:

The congenic Ahr^b1 mouse harbors the Ahr^b1 allele originally derived from B6 mice on a D2 background. This mouse has been used primarily to determine the relevance of genetic background on AHR activity. Because the B6 mouse is commonly used for toxicologic research, congenic D2.B6-Ahr^b1mice are not as widely characterized. There are, however, advantages when wanting to examine the consequences of distinct Ahr alleles on the D2 background. Additionally, this mouse has power when compared to wildtype DBA/2-Ahr^d mice and allows examination of genetic background independent of AHR genotype when compared to congenic B6.D2N-Ahr^d mice and wildtype B6 (Ahr^b1) mice.

Ahr Mutations Induced by Gene Targeting

Given the observation that animal models provide powerful reflections of AHR signaling, both from a toxicological and developmental perspective, early research efforts focused on the development of null and recombinant mouse models using “gene targeting”. The gene targeting approach is dependent upon mechanisms of homologous recombination and is reliant on the ability to culture ESCs that can be genetically manipulated in vitro. While a variety of gene targeting approaches have been employed for hundreds of mouse loci, the majority of Ahr alleles were modified using the positive negative selection approach pioneered by Capecchi and colleagues (Mansour et al., 1988). Because homologous recombination was often performed in derivatives of ESCs from the 129 mouse, the resultant models often require varying degrees of backcrossing onto a standard genetic background such as B6. Initial reports characterizing three Ahr null mutants generated by this protocol, while different in some respects, provided some of the earliest and most compelling evidence for a substantive role for AHR in normal physiology specifically for endpoints related to the hepatic, immune, reproductive, and vascular systems.

Ahr^Δ1 null mouse:

The Ahr^Δ1 mouse harbors a neomycin resistance gene driven by the PGK promoter (Neo^PGK) in place of exon-1 at the Ahr locus (Figure 4, Table 2)(Fernandez-Salguero et al., 1995). While these mice appeared to develop normally in utero, homozygous mutants die or were selectively cannibalized for up to four days following birth. Subsequent study revealed that only 46% of null females would successfully raise pups to weaning (Abbott et al., 1999). Necropsy described lymphocyte infiltration into the lung, gut, and urinary tract. Perhaps most notably, these mice displayed delays and alteration in seeding of peripheral T and B lymphocytes in adolescent mice and a diminished lifespan of these cells in older mice. This observation led to the hypothesis that AHR signaling plays a vital role in seeding, homing, emigration, and lifespan of peripheral T and B lymphocytes. This idea has been supported and expanded upon recently as additional evidence emerges detailing missing or impaired populations of resident immune cells in adult null mice at barrier tissues like the lung, intestine, and skin (Esser & Rannug, 2015; Kiss et al., 2011; Lee et al., 2012; Li et al., 2018; Qiu et al., 2012; Stockinger et al., 2014).

Ahr^Δ2 null mouse:

The Ahr^Δ2 mouse harbors a Neo^PGK cassette in place of exon-2 (Figure 4, Table 2)(J. V. Schmidt et al., 1996). These mice express extensive neonatal hepatic steatosis that resolves by 3 weeks of age, perinatal hepatic peripheral necrosis, smaller livers relative to body weight, altered liver lobe weights, and patent DV that persists through adulthood. The DV is a fetal vascular structure that allows blood to bypass the liver during development, and typically closes shortly after birth. The patent DV is one of the most easily assessed and penetrant AHR-mediated phenotypes (Harstad et al., 2006; Lahvis et al., 2000; Lahvis et al., 2005). Mice null for Ahr exhibit a patent DV with 100% penetrance, though the AHR-driven genes responsible for shunt closer remain unknown. Subsequent examination of this mouse has revealed significant defects in resident immune cell populations in the gut, lung, and skin as well as increased susceptibility to certain diseases including psoriasis, experiment colitis and colorectal cancer, and infections of microorganisms like Citrobacter rodentium and Toxoplasma gondii (Díaz-Díaz et al., 2016; Furue et al., 2019; H. R. Kim et al., 2020; J. J. Kim et al., 2012; Kiss et al., 2011; Qiu et al., 2012; Sanchez et al., 2010; Wagage et al., 2015).

Ahr^LacZ null mouse:

The Ahr^LacZ mouse harbors a LacZ gene with an artificial polyA tail fused to an inverted neomycin-resistant gene driven by the pTK promoter (Neo^pTK) following exon-1 of Ahr (Figure 4, Table 2)(Mimura et al., 1997). This mouse was used to determine the dependence of teratogenic effects of TCDD on AHR signaling. Following TCDD treatment in pregnant dams, null pups were examined for teratogenic responses on palatogenesis and nephrogenesis. This study supported previous studies from other laboratories regarding the highly context-dependent role of AHR (S. Jain et al., 1998; Willey et al., 1998). That is, factors such as receptor number, cell type, and developmental window can differentially influence the outcome of receptor activation. In this report, some TCDD teratogenic endpoints vary depending on amount of receptor. For example, mice with one copy of functional Ahr (Ahr^b1/LacZ) were less sensitive to TCDD-induced cleft palate than mice with two copies of Ahr (Ahr^b1/b1). In contrast, other endpoints such as dilation of renal pelvis was not influenced by haploinsufficiency. This mouse has also been used to examine fertility endpoints such that females were reported to be sub-fertile due to impaired follicular development and ovulation (Baba et al., 2005). The insertion of the LacZ construct at Ahr in this model allows staining as a surrogate for natural AHR expression patterns and has thus been useful in mapping Ahr expression patterns during development (Table 3).

Reconciliation of divergent phenotypes in the three Ahr null animals:

Despite inconsistent reports in initial characterization of these three models, subsequent examination of these alleles has indicated that all phenotypes are in fact quite similar and the variation is due to genetic background, environmental variables, or distinct laboratory emphasis (Lahvis & Bradfield, 1998). Given that each of the three lines were generated using different 129-derived ESCs, it seems likely that early reports employed lines with differing genetic backgrounds due to husbandry patterns used to propagate colonies. The recently described genetic compensation response may also influence expression of the various Ahr null alleles and therefore affect endpoint manifestation (El-Brolosy et al., 2019; Z. Ma et al., 2019). Differences in the environment such as diet, amount of ligand in the diet, bedding, caging, or the presence of pathogens in the facility can influence observed phenotypes. These environmental considerations are particularly important as it is now known that ligands obtained through the diet can influence microbiome and AHR signaling, and it is also likely that differences in pathogens can affect immune output in laboratory mice (Brawner et al., 2019; Leavy, 2011; Schanz et al., 2020). Finally, the three strains have been used in different types of experiments and have thus been examined for different endpoints. Discrepancy in reported phenotype, then, may simply be due to the fact that each laboratory focused on unique aspects of AHR biology. Despite early inconsistencies, many of which have since been relieved, the three Ahr null alleles have been widely useful in studying AHR biology.

