Generation of a Magoh conditional allele in mice

Magoh encodes a core component of the exon junction complex (EJC), which binds mRNA and regulates mRNA metabolism. Magoh is highly expressed in proliferative tissues during development. EJC components have been implicated in several developmental disorders including TAR syndrome, Richieri–Costa–Pereira syndrome, and intellectual disability. Existing germline null Magoh mice are embryonic lethal as homozygotes and perinatal lethal as heterozygotes, precluding detailed analysis of embryonic and postnatal functions. Here, we report the generation of a new genetic tool to dissect temporal and tissue‐specific roles for Magoh in development and adult homeostasis. This Magoh conditional allele has two loxP sites flanking the second exon. Ubiquitous Cre‐mediated deletion of the floxed allele in a heterozygous mouse (Magoh^del/+) causes 50% reduction of both Magoh mRNA and protein. Magoh^del/+ mice exhibit both microcephaly and hypopigmentation, thus phenocopying germline haploinsufficient Magoh mice. Using Emx1‐Cre, we further show that conditional Magoh deletion in neural progenitors during embryonic development also causes microcephaly. We anticipate this novel conditional allele will be a valuable tool for assessing tissue‐specific roles for Magoh in mammalian development and postnatal processes. genesis 52:752–758, 2014. © 2014 Wiley Periodicals, Inc.     

A p57 conditional mutant allele that allows tracking of p57‐expressing cells

p57^Kip2 (p57 ) is a maternally expressed imprinted gene regulating growth arrest which belongs to the CIP/KIP family of cyclin‐dependent kinase inhibitors. While initially identified as a cell cycle arrest protein through inhibition of cyclin and cyclin‐dependent kinase complexes, p57 activity has also been linked to differentiation, apoptosis, and senescence. In addition, p57 has recently been shown to be involved in tumorigenesis and cell fate decisions in stem cells. Yet, p57 function in adult tissues remains poorly characterized due to the perinatal lethality of p57 knock‐out mice. To analyze p57 tissue‐specific activity, we generated a conditional mouse line (p57^FL‐ILZ/+ ) by flanking the coding exons 2–3 by LoxP sites. To track p57‐expressing or mutant cells, the p57^FL‐ILZ allele also contains an IRES‐linked β‐galactosidase reporter inserted in the 3′ UTR of the gene. Here, we show that the β‐galactosidase reporter expression pattern recapitulates p57 tissue specificity during development and in postnatal mice. Furthermore, we crossed the p57^FL‐ILZ/+ mice with PGK‐Cre mice to generate p57^cKO‐ILZ/+ animals with ubiquitous loss of p57. p57^cKO‐ILZ/+ mice display developmental phenotypes analogous to previously described p57 knock‐outs. Thus, p57^FL‐ILZ/+ is a new genetic tool allowing expression and functional conditional analyses of p57.     

Generation of a conditional null allele of NADPH oxidase activator 1 (NOXA1)

NADPH oxidase complexes are multiprotein assemblies that generate reactive oxygen species in a variety of mammalian tissues. The canonical phagocytic oxidase consists of a heterodimeric, enzymatic core comprised of the transmembrane proteins, CYBB andCYBA and is regulated, in part, by an “organizing” function of NCF1 and an “activating” activity of NCF2. In contexts outside of the phagocyte, these regulatory functions may be encoded not only by NCF1 and NCF2, but also alternatively by their respective paralogues, NOXO1 and NOXA1. To allow tissue‐specific dissection of Noxa1 function in mouse, we have generated an allele of Noxa1 suitable for conditional inactivation. Moreover, by crossing Noxa1 conditional allele carriers to B6.129S4‐Meox2^tm1(Cre)Sor/J mice, we have generated first, Noxa1‐null heterozygotes, and ultimately, Noxa1‐null homozygotes. Through the thoughtful use of tissue‐specific, Cre‐expressing mouse strains, the Noxa1 conditional allele will offer insight into the roles of NOXA1 in the variety of tissues in which it is expressed. genesis 48:568–575, 2010. © 2010 Wiley‐Liss, Inc.     

