Generation and characterization of mice with null mutation of the chloride intracellular channel 1 gene

CLIC1 belongs to a family of highly conserved and widely expressed intracellular chloride ion channel proteins existing in both soluble and membrane integrated forms. To study the physiological and biological role of CLIC1 in vivo, we undertook conditional gene targeting to engineer Clic1 gene knock‐out mice. This represents creation of the first gene knock‐out of a vertebrate CLIC protein family member. We first generated a Clic1 Knock‐in (Clic1^FN) allele, followed by Clic1 knock‐out (Clic1^−/−) mice by crossing Clic1^FN allele with TNAP‐cre mice, resulting in germline gene deletion through Cre‐mediated recombination. Mice heterozygous or homozygous for these alleles are viable and fertile and appear normal. However, Clic1^−/− mice show a mild platelet dysfunction characterized by prolonged bleeding times and decreased platelet activation in response to adenosine diphosphate stimulation linked to P2Y₁₂ receptor signaling. genesis 48:127–136, 2010. © 2010 Wiley‐Liss, Inc.     

Generation and characterization of endonuclease G null mice

Endonuclease G (endo G) is one of the most abundant nucleases in eukaryotic cells. It is encoded in the nucleus and imported to the mitochondrial intermembrane space. This nuclease is active on single- and double-stranded DNA. We genetically disrupted the endo G gene in mice without disturbing a conserved, overlapping gene of unknown function that is oriented tail to tail with the endo G gene. In these mice, the production of endo G protein is not detected, and the disruption abolishes the nuclease activity of endo G. The absence of endo G has no effect on mitochondrial DNA copy number, structure, or mutation rate over the first five generations. There is also no obvious effect on nuclear DNA degradation in standard apoptosis assays. The endo G null mice are viable and show no age-related or generational abnormalities anatomically or histologically. We infer that this highly conserved protein has no mitochondrial or apoptosis function that can discerned by the assays described here and that it may have a function yet to be determined. The early embryonic lethality of endo G null mice recently reported by others may be due to the disruption of the gene that overlaps the endo G gene.     

Generation of mice with a conditional Stat1 null allele

Interferons (IFNs) are key cytokines in the innate immune response that also bridge the gap to adaptive immunity. Signaling upon stimulation by IFN type I, II and III is mediated by the Jak-Stat pathway. STAT1 is activated by all three IFN receptor complexes and absence of STAT1 from mice increases their susceptibility to pathogens. In addition, depending on the setting, STAT1 can act as tumor suppressor or oncogene. Here we report the generation and detailed functional characterization of a conditional Stat1 knockout mouse. We show the integrity of the conditional Stat1 locus and report successful in vivo deletion by means of a ubiquitous and a tissue-specific Cre recombinase. The conditional Stat1 null allele represents an important tool for identifying novel and cell-autonomous STAT1 functions in infection and cancer.     

Generation of mice with a conditional null allele of the Jagged2 gene

The Notch signaling pathway is an evolutionarily‐conserved intercellular signaling mechanism, and mutations in its components disrupt embryonic development in many organisms and cause inherited diseases in humans. The Jagged2 (Jag2) gene, which encodes a ligand for Notch pathway receptors, is required for craniofacial, limb, and T cell development. Mice homozygous for a Jag2 null allele die at birth from cleft palate, precluding study of Jag2 function in postnatal and adult mice. We have generated a Jag2 conditional null allele by flanking the first two exons of the Jag2 gene with loxP sites. Cre‐mediated deletion of the Jag2^flox allele generates the Jag2^del2 allele, which behaves genetically as a Jag2 null allele. This Jag2 conditional null allele will enable investigation of Jag2 function in a variety of tissue‐specific contexts. genesis 48:390–393, 2010. © 2010 Wiley‐Liss, Inc.     

Molecular cloning and characterization of an intracellular chloride channel in the proximal tubule cell line, LLC-PK1

CLC5 is an intracellular chloride channel of unknown function, expressed in the renal proximal tubule. The subcellular localization and function of CLC5 were investigated in the LLC-PK1 porcine proximal tubule cell line. We cloned a cDNA for the porcine CLC5 ortholog (pCLC5) that is predicted to encode an 83-kDa protein with 97% amino acid sequence identity to rat and human CLC5. By immunofluorescence, pCLC5 was localized to early endosomes of the apical membrane fluid-phase endocytotic pathway and to the Golgi complex. Xenopus oocytes injected with pCLC5 cRNA exhibited outwardly rectifying whole cell currents with a relative conductance profile (nitrate Cl(-) approximately Br(-) > I(-) > acetate > gluconate) different from that of control oocytes. Acidification of the extracellular medium reversibly inhibited this outward current with a pK(a) of 6.0 and a Hill coefficient of 1. Overexpression of CLC5 in LLC-PK1 cells resulted in morphological changes, including loss of cell-cell contacts and the appearance of multiple prominent vesicles. These findings are consistent with a potential role for CLC5 in the acidification of membrane compartments of both the endocytic and the exocytic pathway and suggest that its function may be important for normal intercellular adhesion and vesicular trafficking.     

