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Biomedical subjects

G M Hansen

Publications and source records attributed to G M Hansen.

At least 19 recordsLinked to original sources
PubMed2007

Gene trap mutagenesis.

Our ability to genetically manipulate the mouse has had a great impact on medical research over the last few decades. Mouse genetics has developed into a powerful tool for dissecting the genetic causes of human disease and identifying potential targets for pharmaceutical intervention. With the recent sequencing of the human and mouse genomes, a large number of novel genes have been identified whose function in normal and disease physiology remains largely unknown. Government-sponsored multinational efforts are underway to analyze the function of all mouse genes through mutagenesis and phenotyping, making the mouse the interpreter of the human genome. A number of technologies are available for the generation of mutant mice, including gene targeting, gene trapping and transposon, chemical or radiation-induced mutagenesis. In this chapter, we review the current status of gene trapping technology, including its applicability to conditional mutagenesis.

Alleles↗
PubMed2004

Comparison of minimal inhibitory and mutant prevention drug concentrations of 4 fluoroquinolones against clinical isolates of methicillin-susceptible and -resistant Staphylococcus aureus.

Staphylococcus aureus remains an important human pathogen affecting both outpatients and those hospitalized. Increasing antimicrobial resistance is global but prevalence rates are variable for different geographical areas. Fluoroquinolones have been used to treat S. aureus infections and the newer quinolones have enhanced in vitro activity against this organism. The mutant prevention concentration (MPC) defines the antimicrobial drug concentration threshold that would require an organism to simultaneously possess two mutations for growth in the presence of the drug. We tested clinical isolates of methicillin-susceptible (MSSA) and methicillin-resistant (MRSA) S. aureus by minimum inhibitory concentration (MIC) and MPC against gatifloxacin, gemifloxacin, levofloxacin and moxifloxacin. For MSSA strains, the rank order of potency based on MIC(90) values were gemifloxacin (0.063 mg/l) = moxifloxacin (0.063 mg/l) > gatifloxacin (0.05 mg/l) = levofloxacin (0.25 mg/l) and by MPC values moxifloxacin (0.25 mg/l) > gemifloxacin (0.5 mg/l) > gatifloxacin (1 mg/l) = levofloxacin (1mg/l). For 87% of the isolates the MPC value was 0.5 mg/l for gatifloxacin. The rank order of potency based on the time the serum drug concentration exceeded the MPC(90), was as follows: moxifloxacin (>24 h) > levofloxacin (>18 h) > gatifloxacin (12 h) > gemifloxacin (9 h). Serum drug concentration remained in excess of the MPC(87) for 24 h for gatifloxacin. Both MIC(90) and MPC(90) values were higher against MRSA strains and the time above the MPC(90) was significantly shorter for all agents.

Anti-Infective Agents↗
PubMed2000

Genetic profile of insertion mutations in mouse leukemias and lymphomas.

Murine leukemia retroviruses (MuLVs) cause leukemia and lymphoma in susceptible strains of mice as a result of insertional mutation of cellular proto-oncogenes or tumor suppressor genes. Using a novel approach to amplify and sequence viral insertion sites, we have sequenced >200 viral insertion sites from which we identify >35 genes altered by viral insertion in four AKXD mouse strains. The class of genes most frequently altered are transcription factors, however, insertions are found near genes involved in signal transduction, cell cycle control, DNA repair, cell division, hematopoietic differentiation, and near many ESTs and novel loci. Many of these mutations identify genes that have not been implicated in cancer. By isolating nearly all the somatic viral insertion mutations contributing to disease in these strains we show that each AKXD strain displays a unique mutation profile, suggesting strain-specific susceptibility to mutations in particular genetic pathways.

Animals↗
PubMed1999

Activation of Hex and mEg5 by retroviral insertion may contribute to mouse B-cell leukemia.

AKXD recombinant inbred mice develop a variety of leukemias and lymphomas due to retrovirally mediated insertional activation of cellular proto-oncogenes. We describe a new retroviral insertion site that is the most frequent genetic alteration in AKXD B-cell leukemias. Multiple genes flank the site of viral insertion, but the expression of just two, Hex and mEg5, is significantly upregulated. Hex is a divergent homeobox gene that is transiently expressed in many hematopoietic lineages, suggesting an involvement in cellular differentiation. mEg5 is a member of the bim-C subfamily of kinesin related proteins that are necessary for spindle formation and stabilization during mitosis. Our data provide the first genetic evidence for the activation of these genes in leukemia, and suggest that unscheduled expression of Hex and mEg5 contributes to the development of B-cell leukemia. In addition, this work highlights the use of genomic approaches for the study of position effect mutations.

