Generation of mice with a novel conditional null allele of the <Emphasis Type="Italic">Sox9</Emphasis> gene

Sox9 is expressed in multiple tissues during mouse development and adulthood. Mutations in the Sox9 gene or changes in expression levels can be attributed to many congenital diseases. Heterozygous loss-of-function mutations in the human SOX9 gene cause Campomelic dysplasia, a semi-lethal skeletal malformation syndrome. Disruption of Sox9 by conventional gene targeting leads to perinatal lethality in heterozygous mice, hence hampering the feasibility to obtain the homozygous Sox9 null mice for in vivo functional studies. In this study, we generated a conditional allele of Sox9 (Sox9 ^tm4.Tlu ) by flanking exon 1 with loxP sites. Homozygous mice for the Sox9 ^tm4.Tlu allele (Sox9 ^flox/flox ) are viable, fertile and indistinguishable from wildtype (WT) mice, indicating that the Sox9 ^tm4.Tlu allele is a fully functional Sox9 allele. Furthermore, we demonstrated that Cre-mediated recombination using a Col2a1-Cre line resulted in specific ablation of Sox9 activity in cartilage tissues.     

Generation of mice with a conditional null fraser syndrome 1 (Fras1) allele

Summary: Fraser syndrome (FS) is an autosomal recessive disease characterized by skin lesions and kidney and upper airway malformations. Fraser syndrome 1 (FRAS1) is an extracellular matrix protein, and FRAS1 homozygous mutations occur in some FS individuals. FRAS1is expressed at the epithelial‐mesenchymal interface in embryonic skin and kidney. blebbed mice have a null Fras1 mutation and phenocopy human FS. Like humans with FS, they exhibit a high fetal and neonatal mortality, precluding studies of FRAS1 functions in later life. We generated conditional Fras1 null allele mice. Cre‐mediated generalized deletion of this allele generated embryonic skin blisters and renal agenesis characteristic of blebbed mice and human FS. Targeted deletion of Fras1 in kidney podocytes circumvented skin blistering, renal agenesis, and early death. FRAS1 expression was downregulated in maturing glomeruli which then became sclerotic. The data are consistent with the hypothesis that locally produced FRAS1 has roles in glomerular maturation and integrity. This conditional allele will facilitate study of possible role for FRAS1 in other tissues such as the skin. genesis 50:892–898, 2012. © 2012 Wiley Periodicals, Inc.     

Intraflagellar transport protein 172 is essential for primary cilia formation and plays a vital role in patterning the mammalian brain

IFT172, also known as Selective Lim-domain Binding protein (SLB), is a component of the intraflagellar transport (IFT) complex. In order to evaluate the biological role of the Ift172 gene, we generated a loss-of-function mutation in the mouse. The resulting Slb mutant embryos die between E12.5 and 13.0, and exhibit severe cranio-facial malformations, failure to close the cranial neural tube, holoprosencephaly, heart edema and extensive hemorrhages. Cilia outgrowth in cells of the neuroepithelium is initiated but the axonemes are severely truncated and do not contain visible microtubules. Morphological and molecular analyses revealed a global brain-patterning defect along the dorsal-ventral (DV) and anterior-posterior (AP) axes. We demonstrate that Ift172 gene function is required for early regulation of Fgf8 at the midbrain-hindbrain boundary and maintenance of the isthmic organizer. In addition, Ift172 is required for proper function of the embryonic node, the early embryonic organizer and for formation of the head organizing center (the anterior mesendoderm, or AME). We propose a model suggesting that forebrain and mid-hindbrain growth and AP patterning depends on the early function of Ift172 at gastrulation. Our data suggest that the formation and function of the node and AME in the mouse embryo relies on an indispensable role of Ift172 in cilia morphogenesis and cilia-mediated signaling.     