Ahr Hypomorphic Mouse (Ahr^fxneo):

The Ahr hypomorph, referred to as Ahr^fxneo, harbors a “floxed” (flanked by loxP recombination sites) Neo^PGK cassette within the second intron (Figure 4, Table 2 (Walisser, Bunger, Glover, & Bradfield, 2004). The Ahr^fxneo allele expresses limited amounts of AHR protein, estimated to be 10–20% expression compared to wildtype counterparts. While extensive studies to explain how the presence of the Neo^PGK cassette leads to hypomorphism are lacking, preliminary evidence from multiple alleles in our own laboratory suggest that the presence of the Neo^PGK transgene reduces RNA expression from multiple loci, independent of direction or position (Bunger et al., 2008; Bunger et al., 2003; Lin et al., 2008; Walisser, Bunger, Glover, & Bradfield, 2004; Walisser et al., 2005). The Ahr^fxneo mouse was generated using homologous recombination in 129 ESCs that harbor the Ahr^d allele (Figure 5). In addition to the Neo^PGK cassette, exon-2 is also floxed with an additional downstream loxP site located within the third intron. This allele results in an AHR of wildtype amino acid sequence, although a significantly lower amount of protein product is produced.

Figure 5.

Phylogenetic tree depicting relationships of induced Ahr models. Many of the induced mutations were generated using 129 Sv ESCs. The models generated from these ESCs all harbor the Ahrd allele at the Ahr locus. The Ahrfxneo mouse serves as the parental strain for the Ahrfx, AhrV375A, AhrNG367R, and AhrTer383 models. The AhrCAIR was generated using B6-derived ESCs and harbors the Ahrb1 allele.

The AHR hypomorph mouse is useful in understanding how low level of receptor and subsequent activation can affect a given endpoint (Table 3). Mice hypomorphic for AHR exhibit a patent DV with ~12% penetrance and a 7-fold lower level of CYP1A1 activity (Walisser, Bunger, Glover, & Bradfield, 2004). Interestingly, hypomorphic pups exposed in utero to TCDD displayed normal DV closure. Similarly, in hypomorphic mice treated with the AHR agonist SU5416, ~59% of mice displayed normal DV closure compared to ~4% of vehicle treated controls (Mezrich et al., 2012). These data indicate that the developmental pathway responsible for DV closure can be activated by high doses of weaker ligands or low doses of potent ligand despite diminished level of receptor. This rescue potential suggests that an endogenous ligand, perhaps in low concentrations or a low affinity molecule, activates AHR at a critical point in development to trigger expression of an unknown battery of genes that leads to proper vascular development and DV closure. Other endpoints observed in the Ahr null models, including the influence of Ahr^fxneo on immune cell populations for example, are not known. This hypomorphic model of AHR may be an interesting opportunity to examine potential gene compensatory mechanisms that affect AHR-mediated endpoints reported in various null models (El-Brolosy et al., 2019; Z. Ma et al., 2019). Overall, this mouse has been an interesting tool used to understand the relationship between receptor number and endpoint and has provided evidence to support the endogenous ligand hypothesis.

Ahr Conditional Mouse (Ahr^fx):

To create a model useful in understanding the cell specific role of the AHR, Cre-lox technology was used to modify the Ahr^fxneo allele into the conditional Ahr^fx mouse (Figure 5)(Walisser et al., 2005). The Ahr^fx mouse was generated from the Ahr^fxneo mouse through a cross to a line harboring the E2a-driven Cre recombinase transgene (Cre^Ella) (Lakso et al., 1996). The cross to Cre^Ella created a partial recombinant allele such that the Neo^PGK cassette is excised while the loxP sites flanking exon-2 remain intact (Figure 4, Table 2)(Holzenberger et al., 2000; Lakso et al., 1996; Walisser et al., 2005). The resulting model harbors the Ahr^d allele with loxP sites flanking exon-2 that can be used in future crosses with other cell specific Cre recombinases (Haas et al., 2016; Metidji et al., 2018; Wheeler et al., 2013). Because the Neo^PGK cassette has been removed, expression levels of AHR^fx are recovered to wildtype levels, except in cases where Ahr^fx is subsequently excised through crosses to other mouse lines harboring Cre recombinases. The loxP sites are located in the introns surrounding exon-2 and do not appear to influence expression.

The Ahr conditional mice were created to elucidate the cell specific role of the AHR (Table 3). The first use of this mouse was to examine the cell specific role of the AHR in developmental and toxic endpoints. To this end, Ahr^fx was crossed to hepatocyte-specific (Cre^Alb) or endothelial-specific (Cre^Tek) Cre recombinase to demonstrate that TCDD toxicity and the closure of the DV require receptor activation in hepatocytes and endothelial cells, respectively (Kisanuki et al., 2001; Postic et al., 1999; Walisser et al., 2005). Subsequent examination of this mouse crossed to various Cre models has unveiled a cell specific role for AHR in lymphocytes, intestinal epithelial cells, keratinocytes, Langerhans cells, and hematopoietic stem cells to name a few (Bennett et al., 2018; Haas et al., 2016; Hong et al., 2020; Li et al., 2018; Metidji et al., 2018) This mouse has been a vital tool in untangling the complicated cell-specific role of AHR.

Ahr Nuclear Localization Mutant (Ahr^nls):

To examine the importance of nuclear and cytosolic events in AHR mediated development and TCDD toxicity, an exon replacement technique was used to generate mice with a mutation in the N-terminal nuclear localization sequence (NLS) of the AHR (Bunger et al., 2003). To accomplish this, mutations were introduced to change the R37-H38-R39 amino acid sequence to A37-G38-S39 (Figure 4, Table 2). These residues were chosen based upon NLS mapping studies and were predicted to disrupt shuttling to the nucleus (Eguchi et al., 1997; Bert N. Fukunaga & Hankinson, 1996; Ikuta et al., 1998). Our own domain mapping studies suggested such mutations would not affect ligand or chaperone binding, but the mutations do disrupt DNA binding in addition to nuclear localization (Dolwick, Swanson, et al., 1993). Like many alleles generated via gene targeting, the Ahr^nls was produced using homologous recombination in 129 ESCs injected into B6 blastocytes (Bouabe & Okkenhaug, 2013; Bunger et al., 2003; Gerlai, 2016). Due to the 129 origins of the allele, the AHR encoded by this mutant harbors the Ahr^d allele and is considered a low responder (Figure 5) (Poland et al., 1994). Despite this fact, the AHR protein encoded by the Ahr^nls allele was shown to bind ligand but remained cytosolic in response to a 1 nm dose of TCDD (Bunger et al., 2003). These early characterization studies support the idea that alteration in the nuclear localization sequence prevents the Ahr^nls mutant from translocating to the nucleus following agonist binding.

