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At least 289 records · Page 16Linked to original sources
PubMed2006

In vivo transduction by intravenous injection of a lentiviral vector expressing human ADA into neonatal ADA gene knockout mice: a novel form of enzyme replacement therapy for ADA deficiency.

Using a mouse model of adenosine deaminase-deficient severe combined immune deficiency syndrome (ADA-deficient SCID), we have developed a noninvasive method of gene transfer for the sustained systemic expression of human ADA as enzyme replacement therapy. The method of delivery is a human immunodeficiency virus 1-based lentiviral vector given systemically by intravenous injection on day 1 to 2 of life. In this article we characterize the biodistribution of the integrated vector, the expression levels of ADA enzyme activity in various tissues, as well as the efficacy of systemic ADA expression to correct the ADA-deficient phenotype in this mouse model. The long-term expression of enzymatically active ADA achieved by this method, primarily from transduction of liver and lung, restored immunologic function and significantly extended survival. These studies illustrate the potential for sustained in vivo production of enzymatically active ADA, as an alternative to therapy by frequent injection of exogenous ADA protein.

Adenosine Deaminase↗
PubMed2004

Targeted gene knockout reveals a role in meiotic recombination for ZHP-3, a Zip3-related protein in Caenorhabditis elegans.

The meiotically expressed Zip3 protein is found conserved from Saccharomyces cerevisiae to humans. In baker's yeast, Zip3p has been implicated in synaptonemal complex (SC) formation, while little is known about the protein's function in multicellular organisms. We report here the successful targeted gene disruption of zhp-3 (K02B12.8), the ZIP3 homolog in the nematode Caenorhabditis elegans. Homozygous zhp-3 knockout worms show normal homologue pairing and SC formation. Also, the timing of appearance and the nuclear localization of the recombination protein Rad-51 seem normal in these animals, suggesting proper initiation of meiotic recombination by DNA double-strand breaks. However, the occurrence of univalents during diplotene indicates that C. elegans ZHP-3 protein is essential for reciprocal recombination between homologous chromosomes and thus chiasma formation. In the absence of ZHP-3, reciprocal recombination is abolished and double-strand breaks seem to be repaired via alternative pathways, leading to achiasmatic chromosomes and the occurrence of univalents during meiosis I. Green fluorescent protein-tagged C. elegans ZHP-3 forms lines between synapsed chromosomes and requires the SC for its proper localization.

Amino Acid Sequence↗
PubMed1996

Cell-type and promoter-context dependent retinoic acid receptor (RAR) redundancies for RAR beta 2 and Hoxa-1 activation in F9 and P19 cells can be artefactually generated by gene knockouts.

By using RAR type (alpha, beta, or gamma)-specific synthetic retinoids and a pan-retinoic X receptor (RXR)-specific ligand, we have investigated the contribution of RARs and RXRs in the activation of RA target genes and the differentiation of embryonal carcinoma cells. We demonstrate cell-type- and promoter context-dependent functional redundancies that differ between the three RAR types for mediating the induction of RARbeta2 and Hoxa-1 in wild-type, RARgamma-/- and RARalpha-/- F9 cells and in P19 cells. The extent of redundancy between RARs is further modulated by the synergistic activation of RXRs with a pan-RXR agonist. We also demonstrate that the expression of RARbeta2 is auto-inducible in RARgamma-/- but not in wild-type F9 cells, indicating that the functional redundancies observed between RARs in gene disruption studies can be artefactually generated. Thus, even though all three RARs can functionally substitute each other for inducing the expression of RA target genes and cell differentiation, one RAR can cell-specifically override the activity of the other RARs. Interestingly, only RARgamma can mediate the retinoic acid-induced differentiation of wild-type F9 cells, whereas the differentiation of P19 cells can be mediated by either RARalpha or RARgamma.

Cell Differentiation↗
PubMed2006

Identification of an endo-beta-1,4-D-xylanase from Magnaporthe grisea by gene knockout analysis, purification, and heterologous expression.