Generation and characterization of mice bearing null alleles of nradd/Nrh2

The Neurotrophin receptor associated death domain gene (Nradd/Nrh2/Plaidd) is a type I transmembrane protein with a unique and short N‐terminal extracellular domain and a transmembrane and intracellular domain that bears high similarity to the p75 neurotrophin receptor (p75NTR/Ngfr). Initial studies suggested that NRADD regulates neurotrophin signaling but very little is known about its physiological roles. We have generated and characterized NRADD conditional and germ‐line null mouse lines. These mice are viable and fertile and dońt show evident abnormalities. However, NRADD deletion results in an increase in the proportion of dorsal root ganglion neurons expressing p75NTR. The NRADD conditional and complete knockout mouse lines generated are new and useful tools to study the physiological roles of NRADD. Birth Defects Research (Part A) 106:605–612, 2016. © 2016 Wiley Periodicals, Inc.     

Isolation and characterization of the human CLC-5 chloride channel gene promoter

The human CLC-5 chloride channel is expressed mainly in the kidney and its mutations cause Dent's disease (a familial renal tubular syndrome with hypercalciuria, tubular proteinuria, rickets, nephrocalcinosis, and eventual renal failure). To gain insight into the regulatory mechanism of CLC-5 expression, a genomic clone that contains the 5'-flanking region of the human CLC-5 gene was isolated and characterized. Two types of 5'-ends of cDNA were isolated by 5'-rapid amplification of cDNA ends, and one of them, approximately 2.1 kbp upstream of ATG-containing exon II, was first identified in human. The major promoter activity was detected in the 5'-flanking region of this newly identified exon Ia. The sequence of the proximal 5'-flanking region contained an activator protein (AP)-1-like site and cAMP-responsive element, but it lacked a TATA box, a GC-rich element, and an SP-1 site. Deletion analysis of the 5'-flanking region showed that the fragments containing the AP-1-like element (TGACTCC) positioned at -38 exhibited high promoter activities in CLC-5 expressing LLC-PK1 cells, but that further deletions not containing this AP-1-like element resulted in a great loss of luciferase activities. Gel-retardation analysis demonstrated the existence of a specific protein binding to this AP-1-like element in LLC-PK1 cells, which seemed to differ from an authentic AP-1. This study clarified the key element of the human CLCN5 promoter, and the mutation in this region could be the cause of Dent's disease.     

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.     

Generation of a conditionally null allele of the laminin alpha1 gene

Laminins are heterotrimeric glycoproteins of the basement membranes. Laminin 1 (alpha1, beta1, gamma1) is the major laminin expressed during early mouse embryogenesis. To gain access to the physiological function of laminin alpha1 chain, we developed a conditionally null allele of its encoding gene (Lama1) using the cre/loxP system. Floxed-allele-carrying mice (Lama1(flox/flox)) display no overt phenotype. Lama1(flox/flox) mice were crossed with transgenic deleter mice (CMV-Cre) to generate Lama1-deficient mice (Lama1(Delta/Delta)). Lama1(Delta/Delta) embryos die during the early postimplantation period after embryonic day 6.5. They lack Reichert's membrane, an extraembryonic basement membrane in which laminin alpha1 is normally highly expressed. In parallel, Lama1(Delta/Delta) embryos display 1) parietal and visceral endoderm differentiation defects with altered expression of cytokeratin 19 and GATA4, respectively, and 2) an induction of apoptosis. This new mouse model is of particular interest as it will allow time- and tissue-specific inactivation of the Lama1 gene in various organs.     

Stress-induced apoptosis associated with null mutation of ADAR1 RNA editing deaminase gene

One type of RNA editing involves the conversion of adenosine residues into inosine in double-stranded RNA through the action of adenosine deaminases acting on RNA (ADAR). A-to-I RNA editing of the coding sequence could result in synthesis of proteins not directly encoded in the genome. ADAR edits also non-coding sequences of target RNAs, such as introns and 3'-untranslated regions, which may affect splicing, translation, and mRNA stability. Three mammalian ADAR gene family members (ADAR1-3) have been identified. Here we investigated phenotypes of mice homozygous for ADAR1 null mutation. Although live ADAR1-/- embryos with normal gross appearance could be recovered up to E11.5, widespread apoptosis was detected in many tissues. Fibroblasts derived from ADAR1-/- embryos were also prone to apoptosis induced by serum deprivation. Our results demonstrate an essential requirement for ADAR1 in embryogenesis and suggest that it functions to promote survival of numerous tissues by editing one or more double-stranded RNAs required for protection against stress-induced apoptosis.     

Oxidation promotes insertion of the CLIC1 chloride intracellular channel into the membrane

Members of the chloride intracellular channel (CLIC) family exist primarily as soluble proteins but can also auto-insert into cellular membranes to form ion channels. While little is known about the process of CLIC membrane insertion, a unique feature of mammalian CLIC1 is its ability to undergo a dramatic structural metamorphosis between a monomeric glutathione-S-transferase homolog and an all-helical dimer upon oxidation in solution. Whether this oxidation-induced metamorphosis facilitates CLIC1 membrane insertion is unclear. In this work, we have sought to characterise the role of oxidation in the process of CLIC1 membrane insertion. We examined how redox conditions modify the ability of CLIC1 to associate with and insert into the membrane using fluorescence quenching studies and a sucrose-loaded vesicle sedimentation assay to measure membrane binding. Our results suggest that oxidation of monomeric CLIC1, in the presence of membranes, promotes insertion into the bilayer more effectively than the oxidised CLIC1 dimer.     

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.