A critical role for the nuclear protein Akirin2 in the formation of mammalian muscle in vivo

Evolutionarily conserved Akirin nuclear proteins interact with chromatin remodeling complexes at gene enhancers and promoters, and have been reported to regulate cell proliferation and differentiation. Of the two mouse Akirin genes, Akirin2 is essential during embryonic development, with known in vivo roles in immune system function and the formation of the cerebral cortex. Here we demonstrate that Akirin2 is critical for mouse myogenesis, a tightly regulated developmental process through which myoblast precursors fuse to form mature skeletal muscle fibers. Loss of Akirin2 in somitic muscle precursor cells via Sim1‐Cre‐mediated excision of a conditional Akirin2 allele results in neonatal lethality. Mutant embryos exhibit a complete lack of forelimb, intercostal, and diaphragm muscles due to extensive apoptosis and loss of Pax3‐positive myoblasts. Severe skeletal defects, including craniofacial abnormalities, disrupted ossification, and rib fusions are also observed, attributable to lack of skeletal muscles as well as patchy Sim1‐Cre activity in the embryonic sclerotome. We further show that Akirin2 levels are tightly regulated during muscle cell differentiation in vitro, and that Akirin2 is required for the proper expression of muscle differentiation factors myogenin and myosin heavy chain. Our results implicate Akirin2 as a major regulator of mammalian muscle formation in vivo.     

Heterozygous deletion of Atbf1 by the Cre‐loxP system in mice causes preweaning mortality

ATBF1 is a large nuclear protein that contains multiple zinc‐finger motifs and four homeodomains. In mammals, ATBF1 regulates differentiation, and its mutation and/or downregulation is involved in tumorigenesis in several organs. To gain more insight into the physiological functions of ATBF1, we generated and validated a conditional allele of mouse Atbf1 in which exons 7 and 8 were flanked by loxP sites (Atbf1^flox). Germline deletion of a single Atbf1 allele was achieved by breeding to EIIa‐cre transgenic mice, and Atbf1 heterozygous mice displayed reduced body weight, preweaning mortality, increased cell proliferation, and attenuated cytokeratin 18 expression, indicating haploinsufficiency of Atbf1. Floxed Atbf1 mice will help us understand such biological processes as neuronal differentiation and tumorigenesis. genesis 1–9, 2012. © 2012 Wiley Periodicals, Inc.     

A mart-1::Cre transgenic line induces recombination in melanocytes and retinal pigment epithelium

The number of transgenic mouse lines expressing Cre in either type of pigment cells (melanocytes and retinal pigment epithelium, RPE) is limited, and the available lines do not always offer sufficient specificity. In this study, we addressed this issue and we report on the generation of a MART‐1::Cre BAC transgenic mouse line, in which the expression of Cre recombinase is controlled by regulatory elements of the pigment cell‐specific gene MART‐1 (mlana). When MART‐1::Cre BAC transgenic mice were bred with the ROSA26‐R reporter line, ß‐galactosidase expression was observed in RPE from E12.5 onwards, and in melanocyte precursors from E17.5, indicating that the MART‐1::Cre line provides Cre recombinase activity in pigment‐producing cells rather than in a particular lineage. In addition, breeding of this mouse line to mice carrying a conditional allele of RBP‐Jκ corroborated the reported phenotypes in both pigment cell lineages, inducing hair greying and microphthalmia. Our results thus suggest, that the MART‐1::Cre line may serve as a novel and useful tool for functional studies in melanocytes and the RPE.genesis 49:403–409, 2011. © 2011 Wiley‐Liss, Inc.     

