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Background: ARF nucleotide-binding site opener (ARNO) is a guanine nucleotide-exchange factor for ADP-ribosylation factor proteins. ARF nucleotide-binding site opener also binds MyD88, and small-molecule inhibition of ARNO reduces inflammation in animal models of inflammatory arthritis and acute inflammation. However, whether genetic deletion of Arno in mice reduces pathologic inflammation has not yet been reported. Furthermore, its role in the nasal cavity has yet to be investigated. Objective: To generate Arno knockout mice and to determine whether genetic loss of ARNO reduces eosinophilic inflammation in the ovalbumin (OVA) murine model of rhinitis. Methods: Arno knockout mice were generated and wild type and knockout littermates were subjected to the OVA-induced mouse model of rhinosinutitis. Eosinophilic inflammation was assessed through immunofluorescent quantification of EMBP+ eosinophils in the septal mucosa and cytokine expression was assessed by quantitative polymerase chain reaction. Results: Arno knockout mice are viable and fertile without any noted deficits. Arno wild type and knockout mice subjected to the OVA-induced model of rhinitis demonstrated an average of 314.5 and 153.8 EMBP+ cells per mm2 septal tissue, respectively ( P < .05). Goblet cells per mm of basal lamina were assessed via Alcian blue and there was no statistically significant difference between Arno wild type and knockout mice. Ovalbumin-induced expression of interleukin-5 (IL-5) was significantly reduced in Arno knockout mice ( P < .05). There was no statistically significant reduction in IL-4, IL-13, or eotaxin-1 expression. Conclusions: These data demonstrate that deletion of Arno reduces eosinophilic inflammation and IL-5 expression in an OVA-induced model of rhinitis.

Hoxa3 was the first Hox gene to be mutated by gene targeting in mice and is required for the development of multiple endoderm and neural crest cell (NCC)-derived structures in the pharyngeal region. Previous studies have shown that the Hoxa3 null mutant lacks third pharyngeal pouch derivatives, the thymus and parathyroids by E18.5, and organ-specific markers are absent or downregulated during initial organogenesis. Our current analysis of the Hoxa3 null mutant shows that organ-specific domains did undergo initial patterning, but the location and timing of key regional markers within the pouch, including Tbx1, Bmp4 and Fgf8, were altered. Expression of the parathyroid marker Gcm2 was initiated but was quickly downregulated and differentiation failed; by contrast, thymus markers were delayed but achieved normal levels, concurrent with complete loss through apoptosis. To determine the cell type-specific roles of Hoxa3 in third pharyngeal pouch development, we analyzed tissue-specific mutants using endoderm and/or NCC-specific Cre drivers. Simultaneous deletion with both drivers resulted in athymia at E18.5, similar to the null. By contrast, the individual tissue-specific Hoxa3 deletions resulted in small, ectopic thymi, although each had a unique phenotype. Hoxa3 was primarily required in NCCs for morphogenesis. In endoderm, Hoxa3 temporally regulated initiation of the thymus program and was required in a cell-autonomous manner for parathyroid differentiation. Furthermore, Hoxa3 was required for survival of third pharyngeal pouch-derived organs, but expression in either tissue was sufficient for this function. These data show that Hoxa3 has multiple complex and tissue-specific functions during patterning, differentiation and morphogenesis of the thymus and parathyroids.

There is emerging evidence that the canonical neural guidance factor netrin can also direct the growth of blood vessels. We deleted the gene encoding UNC5B, a receptor for the netrin family of guidance molecules, specifically within the embryonic endothelium of mice. The result is a profound structural and functional deficiency in the arterioles of the placental labyrinth, which leads first to flow reversal in the umbilical artery and ultimately to embryonic death. As this is the only detectable site of vascular abnormality in the mutant embryos, and because the phenotype cannot be rescued by a wild-type trophectoderm, we propose that UNC5B-mediated signaling is a specific and autonomous component of fetal-placental angiogenesis. Disruption of UNC5B represents a unique example of a mutation that acts solely within the fetal-placental vasculature and one that faithfully recapitulates the structural and physiological characteristics of clinical uteroplacental insufficiency. This pro-angiogenic, but spatially restricted requirement for UNC5B is not unique to murine development, as the knock-down of the Unc5b ortholog in zebrafish similarly results in the specific and highly penetrant absence of the parachordal vessel, the precursor to the lymphatic system.