A knock‐in mouse line conditionally expressing the tumor suppressor WTX/AMER1

WTX/AMER1 is an important developmental regulator, mutations in which have been identified in a proportion of patients suffering from the renal neoplasm Wilms' tumor and in the bone malformation syndrome Osteopathia Striata with Cranial Sclerosis (OSCS). Its cellular functions appear complex and the protein can be found at the membrane, within the cytoplasm and the nucleus. To understand its developmental and cellular function an allelic series for Wtx in the mouse is crucial. Whereas mice carrying a conditional knock out allele for Wtx have been previously reported, a gain‐of‐function mouse model that would allow studying the molecular, cellular and developmental role of Wtx is still missing. Here we describe the generation of a novel mouse strain that permits the conditional activation of WTX expression. Wtx fused to GFP was introduced downstream a stop cassette flanked by loxP sites into the Rosa26 locus by gene targeting. Ectopic WTX expression is reported after crosses with several Cre transgenic mice in different embryonic tissues. Further, functionality of the fusion protein was demonstrated in the context of a Wtx null allele.     

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.     

Hippocampal and Cortical Primary Cilia Are Required for Aversive Memory in Mice

It has been known for decades that neurons throughout the brain possess solitary, immotile, microtubule based appendages called primary cilia. Only recently have studies tried to address the functions of these cilia and our current understanding remains poor. To determine if neuronal cilia have a role in behavior we specifically disrupted ciliogenesis in the cortex and hippocampus of mice through conditional deletion of the Intraflagellar Transport 88 (Ift88) gene. The effects on learning and memory were analyzed using both Morris Water Maze and fear conditioning paradigms. In comparison to wild type controls, cilia mutants displayed deficits in aversive learning and memory and novel object recognition. Furthermore, hippocampal neurons from mutants displayed an altered paired-pulse response, suggesting that loss of IFT88 can alter synaptic properties. A variety of other behavioral tests showed no significant differences between conditional cilia mutants and controls. This type of conditional allele approach could be used to distinguish which behavioral features of ciliopathies arise due to defects in neural development and which result from altered cell physiology. Ultimately, this could lead to an improved understanding of the basis for the cognitive deficits associated with human cilia disorders such as Bardet-Biedl syndrome, and possibly more common ailments including depression and schizophrenia.     

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.     

Generation of Evc2/Limbin global and conditional KO mice and its roles during mineralized tissue formation

Ellis‐van Creveld (EvC) syndrome (OMIM 225500) is an autosomal recessive disease characterized with chondrodysplastic dwarfism in association with abnormalities in oral cavity. Ciliary proteins EVC and EVC2 have been identified as causative genes and they play an important role on Hedgehog signal transduction. We have also identified a causative gene LIMBIN for bovine chondrodysplastic dwarfism (bcd) that is later identified as the bovine ortholog of EVC2. Here, we report generation of conventional and conditional mutant Evc2/Limbin alleles that mimics mutations found in EvC patients and bcd cattle. Resulted homozygous mice showed no ciliary localization of EVC2 and EVC and displayed reduced Hedgehog signaling activity in association with skeletal and oral defects similar to the EvC patients. Cartilage‐specific disruption of Evc2/Limbin resulted in similar but milder skeletal defects, whereas osteoblast‐specific disruption did not cause overt changes in skeletal system. Neural crest‐specific disruption of Evc2/Limbin resulted in defective incisor growth similar to that seen in conventional knockouts; however, differentiation of amelobolasts was relatively normal in the conditional knockouts. These results showcased functions of EVC2/LIMBIN during formation of mineralized tissues. Availability of the conditional allele for this gene should facilitate further detailed analyses of the role of EVC2/LIMBIN in pathogenesis of EvC syndrome. genesis 53:612–626, 2015. © 2015 Wiley Periodicals, Inc.     

