PubMed Health⌕ Search

SEARCH · PubMed Health

Results for “Gene knockout”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 199 records · Page 11Linked to original sources
PubMed2006

Analysis of neural cell function in gene knockout mice: behavior.

A powerful tool to investigative gene function is the ability to create mice with targeted gene mutations. Analysis of the resulting phenotype is sometimes difficult, however, because individual genes have more than one function, and observed effects on complex behaviors are often a result of abnormalities of any of a number of individual processes. One way to address this issue is by examining mice in a battery of behavioral tests to assess the specificity of any observed differences among genotypes. This chapter describes a test battery used to examine metabolic and behavioral phenotypes in mice with mutations in specific glycan-binding proteins and glycosyltransferases genes. Because the potential consequences of these genetic deletions are varied, a large number of assays across a variety of domains was included in the battery. The power and usefulness of this approach is in discovering areas for more detailed investigation.

Animals↗
PubMed2005

[Gene knockout and knockin on the Escherichia coli lac operon loci using pBR322-red system].

pBR322-Red is a newly constructed recombineering plasmid, which contains a part of the pBR322 vector, a series of regulatory elements of lambda-prophage and Red recombination genes. In the beginning, we studied the best working conditions of pBR322-Red, and then modified lac operon in E. coli W3110 chromosome using the plasmid as follow: Firstly, we knockout the lacI gene using Red-mediated recombineering with overlapping single stranded DNA oligonucleotides. Secondly, we substituded the lacA and lacY genes with lacZ, a report gene, by Red-mediated linearized double strands DNA homologous recombination. Finally, we detected the expression of lacZ on these loci for the first time. The results suggested that pBR322-Red system is suitable for modifying W3110 chromosome with various recombination strategies.

Bacteriophage lambda↗
PubMed2002

Deletions are easy detectable in cochlear mitochondrial DNA of Cu/Zn superoxide dismutase gene knockout mice.

OBJECTIVES: To investigate the tissue specificity of reactive oxygen species (ROS) damage to mitochondrial DNA (mtDNA) and to determine whether cochlear mtDNA is a sensitive target for ROS damage. METHODS: 10 Cu/ZnSOD gene (Cu/Zn superoxide dismutase gene, Sod1) knockout mice and 16 wild-type mice were analyzed by nested polymerase chain reaction (PCR). RESULTS: Three deletions were detected in various tissues of Sod1 knockout mice. MtDNA3867bp and mtDNA3726bp deletions were the most visible, and mtDNA4236bp deletion was barely detected in these tissues. There were obvious differences in the ratio of deleted mtDNA/total mtDNA in different tissue. Deleted mtDNA was most abundant in the liver and kidney and less in cochlea, heart and brain. The lowest was in spleen and skin. The ratio in various tissues was 3 - 20 times in Sod1 knockout mice over wild-type mice. In cochlea, the ratio was about 15. CONCLUSIONS: Without the protection of Sod1, ROS can lead to mtDNA deletions in various tissues with significant tissue specificity. Cochlear mtDNA is a sensitive target for ROS damage.

Animals↗
PubMed2004

Metabolic regulation analysis of icd-gene knockout Escherichia coli based on 2D electrophoresis with MALDI-TOF mass spectrometry and enzyme activity measurements.

An integrated study of cell growth characteristics, enzyme activities and protein expression patterns was carried out to investigate how the central metabolism of Escherichia coli changes upon knockout of the isocitrate dehydrogenase (ICDH) gene (icd) in the tricarboxylic acid cycle. Deletion of the icd gene led to reduced specific growth rate and reduced specific glucose consumption rate. The reduced specific growth rate in the icd mutant was due mainly to the lower intracellular ATP/ADP ratio as well as to the lower NADPH/NADP(+) ratio compared with those in the parent strain. However, the specific carbon dioxide evolution rate was found to be higher in the icd mutant strain compared to the parent E. coli. This may be due to the higher activity of 6-phosphogluconate dehydrogenase, phosphoenol pyruvate carboxykinase and NADP(+)-dependent malic enzymes. The glyoxylate pathway was also utilized, as evidenced by the significant upregulation of isocitrate lyase and malate synthase activity in the icd mutant E. coli. The appearance of the glyoxylate pathway caused lower acetate production. Of 21 proteins showing altered expression levels, 17 were successfully identified with the aid of MALDI-TOF mass spectrometry. The results showed that the abolition of ICDH activity significantly affected the respiratory system and electron transport chain, as evidenced by the significant downregulation of proteins encoded by the genes nuoE, nuoH, cydA and cyoA in icd mutant E. coli compared to the parent.