In the developing hindbrain, the functional loss of individual Hox genes has revealed some of their roles in specifying rhombomere (r) identity. However, it is unclear how Hox genes act in concert to confer the unique identity to multiple rhombomeres. Moreover, it remains to be elucidated how these genes interact with other transcriptional programs to specify distinct neuronal lineages within each rhombomere. We demonstrate that in r5, the combined mutation of Hoxa3 and Hoxb3 result in a loss of Pax6- and Olig2-expressing progenitors that give rise to somatic motoneurons of the abducens nucleus. In r6, the absence of any combination of the Hox3 paralogous genes results in ectopic expression of the r4-specific determinant Hoxb1. This ectopic expression in turn results in the differentiation of r4-like facial branchiomotoneurons within this rhombomere. These studies reveal that members of the Hox1 and Hox3 paralogous groups participate in a `Hox code' that is necessary for coordinating both suppression and activation mechanisms that ensure distinction between the multiple rhombomeres in the developing hindbrain.

Approximately one-third of patients with porphyria cutanea tarda (PCT), the most common porphyria in humans, inherit a single mutant allele of the uroporphyrinogen decarboxylase ( URO-D ) gene. PCT associated with URO-D mutations is designated familial PCT. The phenotype is characterized by a photosensitive dermatosis with hepatic accumulation and urinary excretion of uroporphyrin and hepta-carboxylic porphyrins. Most heterozygotes for URO-D mutations do not express a porphyric phenotype unless hepatic siderosis is present. Hemochromatosis gene ( HFE ) mutations are frequently found when the phenotype is expressed. We used homologous recombination to disrupt one allele of murine URO-D . URO-D +/− mice had half-wild type (wt) URO-D protein and enzymatic activity in all tissues but did not accumulate hepatic porphyrins, indicating that half-normal URO-D activity is not rate limiting. When URO-D +/− mice were injected with iron-dextran and given drinking water containing δ-aminolevulinic acid for 21 days, hepatic porphyrins accumulated, and hepatic URO-D activity was reduced to 20% of wt. We bred mice homozygous for an HFE gene disruption ( HFE −/− ) to URO-D +/− mice, generating mice with the URO-D +/− /HFE −/− genotype. These animals developed a porphyric phenotype by 14 weeks of age without ALA supplementation, and URO-D activity was reduced to 14% of wt. These data indicate that iron overload alone is sufficient to reduce URO-D activity to rate-limiting levels in URO-D +/− mice. The URO-D +/− mouse serves as an excellent model of familial PCT and affords the opportunity to define the mechanism by which iron influences URO-D activity.

A procedure is described that directs the self-induced deletion of DNA sequences as they pass through the male germ line of mice. The testes-specific promoter from the angiotensin-converting enzyme gene was used to drive expression of the Cre-recombinase gene. Cre was linked to the selectable marker Neor, and the two genes flanked with loxP elements. This cassette was targeted to the Hoxa3 gene in mouse ES cells that were in turn used to generate chimeric mice. In these chimeras, somatic cells derived from the ES cells retained the cassette, but self-excision occurred in all ES-cell-derived sperm.

The gene causing cystic fibrosis (CF) encodes the CF transmembrane conductance regulator (CFTR), a cAMP-regulated chloride channel. Mutations in this gene result in reduced transepithelial chloride permeability across tissues affected in CF. Consequently, restoring chloride permeability to these tissues may prove therapeutic. Here we report that a combination of forskolin, an adenylate cyclase activator, and milrinone, an inhibitor of class III phosphodiesterases, increases the magnitude of the potential difference across nasal epithelium of mice homozygous for the most common CF mutation, ΔF508, while neither drug alone has a significant effect on potential difference. Transgenic mice lacking CFTR do not respond to the milrinone/forskolin combination, indicating that the effect in ΔF508 mice requires CFTR. These results suggest that, by pharmacological means, at least partial CFTR-mediated electrolyte transport can be restored in vivo to CF tissues expressing ΔF508.