Identification of G Protein-Coupled Receptors (GPCRs) in Primary Cilia and Their Possible Involvement in Body Weight Control

Primary cilia are sensory organelles that harbor various receptors such as G protein-coupled receptors (GPCRs). We analyzed subcellular localization of 138 non-odorant GPCRs. We transfected GPCR expression vectors into NIH3T3 cells, induced ciliogenesis by serum starvation, and observed subcellular localization of GPCRs by immunofluorescent staining. We found that several GPCRs whose ligands are involved in feeding behavior, including prolactin-releasing hormone receptor (PRLHR), neuropeptide FF receptor 1 (NPFFR1), and neuromedin U receptor 1 (NMUR1), localized to the primary cilia. In addition, we found that a short form of dopamine receptor D2 (DRD2S) is efficiently transported to the primary cilia, while a long form of dopamine receptor D2 (DRD2L) is rarely transported to the primary cilia. Using an anti-Prlhr antibody, we found that Prlhr localized to the cilia on the surface of the third ventricle in the vicinity of the hypothalamic periventricular nucleus. We generated the Npy2r-Cre transgenic mouse line in which Cre-recombinase is expressed under the control of the promoter of Npy2r encoding a ciliary GPCR. By mating Npy2r-Cre mice with Ift80 flox mice, we generated Ift80 conditional knockout (CKO) mice in which Npy2r-positive cilia were diminished in number. We found that Ift80 CKO mice exhibited a body weight increase. Our results suggest that Npy2r-positive cilia are important for body weight control.     

Generation of mouse conditional and null alleles of the type III sodium‐dependent phosphate cotransporter PiT‐1

Accelerated vascular calcification occurs in several human diseases including diabetes and chronic kidney disease (CKD). In patients with CKD, vascular calcification is highly correlated with elevated serum phosphate levels. In vitro, elevated concentrations of phosphate induced vascular smooth muscle cell matrix mineralization, and the inorganic phosphate transporter‐1 (PiT‐1), was shown to be required. To determine the in vivo role of PiT‐1, mouse conditional and null alleles were generated. Here we show that the conditional allele, PiT‐1^flox, which has loxP sites flanking exons 3 and 4, is homozygous viable. Cre‐mediated recombination resulted in a null allele that is homozygous lethal. Examination of early embryonic development revealed that the PiT‐1^{Δe3,4/Δe3,4} embryos displayed anemia, a defect in yolk sac vasculature, and arrested growth. Thus, conditional and null PiT‐1 mouse alleles have been successfully generated and PiT‐1 has a necessary, nonredundant role in embryonic development. genesis 47:858–863, 2009. © 2009 Wiley‐Liss, Inc.     

<Emphasis Type="Italic">XPD</Emphasis> Lys<Superscript>751</Superscript>Gln and Asp<Superscript>312</Superscript>Asn polymorphisms and bladder cancer risk: a meta-analysis

Studies on the polymorphisms of Xeroderma Pigmentosum Group D (XPD) have shown inconclusive trends in the risk of bladder cancer. The purpose of this study is to evaluate the role of XPD single nucleotide polymorphisms in bladder cancer susceptibility. We performed a meta-analysis on all available studies, which included 5,368 and 6,683 XPD Lys⁷⁵¹Gln cases and controls and 3,220 and 4,391 Asp³¹²Asn cases and controls, respectively. Overall, Significant risk effects of Lys⁷⁵¹Gln genotype was found under recessive model contrast [Gln/Gln vs. (Gln/Lys + Lys/Lys)] [P = 0.04, OR = 1.12; 95% CI (1.01, 1.26)], and subtle but insignificantly increased risks between Lys⁷⁵¹Gln and bladder cancer were observed under allele contrast (Gln vs. Lys) and homologous contrast (Gln/Gln vs. Lys/Lys) in all subjects. The ⁷⁵¹Gln allele had no significant effect on bladder cancer in all subgroups (Asian, Caucasian and USA). Significant risk effects of Asp³¹²Asn polymorphism on bladder susceptibility were observed in all subjects under all genetic contrasts, however, stratified analyses showed that the ³¹²Asn allele showed different risk effects in USA and Caucasian. The Gln/Gln genotype acts as a risk factor in its association with bladder cancer, and the effect of Lys⁷⁵¹Gln polymorphism on bladder susceptibility should be studied with larger, stratified population; the ³¹²Asn allele has an important role in the etiology of bladder cancer whereas the ethnic background should be carefully concerned in further studies.     