Carbohydrate Metabolism↗
PubMed2001

Mouse gene knockout models for the CLN2 and CLN3 forms of ceroid lipofuscinosis.

The childhood neuronal ceroid-lipofuscinoses (NCLs) are autosomal-recessively inherited neurodegenerative disorders that result in severe cognitive decline and premature death. The genetic bases for a number of different forms of NCL, including those designated CLN2 and CLN3, have now been determined. However, the mechanisms by which the gene defects cause the disease pathology are not known and no effective treatments for these disorders have been developed. To provide tools for studying the mechanisms underlying the disease pathologies and for screening potential therapeutic interventions, work is under way to develop mouse models for the CLN2 and CLN3 disorders. Targeted gene replacement was used to generate mice in which the murine orthologue of the CLN3 gene has been knocked out. Mice that are homozygous for the Cln3 knockout allele develop a number of pathological features similar to those that occur in the human disorder. Among these are accumulation of autofluorescent lysosomal storage bodies, behavioural abnormalities, retinal degeneration, and premature death. On a mixed strain genetic background, the appearance of these symptoms was quite variable, suggesting that other genes can modify the effects of CLN3 mutations. Work to develop a similar mouse gene knockout model for the CLN2 disorder is well under way. Chimaeric mice have been developed with cells that carry an induced mutation in the mouse orthologue of the CLN2 gene that would prevent synthesis of a functional CLN2 protein in mice that are homozygous for the mutation. Mice will be developed that are homozygous for this mutation, and these animals will be evaluated for the development of pathologies similar to those that occur in the human disorder.

Aminopeptidases↗
PubMed2001

A targeted gene knockout in Drosophila.

We previously described a method for targeted homologous recombination at the yellow gene of Drosophila melanogaster. Because only a single gene was targeted, further work was required to show whether the method could be extended to become generally useful for gene modification in Drosophila. We have now used this method to produce a knockout of the autosomal pugilist gene by homologous recombination between the endogenous locus and a 2.5-kb DNA fragment. This was accomplished solely by tracking the altered genetic linkage of an arbitrary marker gene as the targeting DNA moved from chromosome X or 2 to chromosome 3. The results indicate that this method of homologous recombination is likely to be generally useful for Drosophila gene targeting.

Animals↗
PubMed1997

Stem cell factor and c-kit in the mammalian testis: lessons originating from Mother Nature's gene knockouts.

Analysis of mice with naturally occurring mutation in the genes encoding the tyrosine kinase receptor protein, c-kit, and its ligand, stem cell factor (SCF), has implicated these proteins in testicular development and function because of the phenotype of these mice of reduced or absent fertility. Descriptive and functional studies have highlighted the involvement of these proteins in primordial germ cell migration and survival, and in spermatogonial proliferation, survival and adhesion. This review summarises the current knowledge regarding functions of c-kit and SCF in the testis and discusses the implications of information gleaned from other biological systems.

Animals↗
PubMed2004

Effect of zwf gene knockout on the metabolism of Escherichia coli grown on glucose or acetate.

The mutant deficient in glucose-6-phosphate dehydrogenase (G6PDH) was constructed by disrupting zwf gene by one-step inactivation protocol using polymerase chain reaction primers. The knockout of zwf gene was shown to have different influence on the metabolism of Escherichia coli grown on glucose or acetate. The decreased rates of substrate uptake and CO(2) production were found for the mutant grown on acetate, whereas these two rates were increased during the growth on glucose. The metabolic flux analysis based on (13)C-labeling experiments indicates that the metabolism of the mutant grown on glucose is related to the higher flux via tricorboxylic acid (TCA) cycle to generate anabolic reducing equivalents normally provided by the oxidative pentose phosphate pathway. However, the metabolism of the mutant grown on acetate shows a lower flux towards the TCA cycle as compared with the parent strain. The decreased flux through TCA cycle is associated with an increased flux via the glyoxylate shunt, by which the carbon source can bypass the two decarboxylative steps of TCA cycle in which CO(2) is released, thus conserving more carbon for biosynthesis in response to the decreased uptake rate of the carbon source.