β-Catenin has a dual function in cells: fortifying cadherin-based adhesion at the plasma membrane and activating transcription in the nucleus. We found that in melanoma cells, WNT5A stimulated the disruption of N-cadherin and β-catenin complexes by activating the guanosine triphosphatase adenosine diphosphate ribosylation factor 6 (ARF6). Binding of WNT5A to the Frizzled 4–LRP6 (low-density lipoprotein receptor–related protein 6) receptor complex activated ARF6, which liberated β-catenin from N-cadherin, thus increasing the pool of free β-catenin, enhancing β-catenin–mediated transcription, and stimulating invasion. In contrast to WNT5A, the guidance cue SLIT2 and its receptor ROBO1 inhibited ARF6 activation and, accordingly, stabilized the interaction of N-cadherin with β-catenin and reduced transcription and invasion. Thus, ARF6 integrated competing signals in melanoma cells, thereby enabling plasticity in the response to external cues. Moreover, small-molecule inhibition of ARF6 stabilized adherens junctions, blocked β-catenin signaling and invasiveness of melanoma cells in culture, and reduced spontaneous pulmonary metastasis in mice, suggesting that targeting ARF6 may provide a means of inhibiting WNT/β-catenin signaling in cancer.

The most common cause of cystic fibrosis is a mutation that deletes phenylalanine 508 in cystic fibrosis transmembrane conductance regulator (CFTR). The delta F508 protein is misprocessed and degraded rather than traveling to the apical membrane. We used a novel strategy to introduce the delta F508 mutation into the mouse CFTR gene. Affected epithelia from homozygous delta F508 mice lacked CFTR in the apical membrane and were Cl-impermeable. These abnormalities are the same as those observed in patients with delta F508 and suggest that these mice have the same cellular defect. 40% of homozygous delta F508 animals survived into adulthood and displayed several abnormalities found in human disease and in CFTR null mice. These animals should provide an excellent model to investigate pathogenesis and to examine therapies directed at correcting the delta F508 defect.

Mitoferrin1 is 1 of 2 homologous mitochondrial iron transporters and is required for mitochondrial iron delivery in developing erythroid cells. We show that total deletion of Mfrn1 in embryos leads to embryonic lethality. Selective deletion of Mfrn1 in adult hematopoietic tissues leads to severe anemia because of a deficit in erythroblast formation. Deletion of Mfrn1 in hepatocytes has no phenotype or biochemical effect under normal conditions. In the presence of increased porphyrin synthesis, however, deletion of Mfrn1 in hepatocytes results in a decreased ability to convert protoporphyrin IX into heme, leading to protoporphyria, cholestasis, and bridging cirrhosis. Our results show that the activity of mitoferrin1 is required to manage an increase in heme synthesis. The data also show that alterations in heme synthesis within hepatocytes can lead to protoporphyria and hepatotoxicity.

Gene targeting was used to introduce nonselectable genetic changes into chromosomal loci in mouse embryo-derived stem cells. The nonselectable markers were linked to a selectable marker in both insertion- and replacement-type vectors, and the transfer of the two elements to the Hprt locus was assayed. When insertion vectors were used as substrates, the frequency of transfer was highly dependent upon the distance between the nonselectable marker and the double-strand break in the vector. A marker located close to the vector ends was frequently lost, suggesting that a double-strand gap repair activity is involved in vector integration. When replacement vectors were used, cotransfer of a selectable marker and a nonselectable marker 3 kb apart was over 50%, suggesting that recombination between vector and target often occurs near the ends of the vector. To illustrate the use of replacement vectors to transfer specific mutations to the genome, we describe targeting of the delta F508 mutation to the CFTR gene in mouse embryo-derived stem cells.

Mutations were targeted to the Hprt locus in murine embryonic stem cells by using sequence replacement vectors. When the vector was designed such that the mutated sequences were flanked on both sides by several kilobases of DNA homologous to the target locus, replacement of chromosomal sequences with the exogenous DNA occurred with precision. If, on the other hand, the target-homologous DNA on one arm of the vector was reduced to below 1 kb in length, the fidelity of recombination was diminished.

Mice homozygous for the recessive mutation swaying (sw) are characterized by ataxia and hypertonia, attributed to the malformation of anterior regions of the cerebellum. We show that sw is a deletion of a single base pair from the proto-oncogene Wnt-1. The deletion is predicted to cause premature termination of translation, eliminating the carboxy-terminal half of the Wnt-1 protein. Histological examination shows that sw is phenotypically identical to a previously described wnt-1 mutation introduced into mice by gene targeting. Although both mutations in Wnt-1 disrupt primarily the development of the anterior cerebellum, they also exhibit a variability in expressivity such that rostrally adjacent structures in the midbrain and caudally adjacent structures in the posterior cerebellum can also be affected.