The Ahr^nls model was generated as a tool to examine putative cytosolic activity of the AHR that had been reported by a number of laboratories (Table 3) (Blankenship & Matsumura, 1997a, 1997b; Elferink et al., 2001; Enan & Matsumura, 1996; Ge & Elferink, 1998; Klinge et al., 2000; Ohtake et al., 2007; Puga et al., 2000). In early experiments employing this model, the Ahr^nls mice were found to be resistant to TCDD-induced toxicity and exhibit a patent DV through adulthood. These observations suggest the Ahr^nls model is a phenocopy of the Ahr null mouse (Bunger et al., 2003; Lahvis et al., 2005). These data indicate that DV closure and TCDD-mediated hepatotoxicity are both dependent on AHR nuclear translocation and suggest the importance of subsequent ARNT dimerization and AHR-ARNT interactions with genomic AHREs (Figure 1). This model continues to be of use for any experiment designed to isolate nuclear localization dependent events in AHR signaling.

Ahr DNA Binding Mutant (Ahr^dbd):

To better understand the importance of the AHR-ARNT dimer binding to AHREs, the Ahr^dbd model was by mutating the DNA binding domain (DBD) of the AHR (Bunger et al., 2008) using a homologous recombination process similar to that of Ahr^nls described above (Figure 5). In this case, two codons (GS) were inserted between the basic (ending at residue 39) and the helix-loop-helix (beginning at residue 40) domains to shift the basic region of the DBD out of the major groove of DNA such that it would not bind AHREs (Figure 4, Table 2). Similar to the Ahr^nls counterpart, the Ahr^dbd mutant was generated on the low responding Ahr^d allele. This allele was back crossed to B6 for 3 generations for employment in early experiments.

The Ahr^dbd model was created to test the dependence of TCDD toxicity and DV closure on AHRE binding events (Table 3). The role of AHRE binding as an essential part of the signaling pathway is important given that many AHRE-drive genes (e.g., Cyp1a1, Cyp1a2, and Cyp1b1) had not been linked to DV closure or most aspects of TCDD toxicity. Thus, a formal possibility remained that AHR signaling for some endpoints was independent of AHRE binding (Larigot et al., 2018). Examination of the Ahr^dbd construct in vitro revealed that after ligand binding, it retained the ability to translocate to the nucleus and dimerize with ARNT (Bunger et al., 2008). In keeping with its predicted activity, the Ahr^dbd mutant could not activate Cyp1 gene expression or interact with AHREs. Interestingly, this mutation had an unexpected influence on the neighboring NLS and the construct became constitutively nuclear. Yet, in two-hybrid assays, it was shown to maintain its ligand responsiveness and its capacity to dimerize with ARNT (Bunger et al., 2008). The phenotypes observed in the Ahr^dbd mice mimicked those of the Ahr^nls model and the Ahr null model (J. V. Schmidt et al., 1996). That is, these mice exhibit patent DV through adulthood and are resistant to TCDD toxicity. Taken in sum, results with the Ahr^dbd model indicate the DNA binding event is an essential step for certain biological endpoints.

Humanized (Ahr^Hum):

In an effort to gain insight into the mechanisms underlying the human effects of highly toxic agonists like TCDD, a “humanized mouse model” was generated by “knocking in” the human cDNA homologously into the Ahr locus in mice (Figure 4, Table 2)(Moriguchi et al., 2003). This allele, Ahr^Hum, was generated through homologous recombination of the human open reading frame cDNA into exon-1 of the mouse Ahr. The human cDNA was followed by a Neo^pTK cassette in the reverse reading frame for positive selection in 129 derived ESCs (Figure 5)(Mimura et al., 1997; Moriguchi et al., 2003). This allele was then backcrossed onto the B6 genetic background for seven generations.

Given the wide range of responses to xenobiotics in animals, this model was developed in an effort to help inform risk assessment of human TCDD toxicity with the idea that it provided a more relevant model of human signal transduction in response to environmental pollutants. In this regard, position 381 of the human AHR, a position homologous to mouse 375, harbors a V residue and is predicted to confer the low affinity binding phenotype on this receptor form (Dolwick, Schmidt, et al., 1993). Yet, the human AHR also harbors a number of other sequence differences in domains that are less well characterized, and these differences were suspected of playing important roles in human AHR signal transduction that might differ from that in the mouse. Interestingly, in early reports, the Ahr^Hum allele was shown to be less sensitive to TCDD than the Ahr^d allele (Moriguchi et al., 2003). This is a provocative observation that could be an indication that the human receptor is less efficient at signal transduction compared to its mouse counterparts. Alternatively, this minigene approach may also be influenced in unanticipated ways by its lack of introns, the existence of mouse specific pathways that influence signaling, or potentially compromised interactions with mouse cofactors. While the humanized allele may be useful in modeling certain aspects of human AHR signaling in mice, there are a range of unknown interactions that should be considered (Table 3).

Constitutively Active AHR (Ahr^CAIR):

Given the importance of cell specific AHR signaling in developmental processes it regulates and the toxicology of TCDD, constitutively active AHR (CA-AHR) models have proven valuable. Early evidence from domain mapping studies demonstrated that Ahr mutants lacking the PAS B domain constitutively dimerize with ARNT, bind to AHREs, and induce transcription of AHRE-driven genes in a manner that is independent of ligand binding (Andersson et al., 2002; J. McGuire et al., 2001). While transgenic models were initially built to express constitutively actives form of AHR (summarized in Table 4), the approach to generate Ahr^CAIR employed insertion of a minigene lacking PAS B (amino acids 278 to 423) by homologous recombination at the Ahr locus in B6 ESCs (Figure 5) (Ye et al., 2017). This Ahr^CAIR model also harbors a “Flag-Tag” at its N-terminus and a 3’ internal ribosomal entry site (IRES) driving Green Fluorescent Protein (GFP) downstream to monitor expression (Figure 4, Table 2). This hybrid minigene was inserted downstream of a floxed Neo^PGK cassette and within exon-1 allowing for Cre-inducible cell specific expression of the CA-AHR. By inserting this hybrid minigene downstream of a floxed Neo^PGK cassette harboring a tpA transcriptional stop codon, these mice allow cell specific expression of the CA-AHR in specific cell populations using tissue specific Cre promoters in F1 hybrids.