A mouse model to dissect progesterone signaling in the female reproductive tract and mammary gland

Considering the regulatory complexities of progesterone receptor (PR) action throughout the female reproductive axis and mammary gland, we generated a mouse model that enables conditional ablation of PR function in a spatiotemporal specific manner. Exon 2 of the murine PR gene was floxed to generate a conditional PR allele (PR^flox) in mice. Crossing the PR^flox/flox mouse with the ZP3‐cre transgenic demonstrated that the PR^flox allele recombines to a PR null allele (PR^d). Mice homozygous for the recombined null PR allele (PR^d/d) exhibit uterine, ovarian, and mammary gland defects that phenocopy those of our previously described PR knockout (PRKO) model. Therefore, this conditional mouse model for PR ablation represents an invaluable resource with which to further define in a developmental and/or reproductive stage‐specific manner the individual and integrative roles of distinct PR populations resident in multiple progesterone‐responsive target sites. genesis 48:106–113, 2010. © 2009 Wiley‐Liss, Inc.     

Activation loop phosphorylation regulates B‐Raf in vivo and transformation by B‐Raf mutants

Despite being mutated in cancer and RASopathies, the role of the activation segment (AS) has not been addressed for B‐Raf signaling in vivo. Here, we generated a conditional knock‐in mouse allowing the expression of the B‐Raf^AVKA mutant in which the AS phosphoacceptor sites T599 and S602 are replaced by alanine residues. Surprisingly, despite producing a kinase‐impaired protein, the Braf^AVKA allele does not phenocopy the lethality of Braf‐knockout or paradoxically acting knock‐in alleles. However, Braf^AVKA mice display abnormalities in the hematopoietic system, a distinct facial morphology, reduced ERK pathway activity in the brain, and an abnormal gait. This phenotype suggests that maximum B‐Raf activity is required for the proper development, function, and maintenance of certain cell populations. By establishing conditional murine embryonic fibroblast cultures, we further show that MEK/ERK phosphorylation and the immediate early gene response toward growth factors are impaired in the presence of B‐Raf^AVKA. Importantly, alanine substitution of T599/S602 impairs the transformation potential of oncogenic non‐V600E B‐Raf mutants and a fusion protein, suggesting that blocking their phosphorylation could represent an alternative strategy to ATP‐competitive inhibitors.     

Generation of mice with a conditional and reporter allele for Tmem100

Activin receptor‐like kinase 1 (ACVRL1; ALK1) is predominantly expressed in arterial endothelial cells and plays an important role in angiogenesis. ACVRL1 mutations cause hereditary hemorrhagic telangiectasia (HHT), a genetic vascular disorder for which the underlying mechanism is poorly understood. We have found that expression of transmembrane protein 100 (Tmem100) is downregulated in the lung of Acvrl1‐deficient mice; however, its function is unknown. To elucidate the role of Tmem100 in vivo, we generated a conditional knockout allele for Tmem100 in which exon3, containing the entire coding sequence, was flanked by loxP sequences. The targeted allele also possessed a lacZ reporter cassette in intron2 for visualization of Tmem100 expression. We found that Tmem100 was predominantly expressed in arterial endothelial cells of developing embryos. The conditional and reporter allele will be a useful resource to investigate the in vivo role of Tmem100, especially in angiogenesis. genesis 48:673–378, 2010. © 2010 Wiley‐Liss, Inc.     

Conditional and domain‐specific inactivation of the Tsc2 gene in neural progenitor cells