The int-1 proto-oncogene was first identified as a gene activated in virally induced mouse mammary tumours. Expression studies, however, suggest that the normal function of this gene may be in spermatogenesis and in the development of the central nervous system. Genes sharing sequence similarity with int-1 have been found throughout the animal kingdom. For example, int-1 has 54% amino-acid identity to the Drosophila segment polarity gene wingless (wg). Both the int-1 and wg gene products seem to be secreted proteins, presumably involved in cell-cell signalling. We have now explored the function of int-1 in the mouse by disrupting one of the two int-1 alleles in mouse embryo-derived stem cells using positive-negative selection. This cell line was used to generate a chimaeric mouse that transmitted the mutant allele to its progeny. Mice heterozygous for the int-1 null mutation are normal and fertile, whereas mice homozygous for the mutation may exhibit a range of phenotypes from death before birth to survival with severe ataxia. The latter pathology in mice and humans is often associated with defects in the cerebellum. Examination of int-1-/int-1- mice at several stages of embryogenesis revealed severe abnormalities in the development of the mesencephalon and metencephalon indicating a prominent role for the int-1 protein is in the induction of the mesencephalon and cerebellum.

The establishment of anteroposterior (AP) polarity in the early mouse epiblast is crucial for the initiation of gastrulation and the subsequent formation of the embryonic (head to tail) axis. The localization of anterior and posterior determining genes to the appropriate region of the embryo is a dynamic process that underlies this early polarity. Several studies indicate that morphological and molecular markers which define the early AP axis are first aligned along the short axis of the elliptical egg cylinder. Subsequently, just prior to the time of primitive streak formation, a conformational change in the embryo realigns these markers with the long axis. We demonstrate that embryos lacking the signaling factor Wnt3 exhibit defects in this axial realignment. In addition, chimeric analyses and conditional removal of Wnt3 activity reveal that Wnt3 expression in the epiblast is required for induction of the primitive streak and mesoderm whereas activity in the posterior visceral endoderm is dispensable.

Site-specific alteration of the mouse genome has become a routine procedure. The past six years have witnessed the generation and characterization of several hundred novel animals, each harboring an independent mutation in one of the 50 to 100,000 estimated murine genes (1). Genes previ­ ously predicted to be involved in normal development or in the maintenance of adult homeostasis have been targeted for mutation, as have murine homologues of genes implicated in human disease, including some affecting the respiratory system. The phenotypes resulting from these mutations have ranged from the predictable to the surprising-witness the unexpected respiratory defects in animals lacking a func­ tional endothelin-l gene (2), or the relatively benign pulmo­ nary pathology associated with a number of mutant alleles of the CFTR gene (reviewed in 3). The phenotypic charac­ terization of these animals has helped to define biological functions of individual genes and to identify networks of genetic interactions. Rather than attempt to summarize a vast accumulation of data thus far generated, this Perspective will address some of the limitations inherent in this technology, emphasizing how an understanding of these limitations is contributing to a more thorough genetic description of a mammalian organism and its development. The protocol for mutagenesis is by now straightforward: cloned DNA, first mutated in vitro, is introduced into a popu­ lation of pluripotent embryonic stem (ES) cells grown in cul­ ture; those cells in which recombination has replaced the tar­ get, endogenous gene with its mutant homologue are isolated and transplanted into a blastocyst stage mouse embryo; this embryo is implanted and allowed to develop to term, and the resulting chimera (containing cells from the blastocyst as well as ES cells) is then bred to propagate the mutant se­ quences (4, 5). If the mutant allele is a recessive lethal, it can be maintained in the heterozygous state, and a simple genetic cross is used to generate homozygosity. The majority of the currently available mutant animals carry loss-of-func­ tion, or null, alleles of the gene of interest. Not only are such mutations technically the simplest to construct, but the phenotype of the ensuing "knockout" mouse establishes a baseline requirement for the target gene. Initial expectations of this approach are that the time and place of gene action will be defined, that development may

A homopolymer system has been developed to examine the digestion strategies of DNA exonucleases. Escherichia coli exonuclease I and lambda-exonuclease, are processive enzymes. However, T7 exonuclease, spleen exonuclease, E. coli exonuclease III, the 3' leads to 5'-exonuclease of T4 DNA polymerase, and both the 3' leads to 5' and the 5' leads to 3' activity of E. coli DNA polymerase I dissociate frequently from the substrate during the course of digestion. Regions of duplex DNA are a dissociation signal for exonuclease I.