Table 4. Summary of induced mutations at loci known to influence AHR signaling.

Brief summary of ARNT and ARA9 mutations at the respective loci. The parental strain and Ahr allele are noted as well as a brief overview of the influence of these mutations on AHR biology.

Mutant	Name	Ahr allele	Parental Strain	Effect
Arnt hypomorph	Arntfxneo	Ahrb1	129 ES cells backcrossed onto B6	Reduced amount of binding partner (ARNT) available to dimerize with AHR
Arnt conditional	Arntfx	Ahrb1	129 ES cells backcrossed onto B6	Conditional deletion of Arnt
Ara9 hypomorph	Ara9fxneo	Ahrb1	129 ES cells backcrossed onto B6	Reduced amount of ARA9; reduced AHR as a result
Ara9 conditional	Ara9fx	Ahrb1	129 ES cells backcrossed onto B6	Conditional deletion of Ara9

Homozygous mutants with the Neo^PGK-tpA cassette intact (Ahr^CAIR/CAIR) are apparent phenocopies of the Ahr null mouse as determined by deficiencies in the innate lymphoid cells in the gut, though other endpoints such as DV penetrance have not yet been observed (Ye et al., 2017). This mouse model has been particularly useful as heterozygotes harboring one wildtype copy of Ahr^b1 and one Ahr^CAIR allele (Ahr^b1/CAIR) in studies examining expression patterns of resident T regulatory cells in the gut. When crossed to mice harboring a Treg-specific Cre recombinase (Foxp3), Ahr^b1/CAIR mice demonstrate the role of AHR in mediating Treg homing to the gut as well as the protective function AHR serves against T cell-mediated colitis. Future studies employing the Ahr^CAIR mice will be particularly useful in understanding the role of AHR in different immune cells and in contexts where mice are challenged.

Ahr Mutations Induced by Gene Editing

Within the past decade, the generation of model systems has been aided by the advancement of CRISPR-based gene editing techniques (Cong et al., 2013; Gasiunas et al., 2012; Jinek et al., 2012; Mali et al., 2013). Such approaches can be more rapid than the gene targeting method described above and can be readily performed in series to generate a variety of informative alleles including nulls, “knock ins”, and fine gene mutations (Sander & Joung, 2014). Briefly, CRISPR technology is commonly employed in mouse embryos, where the Cas9 endonuclease is directed to the genomic site of interest by a guide RNA. The Cas9 protein then introduces a targeted double stranded break in the DNA at the target site which stimulates cellular repair mechanisms like nonhomologous end joining (NHEJ) or homology directed repair (HDR). The HDR mechanism is often used to incorporate a donor template that harbors the precise mutation, whereas the NHEJ mechanism commonly introduces indels that often result in null alleles. In some cases when a donor template is introduced in an effort to induce HDR, the cell employs NHEJ instead and can introduce unpredictable insertions or deletions (indels) during the repair process (Ceccaldi et al., 2016). Additional advancements in the gene editing field, particularly with the advent of techniques like base editing and prime editing that induce editing without double stranded breaks, further improves the range of mutations and the ease with which these specific mutations may be installed(Anzalone et al., 2019; Gaudelli et al., 2017; Komor et al., 2016). Thus far, three Ahr models have been developed using gene editing and this number will likely increase over the coming years.

High affinity conditional allele (Ahr^V375):

The high affinity conditional allele (Ahr^V375A) was generated to address the sensitivity limitation of the conditional Ahr^fx allele described above (R. H. Wilson et al., 2021). Due to the gene targeting strategy that employed 129 derived ESCs, the Ahr^fx model harbors the low affinity Ahr^d allele, resulting in a model that is insensitive to ligands of moderate or weak potency (Figure 5). Thus, use of Ahr^fx model can require high concentrations of certain ligands like beta-naphthoflavone (BNF) or the use of highly potent ligands like TCDD in order to illicit a response (Kennedy et al., 2014). To address this limitation, the conditional Ahr^fx model was edited using CRISPR-Cas9 to install a point mutation converting V375 to A375 (Figure 4, Table 2). Prior studies show that the A375 resides in a position within the ligand binding pocket that is thought to have significant impact on ligand binding affinity for many AHR ligands (Ema et al., 1994; Poland et al., 1994; Xing et al., 2012). The strategy to edit the Ahr^fx allele from a conditional low responding allele to a conditional high responding allele employed the introduction of a donor template harboring the point mutation that was incorporated into the genome via HDR.

Early characterization of this model indicates that Ahr^V375A is significantly more sensitive to induction of AHR-driven genes including Cyp1a1, Cyp1a2, Cyp1b1, and Ahrr in response to agonists of moderate potency (e.g., BNF). While this model is recently published and has not yet experienced widespread use, it is proposed that the line will be useful in conditional deletion studies designed to identify cell populations responsible for AHR-driven gene expression, development, and toxicity endpoints. Early experiments employing this model crossed to mice harboring the hepatocyte-specific Cre recombinase (Cre^Alb) demonstrate cell specific differences in gene induction in the liver. Specially, mice with Ahr^V375A deleted in hepatocytes (Ahr^V375ACre^Alb) are uninducible for Cyp1a1 and Cyp1b1 but are inducible for Ahrr and Cyp1a2. This observation supports the hypothesis that the Ahr^V375A model can be used to decipher the cell autonomous role of AHR using low doses of relatively weak ligand like BNF. The model should also see use in TCDD toxicology studies, because it maintains the conditional nature, but requires lower doses of the toxic agent as compared to protocols using the Ahr^fx model (Kennedy et al., 2014). Comparison of this model to the Ahr^fx allele offers an in vivo opportunity to examine the role of residue 375 in specific AHR endpoints due to the fact that that the two lines are coisogenic, i.e., the only genetic difference between these models is at residue 375. This improved sensitivity and co-isogenic nature of the Ahr^V375A allele will allow this model to be useful in understanding the cell autonomy of AHR signaling.