Animals↗
PubMed1999

A mouse chromosome 19 genetic map including the Lvis1 viral insertion site.

Somatic insertion mutations, such as those caused by the insertion of a proviral element, can contribute to abnormal cell growth by activating cellular proto-oncogenes or inactivating tumor suppressor genes. Lvis1 is a genomic locus frequently disrupted by viral insertion in AKXD B-cell lymphomas (manuscript submitted for publication). To determine whether insertion mutations at Lvis1 affect a known proto-oncogene or tumor suppressor gene, we mapped Lvis1 to distal mouse chromosome 19. A detailed molecular genetic map of this region was constructed, localizing Lvis1 relative to 20 gene and microsatellite markers, 3 of which have not been mapped in the mouse (Nfkb2, Nlz, and Wnt8b). This analysis revealed that Lvis1 maps between two previously identified viral insertion sites, His2 and Frat1, and does not cosegregate with known gene markers. In addition, our study refines the gene order for distal mouse chromosome 19 and expands the comparative map between mouse chromosome 19 and the human chromosome 10q23-q26 homology region.

Animals↗
PubMed1999

Characterization of a novel alpha-mannosidosis-causing mutation and its use in leukocyte genotyping after bone marrow transplantation.

Alpha-Mannosidosis is a lysosomal storage disorder caused by deficiency of lysosomal alpha-mannosidase (LAMAN). Major symptoms include mental retardation, skeletal changes and recurrent infections. Recently, a successful bone marrow transplantation (BMT) in an alpha-mannosidosis patient was reported. Here we show that this patient was homozygous for a novel mutation, a 1-bp insertion (1197-1198insA) in exon 9 of the LAMAN gene. By using this mutation as a marker, we demonstrate that 1 year post-BMT, the LAMAN genotype of the patient's leukocytes was identical to that of the donor. This method of genotyping blood cells is a fast and accurate way to monitor the colonization of donor bone marrow cells.

Bone Marrow Transplantation↗
PubMed1999

Spectrum of mutations in alpha-mannosidosis.

alpha-Mannosidosis is an autosomal recessive disorder caused by deficiency of lysosomal alpha-mannosidase (LAMAN). The resulting intracellular accumulation of mannose-containing oligosaccharides leads to mental retardation, hearing impairment, skeletal changes, and immunodeficiency. Recently, we reported the first alpha-mannosidosis-causing mutation affecting two Palestinian siblings. In the present study 21 novel mutations and four polymorphic amino acid positions were identified by the screening of 43 patients, from 39 families, mainly of European origin. Disease-causing mutations were identified in 72% of the alleles and included eight splicing, six missense, and three nonsense mutations, as well as two small insertions and two small deletions. In addition, Southern blot analysis indicated rearrangements in some alleles. Most mutations were private or occurred in two or three families, except for a missense mutation resulting in an R750W substitution. This mutation was found in 13 patients, from different European countries, and accounted for 21% of the disease alleles. Although there were clinical variations among the patients, no significant LAMAN activity could be detected in any of the fibroblast cultures. In addition, no correlation between the types of mutations and the clinical manifestations was evident.

DNA Mutational Analysis↗
PubMed1999

Humoral immunity and protection of mice challenged with homotypic or heterotypic parvovirus.

BACKGROUND AND OBJECTIVES: Two serotypes of autonomously replicating parvoviruses infect laboratory mice. Genome regions coding for the nonstructural proteins of minute virus of mice [MVM] and mouse parvovirus [MPV] are almost identical, whereas capsid-coding sequences are divergent. We addressed these questions: Does humoral immunity confer protection from acute infection after challenge with homotypic or heterotypic parvovirus, and if it confers protection against acute MPV infection, does it also protect against persistent MPV infection? METHODS: Infant mice without maternal antibody or antibody to MVM or MPV and young adult mice given normal mouse serum or antibody to MVM or MPV were challenged with homotypic or heterotypic virus. In situ hybridization with target tissues was the indicator of infection. RESULTS: Humoral immunity failed to confer protection against acute heterotypic parvovirus infection. In passive transfer studies, MPV DNA was observed occasionally in lymph nodes, intestine, or the spleen of MPV-challenged mice given homotypic antibody and kept for 6 or 28 days. Variable proportions of mice given MPV antibody and homotypic challenge had viral DNA in lymphoid tissues 56 days after virus inoculation. CONCLUSION: A mouse or colony that has sustained infection with MVM or MPV is probably fully susceptible to infection with the heterotypic virus.

