Inducible nitric oxide synthase gene targeting

Wei, Xiao-qing (1995) Inducible nitric oxide synthase gene targeting. PhD thesis, University of Glasgow.

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Nitric oxide (NO) is a critical mediator of a variety of biological functions, including vascular and muscle relaxation, platelet aggregation, neuronal-cell function, microbicidal and tumoricidal activity, and a range of immunopathologies. NO is derived from L-arginine and molecular oxygen in a reaction catalysed by NO synthase (NOS). Three isoforms of NOS have been identified; neuronal constitutive NOS (ncNOS), endothelial constitutive NOS (ecNOS), and inducible NOS (iNOS). ncNOS and ecNOS are calcium-dependent, present constitutively in a variety of tissues and produce physiological concentrations of NO. However, large amounts NO are produced by iNOS which are expressed in cells such as macrophage after stimulation with a number of cytokines, including interferon-gamma (IFN-γ), tumour necrosis factor-alpha (TNF-α), and bacterial lipopolysaccharide (LPS). Findings of iNOS biological functions are based mainly on experiments using L-arginine analogues such as L-NG monomethyl arginine (L-NMMA) which competitively inhibits NO Synthase. These inhibitors are not NOS isoform selective and have differential bioavailability, and hence often render interpretation of results difficult. To directly define the iNOS biological functions, I constructed a strain of iNOS gene targeted mice. The first step of a gene targeting experiment is typically cDNA cloning and sequencing. A cDNA library was constructed in the vector λ ZAPII using mRNA isolated from J774 macrophages activated with IFN-? and LPS. Six independent positive colonies were found in the screen with both 5' and 3' specific probes and were subcloned in pBluescript. A full-length iNOS cDNA was constructed using the clones isolated from the library. Double strand cDNA sequence analysis showed that the J774 iNOS clone was identical to that of the Raw 264.7 macrophages iNOS cDNA sequence. A replacement-type targeting construct was prepared from 129/sv genomic DNA. A single targeted clone was identified amongst 636 screened after two independent electroporations of the CGR8 embryonic stem cell line. Gene replacement was detected by both 5' and 3' specific external probes. Five of ten chimeras generated gave germ line transmission. No homozygous mice were found by Southern blot analysis with 5' and 3' external probe in the offspring from heterozygous mice (FI) breedings. The iNOS gene had been altered by replacement with gene targeting construct and 5' iNOS gene translocation which could be detected by internal probe Southern blot analysis. Using the internal probe, mutant homozygous, heterozygous and wild-type mice were found in the offspring from heterozygous breedings. The ratio of these three genotypes was 21:47:25. Northern blot analysis with 5' iNOS cDNA probe showed that a large messenger appeared in the macrophages of homozygous mice when the cells were activated with IFN-γ and LPS in vitro. However there was no detectable iNOS protein translation by western blot analysis using monoclonal or polyclonal anti-iNOS antibodies. (Abstract shortened by ProQuest.)

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Supported by funding from the World Health Organization.
Subjects: R Medicine > RC Internal medicine
R Medicine > RM Therapeutics. Pharmacology
Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Life Sciences
Supervisor's Name: Supervisor, Not known
Date of Award: 1995
Depositing User: Miss Elaine P. Anderson
Unique ID: glathesis:1995-8351
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 24 Aug 2017 10:29
Last Modified: 02 Sep 2021 14:11
Thesis DOI: 10.5525/gla.thesis.8351
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