Magnaporthe grisea, a destructive ascomycetous pathogen of rice, secretes cell wall-degrading enzymes into a culture medium containing purified rice cell walls as the sole carbon source. From M. grisea grown under the culture conditions described here, we have identified an expressed sequenced tag, XYL-6, a gene that is also expressed in M. grisea-infected rice leaves 24 h postinoculation with conidia. This gene encodes a protein about 65% similar to endo-beta-1,4-D-glycanases within glycoside hydrolase family GH10. A M. grisea knockout mutant for XYL-6 was created, and it was shown to be as virulent as the parent strain in infecting the rice host. The proteins secreted by the parent strain and by the xyl-6Delta mutant were each fractionated by liquid chromatography, and the collected fractions were assayed for endo-beta-1,4-D-glucanase or endo-beta-1,4-D-xylanase activities. Two protein-containing peaks with endo-beta-1,4-D-xylanase activity secreted by the parent strain are not detectable in the column eluant of the proteins secreted by the mutant. The two endoxylanases (XYL-6alpha and XYL-6beta) from the parent were each purified to homogeneity. N-terminal amino acid sequencing indicated that XYL-6alpha is a fragment of XYL-6beta and that XYL-6beta is identical to the deduced protein sequence encoded by the XYL-6 gene. Finally, XYL-6 was introduced into Pichia pastoris for heterologous expression, which resulted in the purification of a fusion protein, XYL-6H, from the Pichia pastoris culture filtrate. XYL-6H is active in cleaving arabinoxylan. These experiments unequivocally established that the XYL-6 gene encodes a secreted endo-beta-1,4-D-xylanase.

Amino Acid Sequence↗
PubMed1995

Gene knockout mice establish a primary protective role for major histocompatibility complex class II-restricted responses in Chlamydia trachomatis genital tract infection.

Mice with disrupted beta 2-microglobulin (beta 2m-/-), I-A (class II-/-), or CD4 (CD4-/-) genes were examined for their capacity to resolve Chlamydia trachomatis genital tract infection. C57BL/6 and beta 2m-/- mice resolved infection similarly and were culture negative by 4 to 5 weeks following infection. Conversely, major histocompatibility complex (MHC) class II-/- mice failed to resolve infection, and CD4-/- mice showed a significant delay (2 weeks). Secondary challenge of C57BL/6, beta 2m-/-, and CD4-/- mice established that acquired protective immunity, which was characterized by an infection of shortened duration and reduced shedding of infectious organisms, developed. Serological analysis of C57BL/6 and beta 2m-/- mice by enzyme-linked immunosorbent assays revealed no striking differences in the immunoglobulin subclass specificity of the anti-Chlamydia response, although some differences were observed in the magnitude of the immunoglobulin G2a (IgG2a) and IgG2b responses. Class II-/- mice produced lower-titered serum anti-Chlamydia antibodies of all isotypes. The serum antibody responses of CD4-/- mice were similar to those of C57BL/6 mice, except that the anti-Chlamydia IgA response was delayed by approximately 3 weeks. Analysis of vaginal washes for Chlamydia-reactive antibodies revealed the presence of IgG2a, IgG2b, and IgA in C57BL/6 and beta 2m-/- mice and primarily of IgA in CD4-/- mice. Vaginal washes from class II-/- mice were consistently antibody negative. Interestingly, the Chlamydia-specific IgA response in the vaginal washes of CD4-/- mice was delayed, but its appearance coincided with decreased shedding of infectious organisms and resolution of infection. Our results demonstrate that MHC class II-restricted T-cell responses are necessary for the development of protective immunity to Chlamydia genital tract infection and that local (vaginal) anti-Chlamydia IgA antibody coincides with the resolution of infection. A substantive role for MHC class I-restricted T-cell responses in protective immunity to Chlamydia genital tract infection was not confirmed.

Animals↗
PubMed2003

Combined effects of prostaglandin E receptor subtype EP1 and subtype EP4 antagonists on intestinal tumorigenesis in adenomatous polyposis coli gene knockout mice.