Animals↗
PubMed1997

alpha-Mannosidosis: functional cloning of the lysosomal alpha-mannosidase cDNA and identification of a mutation in two affected siblings.

a-Mannosidosis (MIM 248500) is an autosomal recessive lysosomal storage disorder resulting from deficient activity of lysosomal alpha-mannosidase (LAMAN) (EC 3.2.1.24). The disease is characterized by massive intracellular accumulation of mannose-rich oligosaccharides with resulting mental retardation, hearing loss, immune deficiency and skeletal changes. We report here the purification and characterization of human placenta LAMAN. The enzyme is synthesized as a single-chain precursor which is processed into three glycopeptides of 70, 42 and 15 kDa. The 70 kDa peptide is further partially proteolysed into three more peptides that are joined by disulfide bridges. The laman cDNA sequence was assembled from overlapping fragments obtained by PCR on human fibroblast and human lung cDNA. The deduced amino acid sequence contains a putative signal peptide of 48 amino acids followed by a polypeptide sequence of 962 amino acids. Northern blot analyses revealed a single transcript of approximately 3.5 kb present in all tissues examined but at varying levels. Two affected siblings of Palestinian origin were homozygous for a mutation that causes a His-->Leu replacement at a position which is conserved among class 2 alpha-mannosidases from several species.

Amino Acid Sequence↗
PubMed1996

Location of the 9257 and ataxia mutations on mouse chromosome 18.

The location of three mutations on proximal Chromosome (Chr) 18 was determined by analysis of the offspring of several backcrosses. The results demonstrate that ataxia and the insertional mutation TgN9257Mm are separated by less than 1 cM and are located approximately 3 cM from the centromere, while the balding locus is 7 cM more distal. Previous data demonstrated that the twirler locus also maps within 1 cM of ataxia. The corrected locations will contribute to identification of appropriate candidate genes for these mutations. Two polymorphic microsatellite markers for proximal Chr 18 are described, D18Umi1 and D18Umi2. The Lama3 locus encoding the alpha 3 subunit of nicein was mapped distal to ataxia and did not recombine with Tg9257.

Animals↗
PubMed1995

Enzyme-linked immunosorbent assay for detection of antibody to lymphocytic choriomeningitis virus in mouse sera, with recombinant nucleoprotein as antigen.

An enzyme-linked immunosorbent assay (ELISA) was developed for the detection of antibody to lymphocytic choriomeningitis virus (LCMV) in mouse sera. This assay is based on recombinant LCMV nucleoprotein generated in a baculovirus system. Sera from experimentally and naturally infected as well as noninfected mice were tested, and the results were compared with those obtained from an established immunofluorescence assay (IFA) that uses infected cells as antigen. An excellent correlation was found; the ELISA specificity and sensitivity were calculated to be 100 and 95% respectively. Unlike the IFA, this ELISA does not require the handling of infective virus. It eliminates the need to work with a zoonotic agent in the laboratory while allowing effective screening of laboratory mouse populations for LCMV antibody.

Animals↗
PubMed1994

Experimental Borrelia burgdorferi infection in Peromyscus leucopus.

We evaluated the susceptibility of laboratory-reared adult and infant white-footed mice (Peromyscus leucopus) to a known pathogenic isolate of Borrelia burgdorferi (N40). Two-month-old and 3-day-old Peromyscus were inoculated intradermally with 10(6) to 10(7) spirochetes. At 21 days for adults or 30 days for infants post inoculation, mice were killed, and tissues were cultured for spirochetes and examined microscopically. Based on serology and culture, adult mice became infected but did not have any gross or microscopic lesions. Mice inoculated as infants became infected, and also developed carditis and multifocal arthritis. Contact transmission between inoculated infants and their naive mothers was not observed. Age at inoculation appeared to be a critical factor in inducing Lyme borreliosis lesions in Peromyscus leucopus, as in other species.

Animals↗
PubMed1993

Variant responses of mice to Borrelia burgdorferi depending on the site of intradermal inoculation.