Heteroduplexes were prepared from two plasmids, pRH4-14/TK and pRH5-8/TK, containing different amber mutations in the neomycin resistance gene (Neor). The Neor gene was engineered to be expressed in both bacterial and mammalian cells. A functional Neor gene conferred kanamycin resistance to bacteria and resistance to the drug G418 to mammalian cells. In addition, the plasmids contained restriction site polymorphisms which did not confer a selectable phenotype but were used to follow the pattern of correction of mismatched bases in the heteroduplexes. In a direct comparison of the efficiency of transforming mouse LMtk- cells to G418r, the injection of heteroduplexes of pRH4-14/TK-pRH5-8/TK was 10-fold more efficient than the coinjection of pRH4-14/TK and pRH5-8/TK linear plasmid DNA. In fact, injection of 5 to 10 molecules of heteroduplex DNA per cell was as efficient in transforming LMtk- cells to G418r as the injection of 5 to 10 molecules of linear plasmid DNA per cell containing a wild-type Neor gene. To determine the pattern of mismatch repair of the injected heteroduplexes, plasmids were "rescued" from the G418r cell lines. From this analysis we conclude that the generation of wild-type Neor genes from heteroduplex DNA proceeds directly by correction of the mismatched bases, rather than by alternative mechanisms such as recombination between the injected heteroduplexes. Our finding that a cell can efficiently correct mismatched bases when confronted with preformed heteroduplexes suggests that this experimental protocol could be used to study a wide range of DNA repair mechanisms in cultured mammalian cells.

We have examined the mechanism of homologous recombination between plasmid molecules coinjected into cultured mammalian cells. Cell lines containing recombinant DNA molecules were obtained by selecting for the reconstruction of a functional Neor gene from two plasmids that bear different amber mutations in the Neor gene. In addition, these plasmids contain restriction-length polymorphisms within and near the Neor gene. These polymorphisms did not confer a selectable phenotype but were used to identify and categorize selected and nonselected recombinant DNA molecules. The striking conclusion from this analysis is that the predominant mechanism for the exchange of information between coinjected plasmid molecules over short distances (i.e., less than 1 kilobase) proceeds via nonreciprocal homologous recombination. The frequency of homologous recombination between coinjected plasmid molecules in cultured mammalian cells is extremely high, approaching unity. We demonstrate that this high frequency requires neither a high input of plasmid molecules per cell nor a localized high concentration of plasmid DNA within the nucleus. Thus, it appears that plasmid molecules, once introduced into the nucleus, have no difficulty seeking each other out and participating in homologous recombination even in the presence of a vast excess of host DNA sequences. Finally, we show that most of the homologous recombination events occur within a 1-h interval after the introduction of plasmid DNA into the cell nucleus.

We mutated, by gene targeting, the endogenous hypoxanthine phosphoribosyl transferase (HPRT) gene in mouse embryo-derived stem (ES) cells. A specialized construct of the neomycin resistance (neor) gene was introduced into an exon of a cloned fragment of the Hprt gene and used to transfect ES cells. Among the G418r colonies, 1/1000 were also resistant to the base analog 6-thioguanine (6-TG). The G418r, 6-TGr cells were all shown to be Hprt- as the result of homologous recombination with the exogenous, neor-containing, Hprt sequences. We have compared the gene-targeting efficiencies of two classes of neor-Hprt recombinant vectors: those that replace the endogenous sequence with the exogenous sequence and those that insert the exogenous sequence into the endogenous sequence. The targeting efficiencies of both classes of vectors are strongly dependent upon the extent of homology between exogenous and endogenous sequences. The protocol described herein should be useful for targeting mutations into any gene.

We corrected a defective gene residing in the chromosome of a mammalian cell by injecting into the nucleus copies of the same gene carrying a different mutation. We determined how the number, the arrangement, and the chromosomal position of the integrated gene, as well as the number of injected molecules influence the gene-targeting frequency. Recombination between the newly introduced DNA and its chromosomal homolog occurred at a frequency of 1 in 10(3) cells receiving DNA. Correction events were mediated by either double reciprocal recombination or gene conversion. This resulted in sequences in the genome being replaced by sequences of the introduced DNA or, in separate experiments, sequences in the incoming DNA being replaced by chromosomal sequences. Both point mutations and deletion mutations were corrected; however, the nature of the mutation carried by the respective sequence influenced whether the integrated or injected sequence was corrected.