Previous studies have shown that prostaglandin E(2) (PGE(2)) is involved in intestinal carcinogenesis through its binding to the PGE(2) receptor subtypes EP(1) and EP(4) and activation of downstream pathways. ONO-8711 and ONO-AE2-227, prostaglandin E receptor subtype EP(1)- and EP(4)-selective antagonists, respectively, are known to suppress formation of intestinal polyps in adenomatous polyposis coli gene-deficient mice. The present study was designed to investigate the combined effects of EP(1) and EP(4) antagonists on spontaneous polyp formation in APC1309 mice in order to determine the contribution of each receptor to intestinal tumorigenesis. APC1309 mice were treated with 400 ppm of ONO-8711 alone, 400 ppm of ONO-AE2-227 alone or both in combination in the diet for 6 weeks. The mean area of polyps found in the intestine, calculated as the longer diameter x the shorter diameter x pi, was reduced by 12%, 43% (P < 0.01) and 56% (P < 0.01) of the mean control value (8.8 mm(2)) in the ONO-8711 alone, ONO-AE2-227 alone and combination treatment groups, respectively, suggesting clear additive effects of the combination. The same additive tendency for suppression was also observed with respect to the numbers of polyps in the intestine. Polyp size reduction was more remarkable with the EP(4) antagonist, while the number reduction was more pronounced with the EP(1) antagonist. Our results indicate that EP(1) and EP(4) may have separate intrinsic roles and, to some extent, contribute to polyp formation independently. Thus, combination treatment has potential for the chemoprevention of colon carcinogenesis.

Animals↗
PubMedDate not supplied

Probing protein function using a combination of gene knockout and proteome analysis by mass spectrometry.

Recently the determination of the genome sequences of three procaryotes (Haemophilus influenzae, Methanococcus jannaschii and Mycoplasma genitalium) as well as the first eucaryotic genome (Saccharomyces cerevisiae) were completed. Between 40-60% of the genes were found to code for proteins to which no function could be assigned. We describe an approach which combines proteome analysis (mapping of expressed proteins isolated by two-dimensional polyacrylamide gel electrophoresis to the genome) with genetic manipulations to study the complex pattern of protein regulation occurring in Escherichia coli in response to sulfate starvation. We have previously described the upregulation of eight spots on two-dimensional (2-D) gels in response to sulfate starvation and the assignment of six of these to entries in the E. coli genome sequence (Quadroni et al., Eur. J. Biochem. 1996, 239, 773-781). Here we describe the identification of the remaining two proteins which are encoded in a sulfate-controlled operon in the 21.5' region of the E. coli genome. Upregulated protein spots were cut from multiple 2-D gels collected and run on a modified funnel gel to concentrate the proteins and remove the sodium dodecyl sulfate before digestion. The peptide masses obtained from the digests were used to search the SwissProt database or a six-frame translation of the EMBL DNA database using a peptide mass fingerprinting algorithm. A digest can be reanalyzed after deuterium exchange to obtain a second, orthogonal data set to increase the confidence level of protein identification. The digests of the remaining unidentified proteins were used for peptide fragment generation using either post-source decay in a matrix-assisted laser desorption ionization (MALDI) time-of-flight mass spectrometer or collision-induced dissociation (CID) coupled mass spectrometry (MS/MS) with triple stage quadrupole or ion trap mass spectrometers. The spectra were used as peptide fragment fingerprints to search the SwissProt and EMBL databases.

Amino Acid Sequence↗
PubMed1999

A mouse gene knockout model for juvenile ceroid-lipofuscinosis (Batten disease).

The human hereditary ceroid-lipofuscinoses are a group of autosomal recessively inherited diseases characterized by massive accumulations of autofluorescent lysosomal storage bodies in the cells of many tissues and by neuronal degeneration throughout the central nervous system. There are a number of clinically and genetically distinct forms of ceroid-lipofuscinosis, the most common of which is the juvenile type, also known as Batten disease and CLN3. To study the mechanisms that lead to pathology in CLN3 and to evaluate potential therapies, a mouse model has been generated by targeted disruption of the mouse ortholog of the CLN3 gene (Cln3). As in affected humans, mice homozygous for the disrupted Cln3 allele show accumulation of autofluorescent storage material in neurons and other cell types. The storage material consists of membrane-bounded intracellular inclusions with ultrastructural features typical of the ceroid-lipofuscinoses. The accumulation of this storage material validates the Cln3 knockout mice as a model for the human disorder.