C3H/He mice inoculated intradermally at one of two sites with Borrelia burgdorferi responded differently to infection. Shoulder-inoculated mice developed spirochetemia, B. burgdorferi-specific antibody, and arthritis earlier than foot-inoculated mice. Lymphocyte populations derived from spleen tissue were elevated in the shoulder- but not the foot-inoculated mice, and those from lymph nodes were increased in both groups. Lymphocytes derived from blood and spleen tissue showed impaired proliferative responses to all mitogens for shoulder-inoculated mice only, whereas proliferation of lymph node cells was not affected, regardless of route. These results demonstrate that the site of initial B. burgdorferi inoculation is an important determinant in the pathogenesis of B. burgdorferi infection.

Animals↗
PubMed1993

A serologic survey for viruses and Mycoplasma pulmonis among wild house mice (Mus domesticus) in southeastern Australia.

Plasma samples from 267 wild house mice (Mus domesticus) trapped at 14 sites in southeastern Australia were screened for antibody to 14 viruses normally associated with laboratory-reared rodents and to Mycoplasma pulmonis. Serologic prevalence was high for murine cytomegalovirus (99%, n = 94), murine coronavirus (95%), and murine rotavirus (74%). Samples from mice collected at all sites contained antibody to these viruses. The serologic prevalence was lower for mouse adenovirus, strain K87 (37%), parvovirus (33%), and reovirus type 3 (28%), with substantial site-to-site variation. Plasma from mice collected at 12 sites contained mouse adenovirus or reovirus antibody, and samples from mice at eight sites contained parvovirus antibody. Parvovirus-antibody positive mice were typically from high density populations or from low density populations that had recently declined from high density. Antibody to lymphocytic choriomeningitis virus (LCMV) and Sendai virus occurred at only three sites, and the serologic prevalence was very low (9.6% and 1.8%, respectively). All of the LCMV-positive mice were from northeastern New South Wales. The presence of this zoonotic virus in a mouse plague-prone region raises questions about human health risks resulting from cohabitation with large numbers of mice. It appeared that mouse populations at high density or declining from high density had higher prevalence of viral antibody than populations that had been at low or moderate density for some time. Thus, viral epizootics may occur among high-density populations and may be responsible for or precipitate declines in mouse density. These data raise the possibility of rodent viruses having potential as biological control agents.

Animals↗
PubMed1990

Lyme borreliosis in selected strains and ages of laboratory mice.

The susceptibility of laboratory mice to Borrelia burgdorferi was evaluated for selected genotypes and ages. C3H/He, SWR, C57BL/6, SJL, and BALB/c mice inoculated at age 3 days developed uniformly severe polyarthritis at 30 days after intraperitoneal inoculation. Mice inoculated at age 3 weeks also developed polyarthritis, but severity was influenced by genotype, with C3H/He and SWR mice the most severely affected. Susceptible strains developed higher IgG ELISA antibody titers to B. burgdorferi than did resistant mice. Adult (12 weeks) C3H/He mice were also susceptible, but arthritis was not as severe as in those inoculated at age 3 weeks. SKH (hairless) mice developed polyarthritis but not skin disease when inoculated intradermally. Carditis occurred frequently among C3H/He, BALB/c, and hairless mice and in some SWR mice but not in C57BL/6 or SJL mice. This study demonstrates that severity of Lyme borreliosis is age- and genotype-dependent and that laboratory mice are a potentially valuable model.

Animals↗
PubMed1990

Experimental chronic Lyme borreliosis in Lewis rats.

The course of Lyme borreliosis in LEW/N rats inoculated intraperitoneally as infants with 10(6) Borrelia burgdorferi was followed for 360 days. Spirochetes were detected in the blood through 30 days, in the brain through 60 days, and persisted in the spleen, liver, kidneys and articular tissue through 360 days. Acute exudative arthritis, tendonitis, and bursitis were evident in multiple joints by day 30. Arthritis regressed thereafter but capsular fibrosis and lymphoplasmacytic infiltrates persisted throughout the study. Several rats developed exacerbations of acute arthritis within days 180-360, a pattern similar to that encountered in human Lyme disease. Rats had a high prevalence of nonsuppurative myocarditis and vasculitis during days 90-360. Spirochetes were visualized by microscopy in joints and other tissues during the first month of infection, but were seen only sporadically thereafter. All rats seroconverted to B. burgdorferi by day 30. IgM titers persisted and IgG titers rose progressively through day 360. Immunoblots revealed IgM reactivity to a single 41 kDa protein until 360 days, when reactivity to a 60 kDa protein emerged. IgG reactivity occurred against progressively more proteins with time, indicating continued antigenic stimulation. Chronic and recurrent arthritic lesions and myocardial involvement suggest that the rat is a reliable model for further investigation.

Animals↗