Tuberous sclerosis complex (TSC) is a genetic disease characterized by multiorgan benign tumors as well as neurological manifestations. Epilepsy and autism are two of the more prevalent neurological complications and are usually severe. TSC is caused by mutations in either the TSC1 (encodes hamartin) or the TSC2 (encodes tuberin) genes with TSC2 mutations being associated with worse outcomes. Tuberin contains a highly conserved GTPase‐activating protein (GAP) domain that indirectly inhibits mammalian target of rapamycin complex 1 (mTORC1). mTORC1 dysregulation is currently thought to cause much of the pathogenesis in TSC but mTORC1‐independent mechanisms may also contribute. We generated a novel conditional allele of Tsc2 by flanking exons 36 and 37 with loxP sites. Mice homozygous for this knock‐in Tsc2 allele are viable and fertile with normal appearing growth and development. Exposure to Cre recombinase then creates an in‐frame deletion involving critical residues of the GAP domain. Homozygous conditional mutant mice generated using Emx1^Cre have increased cortical mTORC1 signaling, severe developmental brain anomalies, seizures, and die within 3 weeks. We found that the normal levels of the mutant Tsc2 mRNA, though GAP‐deficient tuberin protein, appear unstable and rapidly degraded. This novel animal model will allow further study of tuberin function including the requirement of the GAP domain for protein stability. genesis 51:284–292. © 2013 Wiley Periodicals, Inc.     

A conditional allele of Rspo3 reveals redundant function of R‐spondins during mouse limb development

Summary: R‐spondins are secreted ligands that bind cell surface receptors and activate Wnt/β‐catenin signaling. Human mutations and gene inactivation studies in mice have revealed a role for these four proteins (RSPO1‐4) in diverse developmental processes ranging from sex determination to limb development. Among the genes coding for R‐spondins, only inactivation of Rspo3 shows early embryonic lethality (E10.5 in mice). Therefore, a conditional allele of this gene is necessary to understand the function of R‐spondins throughout murine development. To address this need, we have produced an allele in which loxP sites flank exons 2–4 of Rspo3, allowing tissue‐specific deletion of these exons in the presence of Cre recombinase. We used these mice to investigate the role of Rspo3 during limb development and found that limbs ultimately developed normally in the absence of Rspo3 function. However, severe hindlimb truncations resulted when Rspo3 and Rspo2 mutations were combined, demonstrating redundant function of these genes. genesis 50:741–749, 2012. © 2012 Wiley Periodicals, Inc.     

Generation of mice with a conditional null allele for Wntless

The Wnt‐signaling pathway is necessary in a variety of developmental processes and has been implicated in numerous pathologies. Wntless (Wls) binds to Wnt proteins and facilitates Wnt sorting and secretion. Conventional deletion of Wls results in early fetal lethality due to defects in body axis establishment. To gain insight into the function of Wls in later stages of development, we have generated a conditional null allele. Homozygous germline deletion of Wls confirmed prenatal lethality and failure of embryonic axis formation. Deletion of Wls using Wnt1‐cre phenocopied Wnt1 null abnormalities in the midbrain and hindbrain. In addition, conditional deletion of Wls in pancreatic precursor cells resulted in pancreatic hypoplasia similar to that previously observed after conditional β‐catenin deletion. This Wls conditional null allele will be valuable in detecting novel Wnt functions in development and disease. genesis 48:554–558, 2010. © 2010 Wiley‐Liss, Inc.     

Generation of an OMgp allelic series in mice

The very limited ability to regenerate axons after injury in the mature mammalian central nervous system (CNS) has been partly attributed to the growth restrictive nature of CNS myelin. Oligodendrocyte myelin glycoprotein (OMgp) was identified as a major myelin‐derived inhibitor of axon growth. However, its role in axon regeneration in vivo is poorly understood. Here we describe the generation and molecular characterization of an OMgp allelic series. With a single gene targeting event and Cre/FLP mediated recombination, we generated an OMgp null allele with a LacZ reporter, one without a reporter gene, and an OMgp conditional allele. This allelic series will aid in the study of OMgp in adult CNS axon regeneration using mouse models of spinal cord injury. The conditional allele will overcome developmental compensation when employed with an inducible Cre, and allows for the study of temporal and tissue/cell type‐specific roles of OMgp in CNS injury‐induced axonal plasticity. genesis 47:751–756, 2009. © 2009 Wiley‐Liss, Inc.