The Ahr^NG367R:

The Ahr^NG367R mutant was generated serendipitously using CRISPR-Cas9 during the attempt to generate the Ahr^V375A allele from the Ahr^fx mouse described above (Figure 5) (Ceccaldi et al., 2016; R. H. Wilson et al., 2021). It has been reported that unpredictable insertions or deletions may occur following a Cas9-induced double stranded break when the cell employs NHEJ to repair the double stranded break in the DNA (Ceccaldi et al., 2016). In this case, the Cas9-induced double stranded break appears to have undergone NHEJ resulting in a three base pair deletion spanning the codons for residues 367 and 368 (Figure 4, Table 2). This deletion resulted in a condensation of residues N367 and G368 into R367. Because the mutation is in frame, the predicted sequence of the protein beyond codons 367 and 368 remains undisrupted. Based on previous domain mapping studies, this mutation occurs in the PAS B domain, near a region predicted to encode the ligand and chaperone binding domains (Figure 2, Figure 4) (Dolwick, Swanson, et al., 1993; Fukunaga & Hankinson, 1996). Preliminary modeling studies suggest that the PAS B domain may be misfolded due to the removal of a glycine residue (G368) that is critical to the proper folding of this region (R. H. Wilson et al., 2021). This prediction is supported by the observation that mice homozygous for this allele are a functional phenocopy of the null in hepatovascular development and gene induction studies (R. H. Wilson et al., 2021). Despite appearing phenotypically and functionally null, the AHR^NG367R protein is expressed (R. H. Wilson et al., 2021). The experimental value of this model beyond providing structural insight of the AHR and PAS B domain remains to be determined and will ultimately be dependent upon additional characterization. Yet, it is interesting to note that Ahr^NG367R provides a novel null allele and that the mutant is conditional given its derivation from Ahr^fx. These genetic characteristics also make the Ahr^NG367R mouse co-isogenic with Ahr^fx and Ahr^V375A, making it a potentially useful tool when used as part of this allelic series.

Truncated (Ahr^Ter383):

Like the Ahr^NG367R allele described above, the Ahr^Ter383 mutant appears to have been generated as an artifact of CRISPR-mediated NHEJ during the effort to generate Ahr^V375A (Figure 5)(Ceccaldi et al., 2016; R. H. Wilson et al., 2021). That is, the Ahr^Ter383 allele is the apparent consequence of a double stranded break induced by Cas9 endonuclease that was repaired through NHEJ mechanisms that inserted a single nucleotide, resulting in a frameshift within codon 371 and a subsequent premature stop codon at codon 383 (Figure 4, Table 2). Due to the proximity of the frame-shift mutations to the LBD, AHR^Ter383 is not expected to bind ligand, though the NLS, DBD, and ARNT binding domains remain intact and are expressed at 37 kDa, as demonstrated in protein expression experiments (Dolwick, Swanson, et al., 1993; R. H. Wilson et al., 2021; Xing et al., 2012)). Functional in vivo studies indicate that the resultant AHR^Ter383 protein displays antimorphic activity for the DV endpoint. In support of this idea are the observations that mice heterozygous for this allele and the Ahr^b1 allele (Ahr^Ter383/b1) exhibit patent DV with 85% penetrance (R. H. Wilson et al., 2021). Yet, excision of the Ahr^Ter383 allele in the endothelial compartment (Cre^Cdh5 ) of Ahr^Ter383Cre^Cdh5 mice rescues the patent DV phenotype (16%) (R. H. Wilson et al., 2021). Interestingly, heterozygous mice display AHR-driven gene induction for Cyp1a1, Cyp1a2, Cyp1b1 and Ahrr at levels comparable to haploinsufficient Ahr mice (Ahr^Δ2/b1). This observation suggests that the Ahr^Ter383 allele is not an antimorph for gene induction but is antimorphic for DV closure.

The lack of a TAD in the AHR^Ter383 mutant potentially mediates the selective antimorphic behavior such that the absence of cofactor binding that typically occurs in the TAD may be sufficient to block gene induction for the DV endpoint but not for induction of the Cyp1s or Ahrr. Given the observation that ARNT has an intact TAD, it is possible that ARNT can recruit cofactors required for gene induction in certain contexts. The missing TAD in the AHR^Ter383 mutant is therefore an interesting observation, the consequences of which warrant additional study. Finally, it is important to note that mice homozygous for this allele are a phenocopy of null mice for the DV endpoint and may be useful in identifying other ARNT dependent signaling events mediated by partners such as hypoxia inducible factors (HIFs) (Chan et al., 1999; Gradin et al., 1996; McIntosh et al., 2010). While the mechanism of AHR^Ter383 is unknown, mice harboring this allele exhibit interesting phenotypes and additional study of this constract mayb provide insight into related pathways.

ARNT and Chaperone Mutants

The various approaches described above have yielded numerous alleles of the Ahr, each informative and useful it its own way. A parallel approach to understand AHR signal transduction in vivo has been to create recombinant alleles at other loci known to influence upstream signaling events. In reviewing these alleles, we will follow our direction noted above to focus on those alleles generated at the locus, leaving for another day a description of transgenic approaches, which although powerful and informative, generate events that reside at locations within the genome that are largely unknown.

Arnt null mutants:

The obligate binding partner of the AHR, ARNT, has been modified in several ways to further elucidate the role of this protein in PAS sensor signaling (Figure 7, Table 5). Initial studies focused on generating Arnt null models (Kozak et al., 1997; Maltepe et al., 1997). Two such models have been generated independently using ESCs from 129 mice. One model was developed through a gene targeting approach that created a disruption of the bHLH domain with the insertion of a Neo^PGK cassette (Maltepe et al., 1997). In another, homologous recombination was used to remove the bHLH exon (Kozak et al., 1997). As homozygotes, both models were found to die in utero between embryonic days 9.5 and 10.5, while heterozygous littermates survived. This early embryonic lethality was soon reveled to be a function of the fact that ARNT had multiple dimerization partners in addition to the AHR. Notably, ARNT is now known to dimerize with HIFs, and through these interactions is essential in biological processes including the hypoxia response, glucose metabolism, blood cell development, and angiogenesis (Gradin et al., 1996; McIntosh et al., 2010). Because Arnt null animals are embryonic lethal and do not produce viable offspring, their use has proven more common in developing an understanding of non-AHR pathways. Such studies are not reviewed here.

Figure 7.