Animals↗
PubMed1995

apo B gene knockout in mice results in embryonic lethality in homozygotes and neural tube defects, male infertility, and reduced HDL cholesterol ester and apo A-I transport rates in heterozygotes.

apo B is a structural constituent of several classes of lipoprotein particles, including chylomicrons, VLDL, and LDL. To better understand the role of apo B in the body, we have used gene targeting in embryonic stem cells to create a null apo B allele in the mouse. Homozygous apo B deficiency led to embryonic lethality, with resorption of all embryos by gestational day 9. Heterozygotes showed an increased tendency to intrauterine death with some fetuses having incomplete neural tube closure and some live-born heterozygotes developing hydrocephalus. The majority of male heterozygotes were sterile, although the genitourinary system and sperm were grossly normal. Viable heterozygotes had normal triglycerides, but total, LDL, and HDL cholesterol levels were decreased by 37, 37, and 39%, respectively. Hepatic and intestinal apo B mRNA levels were decreased in heterozygotes, presumably contributing to the decreased LDL levels through decreased synthesis of apo B-containing lipoproteins. Kinetic studies indicated that heterozygotes had decreased transport rates of HDL cholesterol ester and apo A-I. As liver and intestinal apo A-I mRNA levels were unchanged, the mechanism for decreased apo A-I transport must be posttranscriptional. Heterozygotes also had normal cholesterol absorption and a normal response of the plasma lipoprotein pattern to chronic consumption of a high fat, high cholesterol, Western-type diet. In summary, we report a mouse model for apo B deficiency with several phenotypic features that were unexpected based on clinical studies of apo B-deficient humans, such as embryonic lethality in homozygotes and neural tube closure defects, male infertility, and a major defect in HDL production in heterozygotes. This model presents an opportunity to study the mechanisms underlying these phenotypic changes.

Alleles↗
PubMed2003

Family 1 G protein-coupled receptor function in the CNS. Insights from gene knockout mice.

Family 1 G protein-coupled receptors (GPCRs) are activated by a large number of ligands including photons, odorants, neurotransmitters and hormones and are involved in a wide variety of central and peripheral functions. Due to their wide distribution in the central nervous system (CNS), family 1 GPCRs play a major role in the regulation of neuronal activity and behaviour. In general, the lack of selective ligands for each member of the GPCR subfamilies has made it difficult to assign specific central functions to each receptor subtype. Advances in gene targeting techniques have allowed the inactivation of receptor genes in the mouse through homologous recombination leading to the generation of mouse 'knockout' models lacking one or more GPCRs. In this review, we have listed the family 1 GPCR knockout models produced in the past decade and we have summarized the findings obtained from studies on these mice with respect to CNS function.

Animals↗
PubMed2006

Generation of gene knockouts and mutant models in the laboratory rat by ENU-driven target-selected mutagenesis.

OBJECTIVE: The rat is one of the most important model organisms for biomedical and pharmacological research. However, the generation of novel models for studying specific aspects of human diseases largely depends on selection for specific traits using existing rat strains, thereby solely depending on naturally occurring variation. This study aims to provide the tools to manipulate the rat genome in a more directed way. METHODS: We developed robust, automated, and scaleable reverse genetic methodology based on ENU (N-ethyl-N-nitrosourea)-driven target-selected mutagenesis. Optimal mutagenesis conditions have been determined in three different rat strains and a universal, rapid, and cost-effective dideoxy resequencing-based screening setup was established for mutation discovery. The effectiveness of the approach is illustrated by the identification of 120 induced mutations in a set of genes of interest, including six that result in unique rat knockout models due to the introduction of premature stop codons. In addition, 56 mutations were found that change amino acids, including critical residues in transmembrane domains of receptors and channels. CONCLUSIONS: The approach described here allows for the systematic generation of knockout and protein function altering alleles in the rat. The resulting induced rat models will be powerful tools for studying many aspects of a wide variety of human diseases.

Alkylating Agents↗
PubMed2005

Alteration of vascular urotensin II receptor in mice with apolipoprotein E gene knockout.