Sex-specific effect of the <Emphasis Type="Italic">TP53</Emphasis> PIN3 polymorphism on cancer risk in a cohort study of <Emphasis Type="Italic">TP53</Emphasis> germline mutation carriers

Germline mutations in the tumor suppressor gene TP53 occur in the majority of families with Li-Fraumeni syndrome, who are at an increased risk for a wide spectrum of early onset cancers. Several genetic polymorphisms in TP53 modify its effect on cancer risk. While some studies indicate that the TP53 PIN3 deletion allele (D) accelerate tumor onset in carriers with TP53 germline mutations, other studies have shown that the TP53 PIN3 insertion allele (I) confers a significantly higher risk of developing cancer than D allele. To further determine the effects of the TP53 PIN3 polymorphism on cancer development among TP53 germline mutations and to evaluate if those are differenence between male and female carriers, we studied a total of 152 germline mutation carriers with available DNA samples that can be used for genotyping. Our results indicate that the TP53 PIN3 polymorphism has a sex-specific effect on the risk of cancer in TP53 mutation carriers, conferring cancer risk in men (P = 0.0041) but not women with DI or II genotypes.     

Effects of spontaneous <Emphasis Type="Italic">Kitl</Emphasis><Superscript>Steel</Superscript> mutations on survival and red blood cells of mice

Abstract Kit ligand (Kitl), which is a member of the helical cytokine superfamily, is encoded by the Steel (Sl) locus of mice and is essential for the development of hematopoietic cells, germ cells, and melanocytes. A large series of Kitl ^Sl alleles has been described, including some that arose spontaneously and others that were induced by either chemical or radiation mutagenesis. Here we describe the nucleotide sequence alterations in two spontaneous Kitl ^Sl alleles. The Kitl ^Sl-18R allele has a point mutation that introduces a premature termination codon, and the encoded protein is expected to be null functionally. The Kitl ^Sl-5R allele has an in-frame deletion that results in deletion of amino acids at position 31 and 32 of Kitl. While both mutations exert severe effects on blood cells and survival of homozygous mice, these effects are slightly milder than those of a previously characterized spontaneous deletion allele, Kitl ^Sl-gb. Examination of the survival of compound heterozygotes provided strong genetic evidence that the Kitl ^Sl-18R and Kitl ^Sl-5R mutants are null functionally for mouse survival.     

Generation and validation of mice carrying a conditional allele of the epidermal growth factor receptor

The epidermal growth factor receptor (EGFR) is important for normal homeostasis in a variety of tissues and, when abnormally expressed or mutated, contributes to the development of many diseases. However, in vivo functional studies are hindered by the lack of adult mice lacking EGFR because of the pre‐ and postnatal lethality of EGFR deficient mice. We generated a conditional allele of Egfr (Egfr^tm1Dwt) by flanking exon 3 with loxP sites in order to investigate tissue‐specific functions of this widely expressed receptor tyrosine kinase. The activity of the Egfr^tm1Dwt allele is indistinguishable from wildtype Egfr. Conversely, the Egfr^Δ allele, generated by Cre‐mediated deletion of exon 3 using the germline EIIa‐Cre transgenic line, functions as a null allele. Egfr^Δ/Δ embryos that have complete ablation of EGFR activity and die at mid‐gestation with placental defects identical to those reported for mice homozygous for the Egfr^tm1Mag null allele. We also inactivated the Egfr^tm1Dwt allele tissue‐specifically in the skin epithelium using the K14‐Cre transgenic line. These mice were viable but exhibited wavy coat hair remarkably similar to mice homozygous for the Egfr^wa2 hypomorphic allele or heterozygous for the Egfr^Wa5 antimorphic allele. These results suggest that the hairless phenotype of Egfr nullizygous mice is not solely due to absence of EGFR in the epithelium, but that EGFR activity is required also in skin stromal cells for normal hair morphogenesis. This new mouse model should have wide utility to inactivate Egfr conditionally for functional analysis of EGFR in adult tissues and disease states. genesis 47:85–92, 2009. © 2008 Wiley‐Liss, Inc.     