Gene maps of Ara9 mutants. The Ara9fxneo harbors two loxP site (depicted by red triangles), two of which flank exons 3 through 6. The Flp sites are shown as yellow triangles and flank a NeoPGK cassette in the intron following exon-6. The Ara9fx mouse harbors two loxP sites flanking exons 3–6.

Table 5. Summary of selected Transgenic Ahr models.

Non-exhaustive list of Ahr transgenic models and references.

Transgenic Mouse Model	Tissue-specific	Reference
Constitutively active AHR	Keratin 14	Tauchi 2005 (Tauchi et al. 2005)
	CD2	Nohara 2005 (Nohara et al. 2005)
	Modified simian virus 40 promoter with IgH intron enhancer	Andersson 2002 (Andersson et al. 2002)
	Immunoglobulins heavy chain	Moennikes 2004 (Moennikes et al. 2004)
	Ubiquitous	Brunnberg 2011, McGuire 2001 (Brunnberg et al. ; McGuire et al. 2001)
	Tetracycline active; Fatty acid binding protein	Lee 2010 (Lee et al.)
Humanized AHR	Liver	Flaveny 2009 (Flaveny et al. 2009)

Arnt hypomorph (Arnt^fxneo):

In an effort to develop models for use in AHR signal transduction, strategies were employed to bypass the embryonic lethality. In one approach, the Arnt allele was made hypomorphic or “low expressing” (Arnt^fxneo) using a similar approach as the AHR hypomorph (Walisser, Bunger, Glover, Harstad, et al., 2004). This was accomplished using homologous recombination in 129 ESCs through the insertion of a floxed Neo^PGK cassette downstream of exon-6 (the bHLH encoding exon) (Figure 6, Table 5). The target vector also introduced loxP sites flanking exon-6, such that the allele harbors three loxP sites (loxP-exon6-loxP-neo-loxP) allowing selective deletion of either the Neo^PGK cassette or exon-6 upon crosses with appropriate Cre expressing strains. Importantly, the ARNT hypomorphic expression (approximately 10–20% of normal) is high enough to override the embryonic lethality step resulting in live progeny in nearly normal litter sizes.

Figure 6.

Gene maps of Arnt mutants. The blue exons encode for the bHLH and the green exons encode the PAS domain. The maps are abbreviated at exon-8. The Arntfxneo harbors three loxP (depicted by red triangles) sites, two of which flank exon-6 and one downstream of a NeoPGK cassette. The Arntfx mouse harbors two loxP sites flanking exon-6.

This hypomorphic Arnt model has been used to determine the requirement of ARNT for the AHR-driven adaptive, toxic, and development pathways. The ARNT hypomorph mouse exhibits a patent DV with a penetrance approaching that of the Ahr^Δ2 null mouse (Lahvis et al., 2005; Walisser, Bunger, Glover, & Bradfield, 2004; Walisser, Bunger, Glover, Harstad, et al., 2004). Additionally, this model has an attenuated response to TCDD-induced hepatotoxicity and thymic involution. These mice, however, exhibit EROD activity at levels consistent with wildtype and heterozygous mice. Such observations highlight the importance of ARNT for the AHR-mediated developmental and toxic endpoints and suggest that diminished levels of ARNT do not impair adaptive signaling at the examined dose of TCDD. These data hint at the idea that various AHR-driven processes, such as induction of adaptive metabolism, require smaller amounts of AHR:ARNT dimers than other endpoints like TCDD toxicity and vascular development. Interestingly, the patent DV can be rescued in these animals when treating pregnant dams with TCDD at E12. This observation has been used as support for the idea that activation of AHR by an endogenous ligand at a critical developmental period may be required to induce transcription of genes required for DV closure. This model has been useful in testing “cross talk” ideas in which AHR-driven endpoints- specifically that of TCDD toxicity- are proposed to be independent of AHR:ARNT transcriptional events and instead work through interactions between AHR and other proteins such as c-myc, retinoblastoma, NFKB, and KLF6 (Ge & Elferink, 1998; Puga et al., 2000; Reiners & Clift, 1999; S. R. Wilson et al., 2013). Early observations defining the requirement of ARNT to dissociate Hsp90 from AHR further supports the requirement of ARNT for AHR pathway function even in instances where AHR dimerizes with other partners (Jacqueline McGuire et al., 1994).

ARNT conditional (Arnt^fx):

The ARNT conditional mouse (Arnt^fx) was generated from a cross with the ARNT hypomorph and a mouse harboring the Cre^Ella transgene (Holzenberger et al., 2000; Manabu Nukaya et al., 2010). This approach was similar to the process used in generation of the Ahr^fx conditional mouse noted above (Walisser et al., 2005). Because of the low-level expression of Cre, various combinations of loxP mediated excision occur in the germ line. Thus, through screening of offspring, this cross allowed for identification of a line where excision of the Neo^PGK cassette was completed but a floxed exon-6 remained in the germ line allowing later cell specific deletion (Figure 6). This model has been used to determine the requirement of Arnt in hepatocytes for the dioxin induced hepatotoxicity and adaptive endpoints through crosses with Cre^alb (Manabu Nukaya et al., 2010). One advantage of this mode is that it allows the use of a responsive Ahr^b1 allele and this lower doses of TCDD exposure. As is the case with AHR, ARNT expression in hepatocytes is not required for DV closure. Together, these studies highlight the requirement of ARNT and AHR:ARNT transcriptional activity for many AHR-mediated endpoints. Deletion of Arnt from certain cell types mimics the consequences of cell specific deletion of Ahr, indicating both ARNT and AHR are required for TCDD-induced hepatotoxicity and induction of the adaptive response.

Ara9 hypomorph (Ara9^fxneo):

The ARA9 hypomorph (Ara9^fxneo) is a mouse model that expresses limited amounts of the chaperone protein, Ah receptor associated 9 (ARA9) (also known as Ah Receptor interacting protein, AIP, or Hepatitis B Virus X associated protein, XAP2) (Figure 7) (LaPres et al., 2000; Lin et al., 2008). This mouse was generated using homologous recombination in 129 ESCs to a generate an allele with loxP sites flanking exons-3 through 6 and Frt (Flippase Recognition target) sites flanking the Neo^PGK cassette located immediately downstream of the 3’ loxP sites (Figure 8). The Frt sites allow deletion of the Neo^PGK cassette while the loxP sites allow deletion of the exons upon crosses with transgenic strains harboring the appropriate recombinase (Flp or Cre). Historically, mice harboring this allele were backcrossed onto B6 mice, and therefore express the Ahr^b1 allele.