Urotensin II (U-II), originally identified as a fish neuropeptide, exerts a broad spectrum of biological functions and is considered to be the most potent vasoconstrictor in mammals. Recently the intense staining of U-II-like immunoreactivity within coronary atheroma and the effect of mitogenic in vascular smooth muscle cells (VSMCs) suggest that U-II is possibly a proatherogenic factor in the pathogenesis of cardiovascular diseases (CVD). In the present study, we analyzed the gene expression of U-II and GPR14, a high-affinity receptor for U-II, in aorta of apolipoprotein E (apoE) -/- mutant mice by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). Compared with wild-type mice at the same age group, GPR14 mRNA level is significant increase in aorta of apoE -/- mice at age of 18, 28 and 38 weeks, respectively. The increased GPR14 mRNA level was 54.2, 50.0 and 97.0% in the three age groups, respectively. We did not detect U-II mRNA expression in aorta either in apoE -/- mice or in wild-type mice. In 28-week mice, ligand-binding assay showed that maximum binding capacity (Bmax) of 125I-U-II aorta from apoE -/- mice was increased by 64%, while the affinity of the binding did not change compared with that of wild-type mice. These results indicate that the up-regulation of vascular U-II receptor (UT), GPR14 might be involved in the pathogenesis of atherosclerosis.

Age Factors↗
PubMed1997

Gene knockout of the alpha6 subunit of the gamma-aminobutyric acid type A receptor: lack of effect on responses to ethanol, pentobarbital, and general anesthetics.

The alpha6 subunit of the gamma-aminobutyric acid type A receptor (GABA(A)-R) has been implicated in mediating the intoxicating effects of ethanol and the motor ataxic effects of general anesthetics. To test this hypothesis, we used gene targeting in embryonic stem cells to create mice lacking a functional alpha6 gene. Homozygous mice are viable and fertile and have grossly normal cerebellar cytoarchitecture. Northern blot and reverse transcriptase-polymerase chain reaction analyses demonstrated that the targeting event disrupted production of functional alpha6 mRNA. Autoradiography of histological sections of adult brains demonstrated that diazepam-insensitive binding of [3H]Ro15-4513 to the cerebellar granule cell layer of wild-type mice was completely absent in homozygous mice. Cerebellar GABA(A)-R density was unchanged in the mutant mice; however, the apparent affinity for muscimol was markedly reduced. Sleep time response to injection of ethanol after pretreatment with vehicle or Ro15-4513 did not differ between genotypes. Sleep time response to injection of pentobarbital and loss of righting reflex and response to tail clamp stimulus in mice anesthetized with volatile anesthetics also did not differ between genotypes. Thus, the alpha6 subunit of the GABA(A)-R is not required for normal development, viability, and fertility and does not seem to be a critical or unique component of the neuronal pathway mediating the hypnotic effect of ethanol and its antagonism by Ro15-4513 in mice. Similarly, the alpha6 subunit does not seem to be involved in the behavioral responses to general anesthetics or pentobarbital.

Anesthetics, Inhalation↗
PubMed1999

Gene knockouts reveal separate functions for two cytoplasmic dyneins in Tetrahymena thermophila.

In many organisms, there are multiple isoforms of cytoplasmic dynein heavy chains, and division of labor among the isoforms would provide a mechanism to regulate dynein function. The targeted disruption of somatic genes in Tetrahymena thermophila presents the opportunity to determine the contributions of individual dynein isoforms in a single cell that expresses multiple dynein heavy chain genes. Substantial portions of two Tetrahymena cytoplasmic dynein heavy chain genes were cloned, and their motor domains were sequenced. Tetrahymena DYH1 encodes the ubiquitous cytoplasmic dynein Dyh1, and DYH2 encodes a second cytoplasmic dynein isoform, Dyh2. The disruption of DYH1, but not DYH2, resulted in cells with two detectable defects: 1) phagocytic activity was inhibited, and 2) the cells failed to distribute their chromosomes correctly during micronuclear mitosis. In contrast, the disruption of DYH2 resulted in a loss of regulation of cell size and cell shape and in the apparent inability of the cells to repair their cortical cytoskeletons. We conclude that the two dyneins perform separate tasks in Tetrahymena.

Amino Acid Sequence↗
PubMed2003

Optknock: a bilevel programming framework for identifying gene knockout strategies for microbial strain optimization.