Generation of a conditional mouse model to target Acvr1b disruption in adult tissues

Alk4 is a type I receptor that belongs to the transforming growth factor‐beta (TGF‐β) family. It takes part in the signaling of TGF‐β ligands such as Activins, Gdfs, and Nodal that had been demonstrated to participate in numerous mechanisms ranging from early embryonic development to adult‐tissue homeostasis. Evidences indicate that Alk4 is a key regulator of many embryonic processes, but little is known about its signaling in adult tissues and in pathological conditions where Alk4 mutations had been reported. Conventional deletion of Alk4 gene (Acvr1b) results in early embryonic lethality prior gastrulation, which has precluded study of Alk4 functions in postnatal and adult mice. To circumvent this problem, we have generated a conditional Acvr1b floxed‐allele by flanking the fifth and sixth exons of the Acvr1b gene with loxP sites. Cre‐mediated deletion of the floxed allele generates a deleted allele, which behaves as an Acvr1b null allele leading to embryonic lethality in homozygous mutant animals. A tamoxifen‐inducible approach to target disruption of Acvr1b specifically in adult tissues was used and proved to be efficient for studying Alk4 functions in various organs. We report, therefore, a novel conditional model allowing investigation of biological role played by Alk4 in a variety of tissue‐specific contexts. genesis 51:120–127, 2013. © 2012 Wiley Periodicals, Inc.     

Cytochrome <Emphasis Type="Italic">b</Emphasis><Subscript>5</Subscript> null mouse: a new model for studying inherited skin disorders and the role of unsaturated fatty acids in normal homeostasis

Microsomal cytochrome b ₅ is a ubiquitous, 15.2 kDa haemoprotein implicated in a number of cellular processes such as fatty acid desaturation, drug metabolism, steroid hormone biosynthesis and methaemoglobin reduction. As a consequence of these functions this protein has been considered essential for life. Most of the ascribed functions of cytochrome b ₅, however, stem from in vitro studies and for this reason we have carried out a germline deletion of this enzyme. We have unexpectedly found that cytochrome b ₅ null mice were viable and fertile, with pups being born at expected Mendelian ratios. However, a number of intriguing phenotypes were identified, including altered drug metabolism, methaemoglobinemia and disrupted steroid hormone homeostasis. In addition to these previously identified roles for this protein, cytochrome b ₅ null mice displayed skin defects closely resembling those observed in autosomal recessive congenital ichthyosis and retardation of neonatal development, indicating that this protein, possibly as a consequence of its role in the de novo biosynthesis of unsaturated fatty acids, plays a central role in skin development and neonatal nutrition. Results from fatty acid profile analysis of several tissues suggest that cytochrome b ₅ plays a role controlling saturated/unsaturated homeostasis. These data demonstrate that regional concentrations of unsaturated fatty acids are controlled by endogenous metabolic pathways and not by diet alone.     

<Emphasis Type="Italic">Slc11a1</Emphasis> (<Emphasis Type="Italic">Nramp1</Emphasis>) alleles interact with acute inflammation loci to modulate wound-healing traits in mice

Lines of mice were obtained by selective breeding for maximum (AIRmax) or minimum (AIRmin) acute inflammation. They present distinct neutrophil influx and show frequency disequilibrium of the solute carrier family 11a member 1 (Slc11a1) alleles. This gene is involved in ion transport at the endosomes within macrophages and neutrophils, interfering in their activation. Homozygous AIRmax and AIRmin sublines for the Slc11a1 gene were produced to examine the interaction of this gene with the acute inflammatory loci. The present work investigated wound-healing traits in AIRmax and AIRmin mice, in F₁ and F₂ intercrosses, and in Slc11a1 sublines. Two-millimeter ear punches were made in the mice and hole closure was measured during 40 days. AIRmax mice demonstrated significant tissue repair while AIRmin mice did not. Significant differences between the responses of male and female mice were also observed. Wound-healing traits demonstrated a correlation with neutrophil influx in F₂ populations. AIRmax ^SS showed higher ear-wound closure than AIRmax ^RR mice, suggesting that the Slc11a1 S allele favored ear tissue repair. QTL analysis has detected two inflammatory loci modulating ear wound healing on chromosomes 1 and 14. These results suggest the involvement of the acute inflammation modifier QTL in the wound-healing phenotype.     

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.