Figure 8.

Rat Ahr allele structure and protein maps. A. Gene structure of rat Ahr from Srague-Dawley and Long Evans rats (rAhrSD and rAhrLE) and Han Wistar (rAhrHW). Exons encoding the bHLH are blue, PAS encoding exons are green, and yellow exons encode the TAD. In rAhrSD and rAhrLE rats, the splice donor site (y) splices to the acceptor site (z) at exon 10 and 11 boundaries, respectively. In rAhrHW, there is a G!A mutation in the donor splice site (y), which causes the cell to search elsewhere for cryptic splice sites (a, b, c). One splice site (a) is −129 base pairs from the exon-10 boundary and encodes for the HWdv mRNA. Two other splice sites (b and c) are located in the intron and encode for the HWsiv and HWliv, respectively. B. Protein maps of rat AHR. The rAHRSD encodes a wildtype protein where exon 10 and 11 are joined through the splicing at sites y and z, creating the y-z junction. The rAhrSD animals also encode a Valine (V) at residue 507 in exon 10. The rAhrHW splice variants encode two protein products. The HWdv encodes a protein where cryptic splice site a is used as the new splice donor site and is joined to the z splice donor site at exon-11 (a-z). This results in a protein product that is missing 43 amino acids from exon-10. The HWsiv and HWliv encode for the same protein product which encodes for all of exon-10 and reads through for 7 additional amino acids into the intron (shown in black). Translation is terminated due to the recognition of a new stop codon in the intron which is why HWsic and HWliv encode for the same protein product. Therefore, this protein variant lacks all of exon-11. All rAHRHW proteins harbor an Alanine (A) at residue 507 in exon-10. C. Gene structure of the Ahr null rat. The bHLH was targeted using zinc finger nuclea gene editing and resulted in two alleles, one of which harbors a 2 base pair deletion and one that has a 29 base pair deletion. Both mutations lead to a premature stop codon in exon-2.

Like the ARNT hypomorphic model described above, the Ara9 hypomorph was generated because deletion of the gene resulted in embryonic lethality and this limited its utility for AHR studies. Because the model is suspected to serve a chaperone role, it is not surprising that markedly reduced levels of its AHR client are observed in most if not all tissues (approximately 10–20% of normal expression levels). Essentially, Ara9 hypomorphs are also Ahr hypomorphs (LaPres et al., 2000). Thus, it is not surprising that homozygous Ara9^fxneo mice exhibit a patent DV with 83% penetrance while heterozygous animals exhibit a 10% penetrance of DV. This model had been useful in creating graded expression of ARA9 protein which correlates to a graded expression of AHR. When pregnant dams are treated with TCDD, again the shunt is rescued, providing further support for the idea that activation of AHR by a high affinity ligand can sufficiently activate the AHR developmental pathway. This model may be useful in manipulating levels of AHR protein in order to further elucidate the consequences of varying levels of AHR protein (Table 3).

Ara9 conditional (Ara9^fx):

The Ara9 conditional mouse model (Ara9^fx) was generated from the Ara9^fxneo through crosses with mice expressing Flp recombinase under the control of the ROSA26S promoter (Flp^ROSA26S)(Farley et al., 2000). This allowed excision of the Neo^PGK cassette while maintaining expression of the loxP sites flanking exons-3 though 6 (Figure 7) (M. Nukaya et al., 2010). Offspring with the Neo^PGK cassette excised were employed to remove the cis-effects of the Neo^PGK insertion and release its inhibitory activity on expression. Mice homozygous for the Ara9^fx/fx allele developed normally and did not display apparent liver or cardiac development present in the hypomorph. The Ara9^fx animals can be crossed to cell specific Cre recombinase models to excise Ara9 in targeted cell types.

As an example of the utility of the conditional Ara9^fx model, a cross to Cre^Alb transgenic line has been used to show that ARA9 expression in hepatocytes is required for TCDD-induced hepatic damage (M. Nukaya et al., 2010). This is despite the fact that Ara9^fx/fxCre^Alb mice treated with TCDD display normal induction of TCDD induced Cyp1a1 and Cyp1a2 activity. Interestingly, in Ara9^fx/fxCre^alb mice, Cyp1b1 and Ahrr expression is nearly abolished while Ara9^fx/fx animals exhibit normal, highly inducible levels of expression. These experiments support the hypothesis that different DRE-driven genes respond differentially to varying levels of AHR (Table 5). Overall, this model has brought to light the important observation that not all AHRE-driven genes behave the same in models with altered levels of AHR.

RAT MODELS

Naturally Occurring Polymorphisms in the Rat Aryl Hydrocarbon Receptor

While the natural genetics of mice and rats follow the same principles, practically, it is important to note that rat stains are more commonly used as outbred stocks where more genetic variability can be expected (Brekke et al., 2018; Wildt et al., 2008). Like mice, naturally occurring polymorphisms at the rat Ahr locus have provided models that have shed light on AHR signal transduction (Pohjanvirta et al., 1987; Pohjanvirta et al., 1999). Importantly, the rat and mouse polymorphisms have taught us about different aspects of signaling. Unlike the mouse, the molecular underpinnings of AHR signaling differences in rat strains are not due to ligand binding affinity but are due to polymorphisms that influence transactivation (Pohjanvirta et al., 1998).

The most well characterized rat polymorphism is observed upon comparison of the Han-Wistar (HW), with more commonly used outbred stocks such as the Long-Evans (LE) and Sprague-Dawley (SD). By comparison, the HW stock is about 1000-times less sensitive to certain toxic endpoints of TCDD exposure than are either the SD or LE stocks (Pohjanvirta et al., 1988; Pohjanvirta et al.). Unlike mice where much of the differential sensitivity to TCDD is attributed to the polymorphism in the PAS B domain, the reported differential sensitivity in rats is the consequence of a point mutation at the exon-intron boundary (splice donor site) between exon-10 and 11 (Figure 8)(Pohjanvirta et al., 1998). The result is the generation of three alternative splice variants and two truncated protein products (Moffat et al., 2007; Okey et al., 2005; Pohjanvirta et al., 1998). The three splice variants include a deletion variant (HWdv; the deletion of 129 bases from NT 2326–2454), a “shorter insertion variant” (HWsiv; the insertion of 29 bases between NT 2454 and 2455), and a “longer insertion variant” (HWliv; an insertion of 134 bases between NT 2454 and 2455) (Pohjanvirta et al., 1988). The HWdv deletion employs a cryptic upstream 5’ site within exon-10 that splices to the donor site of exon-11. This event results in an internal loss of 43 amino acids (766 to 808). The HWsiv and the HWliv transcripts employ cryptic donor splice sites within the intron downstream of exon-10. Both lead to read-through into the intron and use of a premature stop codon after addition of seven novel amino acids. The result is a protein that also lacks amino acids from exon-11, producing a net loss of 38 amino acids.