The advent of genome-scale models of metabolism has laid the foundation for the development of computational procedures for suggesting genetic manipulations that lead to overproduction. In this work, the computational OptKnock framework is introduced for suggesting gene deletion strategies leading to the overproduction of chemicals or biochemicals in E. coli. This is accomplished by ensuring that a drain towards growth resources (i.e., carbon, redox potential, and energy) must be accompanied, due to stoichiometry, by the production of a desired product. Computational results for gene deletions for succinate, lactate, and 1,3-propanediol (PDO) production are in good agreement with mutant strains published in the literature. While some of the suggested deletion strategies are straightforward and involve eliminating competing reaction pathways, many others suggest complex and nonintuitive mechanisms of compensating for the removed functionalities. Finally, the OptKnock procedure, by coupling biomass formation with chemical production, hints at a growth selection/adaptation system for indirectly evolving overproducing mutants.

Algorithms↗
PubMed2003

The role of nitric oxide synthase isoforms in extrahepatic portal hypertension: studies in gene-knockout mice.

BACKGROUND & AIMS: Considerable debate exists concerning which isoform of nitric oxide synthase (NOS) is responsible for the increased production of NO in PHT. We used the portal vein ligation model of PHT in wild-type and eNOS- or iNOS-knockout mice to definitively determine the contribution of these isoforms in the development of PHT. METHODS: The portal vein of wild-type mice, or those with targeted mutations in the nos2 gene (iNOS) or the nos3 gene (eNOS), was ligated and portal venous pressure (Ppv), abdominal aortic blood flow (Qao), and portosystemic shunt determined 2 weeks later. RESULTS: In wild-type mice, as compared with sham-operated controls, portal vein ligation (PVL) resulted in a time-dependent increase in Ppv (7.72 +/- 0.37 vs 17.57 +/- 0.51 cmH(2)O, at 14 days) concomitant with a significant increase in Qao (0.12 +/- 0.003 vs 0.227 +/- 0.005 mL/min/g) and portosystemic shunt (0.47% +/- 0.01% vs 84.13% +/- 0.09% shunt). Likewise, PVL in iNOS-deficient mice resulted in similar increases in Ppv, Qao, and shunt development. In contrast, after PVL in eNOS-deficient animals, there was no significant change in Ppv (7.52 +/- 0.22 vs 8.07 +/- 0.4 cmH(2)0) or Qao (0.111 +/- 0.01 vs 0.14 +/-.023 mL/min/g). However, eNOS (-/-) mice did develop a substantial portosystemic shunt (0.33% +/- 0.005% vs 84.53% +/- 0.19% shunt), comparable to that seen in wild-type animals after PVL. CONCLUSIONS: These data support a key role for eNOS, rather than iNOS, in the pathogenesis of PHT.

Animals↗
PubMed1999

Effect of saline infusion on kidney and collecting duct function in atrial natriuretic peptide (ANP) gene "knockout" mice.

Atrial natriuretic peptide (ANP) is thought to play a role in renal regulation of salt balance by reducing tubular reabsorption of sodium and chloride. Therefore, in the chronic absence of this hormone, a defect of salt excretion should be evident. We used an ANP gene deletion model to test this premise. F2 homozygous mutant mice (-/-) and their wild-type littermates (+/+) were fed an 8% NaCl diet prior to an acute infusion of isotonic saline. Arterial blood pressures, renal excretions of salt and water, as well as collecting duct transport of fluid and electrolytes were measured. Pressures were significantly higher in -/- compared with +/+ mice (139 +/- 4 vs. 101 +/- 2 mmHg; 1 mmHg = 133.3 Pa). There was no difference in glomerular filtration rate (-/- = 0.84 +/- 0.06; +/+ = 0.81 +/- 0.04 mL x min(-1) x g(-1) kidney weight). In the collecting duct, sodium and chloride reabsorptions were significantly higher in the -/- group than in the +/+ group. As a result, natriuresis and chloruresis were relatively reduced (U(Na)V: -/- = 8.6 +/- 1.1; +/+ = 14.0 +/- 1.1; U(Cl)V: -/- = 10.1 +/- 1.4; +/+ = 16.0 +/- 1.1 micromol x min(-1) x g(-1) kidney weight). We conclude that the absence of endogenous ANP activity in mice on a high-salt diet subjected to acute saline infusion causes inappropriately high reabsorption of sodium and chloride in the medullary collecting duct, resulting in a relative defect in renal excretory capacity for salt.

Absorption↗