This C-terminal half of the AHR is a region that corresponds to domains responsible for transactivation of target genes (Sanjay Jain et al., 1994; Q. Ma et al., 1995). Importantly, the rat AHR proteins from sensitive (LE) and resistant (HW) strains bind TCDD with similar affinities (Okey et al., 2005; Pohjanvirta & Tuomisto, 1994). Experimental evidence suggests that the disruption of transcriptional activity gives rise to the altered signaling response between HW and other rat stocks (Pohjanvirta et al., 1998). Interestingly, this disruption in the transactivation domain does not alter induction of many typical AHRE-driven genes, such as Cyp1a1(Okey et al., 2005; Pohjanvirta et al., 1999). In contrast, it has been proposed to disrupt an unidentified battery of genes required for certain toxicity endpoints (e.g., lethality)(Okey et al., 2005). Another potential contributor to differential sensitivity in this rat model is through an influence on receptor density. In this regard, in many species, the C-termini also appears to influence receptor stability and thus indirectly may influence receptor expression at many sites (Poland et al., 1994). Thus, in rats, receptor density may be a secondary factor when considering differential responsiveness between stocks, as HW srats have lower receptor density than LE rats.

Induced mutations in the Rat

Rat null:

The rat is an important model in both toxicology and physiology, frequently offering experimental avenues not possible in mice or in vitro. Because of this reason, an Ahr null rat was made on an outbred SD background using the zinc finger nuclease (ZNF) gene editing technology (Harrill et al., 2013). The ZNF techniques preceded CRSISPR but work similarly in that the ZNF creates targeted double stranded breaks in the sequence of interest with a common outcome being the generation of a frameshift or nonsense mutation creating a null allele (Carroll, 2011). In the case of rat Ahr, exon-2, which encodes for the bHLH, was targeted in order to compare this model to previously generated null mice (Harrill et al., 2013; J. V. Schmidt et al., 1996). Two mutations arose from the process, one was a 2 base-pair deletion while the other was a 29 base pair deletion, both of which were predicted, and confirmed by western blot and quantitative PCR, to yield a bona-fide null allele.

Perhaps one of the most interesting findings from the Ahr null rat model is the observation that null rats display different physiological and developmental phenotypes as compared to the mouse null (Harrill et al., 2013). For example, rats null for AHR proteins do not display the vascular defects, such as a patent DV and persistent hyaloid arteries, common in mouse null models (J. V. Schmidt et al., 1996). This is despite the fact that, in both models, AHRE-driven gene expression is blocked (Harrill et al., 2013; J. V. Schmidt et al., 1996). Conversely, null rats display phenotypes that have not been reported in mice, such as hydronephrosis and pathologies in the urinary tract such as bilateral ureter dilation and uroepithelial degeneration. In sum these interesting findings suggest each genome harbors multiple modifiers of AHR signal transduction that may be mapped by genetic means.

CONCLUDING REMARKS

Mouse and rat models have been essential tools in the effort to better understand AHR biology. These models have aided in the understanding of certain AHR mediated processes including dioxin toxicity, adaptive metabolism, development of the vasculature, and modulation of the immune system. With the recent advancements in gene editing technology, it is likely that the creation and availability of additional genetic tools to answer biologically relevant questions will expand in the coming years. Combining these new tools with those that have been created in the past can be useful in examining the role of the AHR in complex biological processes include barrier physiology, the orchestration of immune responses, vascular development, and toxicity of certain AHR ligands. The generation and characterization of new models will also allow the scientific community to reconcile discrepancies observed in existing models contributing to a more complete and accurate understanding of AHR biology in a range of species, including humans. We wrote this review with the intention of providing a resource that compiles the various models that have been and will likely continue to be essential in answering important questions in AHR biology. As more models become available, we expect they will be useful additions to these allelic series.

GLOSSARY OF TERMS

AHR

Aryl hydrocarbon receptor

PAS

Per-ARNT-SIM

PAHs

polycyclic aromatic hydrocarbons

BaP

benzo(a)pyrene

Cyp1a1

cytochromes P450 1a1

Cyp1a2

cytochromes P450 1a2

Cyp1b1

cytochromes P450 1b1

TCDD

2,3,7,8-tetrachlorodibenzo-p-dioxin

bHLH

basic helix-loop-helix

ARNT

aryl hydrocarbon nuclear translocator

AHRE

AHR-ARNT responsive elements; also known as dioxin response elements (DREs) or xenobiotic response elements (XREs)

HSP90

Heat Shock Protein 90

ARA9

Ah Receptor Associated 9, also known as Ah Receptor interacting protein (AIP) or Hepatitis B Virus X associated protein (XAP2)

AHRR

Ah Receptor Repressor

TAD

Transactivation domain

B6

C57BL/6 laboratory mouse strain

D2

DBA/2 laboratory mouse strain

129

129Sv laboratory mouse strain

DV

ductus venosus

ESCs

Embryonic stem cells

Neo^PGK

Neomycin cassette driven by PGK promoter

Cre^Ella

Cre recombinase driven by E2a promoter

Cre^Alb

Cre recombinase driven by Albumin (hepatocyte) promoter

Cre^Tek

Cre recombinase driven by Tek (endothelial) promoter

Neo^pTK

Neomycin cassette driven by pTK promoter

NLS

nuclear localization sequence

DBD

DNA Binding Domain

CA-AHR

Constitutively active AHR

CAIR

Constitutively active AHR with IRES-GFP

IRES

internal ribosomal entry site

GFP

Green florescent protein

Treg

Regulatory T cell

Foxp3

Forkhead Box protein 3

NHEJ

Nonhomologous end joining

HDR

Homology directed repair

BNF

beta naphthoflavone

Cre^Cdh5

Cre recombinase driven by Cdh5 (endothelial) promoter

HIF

Hypoxia inducible factor

Frt

Flippase Recognition target

ZNF

zinc finger nuclease

HW

Han-Wistar rat stock

LE

Long-Evans outbred rat stock

SD

Sprague-Dawley outbred rat stock