Acetates↗
PubMed2001

Insights into molecular mechanisms of contact hypersensitivity gained from gene knockout studies.

Contact hypersensitivity (CHS), a dendritic-cell (DC)-dependent, T-cell-mediated skin immune response to reactive haptens, has been a subject of intense research for many years. The molecular mechanisms underlying CHS are complicated and are not fully understood. During the past few years, varieties of gene-targeted knockout mice have been used in the study of CHS. Such studies have contributed significantly to our understanding of the mechanisms responsible for the initiation of CHS. This review focuses on insights into molecular requirements for CHS gained from knockout studies.

Animals↗
PubMed2002

Exploring the opioid system by gene knockout.

The endogenous opioid system consists of three opioid peptide precursor genes encoding enkephalins (preproenkephalin, Penk), dynorphins (preprodynorphin, Pdyn) and beta-endorphin (betaend), proopiomelanocortin (POMC) and three receptor genes encoding mu-opiod receptor (MOR), delta-opiod receptor (DOR) and kappa-opiod receptor (KOR). In the past years, all six genes have been inactivated in mice by homologous recombination. The analysis of spontaneous behavior in mutant mice has demonstrated significant and distinct roles of each gene in modulating locomotion, pain perception and emotional behaviors. The observation of opposing phenotypes of MOR- and DOR-deficient mice in several behaviors highlights unexpected roles for DOR to be further explored genetically and using more specific delta compounds. The analysis of responses of mutant mice to exogenous opiates has definitely clarified the essential role of MOR in both morphine analgesia and addiction, and demonstrated that DOR and KOR remain promising targets for pain treatment. These studies also show that prototypic DOR agonists partially require MOR for their biological activity and provide some support for the postulated mu-delta interactions in vivo. Finally, data confirm and define a role for several genes of the opioid system in responses to other drugs of abuse, and the triple opioid receptor knockout mutant allows exploring non-classical opioid pharmacology. In summary, the study of null mutant mice has extended our previous knowledge of the opioid system by identifying the molecular players in opioid pharmacology and physiology. Future studies should involve parallel behavioral analysis of mice lacking receptors and peptides and will benefit from more sophisticated gene targeting approaches, including site-directed and anatomically-restricted mutations.

Animals↗
PubMed1999

What do mouse gene knockouts tell us about the structure and function of the red cell membrane?

Recent development of knockout mice with targeted deletion of specific genes encoding various red cell membrane proteins has added valuable armamentarium to red cell membrane structure-function studies. In this chapter we will summarize the various recent developments regarding the structure and function of the red cell membrane derived from studies using knockout mice. In addition to being expressed in red cells, all major red cell membrane proteins are also expressed in cells of various tissues. The potential use of knockout mice to decipher the biological functions of red cell membrane proteins in non-erythroid cells is also explored.

Anemia, Hemolytic↗
PubMed1999

Neuronal and endothelial nitric oxide synthase gene knockout mice.

Targeted disruption of the neuronal nitric oxide synthase (nNOS) and endothelial nitric oxide synthase (eNOS) genes has led to knockout mice that lack these isoforms. These animal models have been useful to study the roles of nitric oxide (NO) in physiologic processes. nNOS knockout mice have enlarged stomachs and defects in the inhibitory junction potential involved in gastrointestinal motility. eNOS knockout mice are hypertensive and lack endothelium-derived relaxing factor activity. When these animals are subjected to models of focal ischemia, the nNOS mutant mice develop smaller infarcts, consistent with a role for nNOS in neurotoxicity following cerebral ischemia. In contrast, eNOS mutant mice develop larger infarcts, and show a more pronounced hemodynamic effect of vascular occlusion. The knockout mice also show that nNOS and eNOS isoforms differentially modulate the release of neurotransmitters in various regions of the brain. eNOS knockout mice respond to vessel injury with greater neointimal proliferation, confirming that reduced NO levels seen in endothelial dysfunction change the vessel response to injury. Furthermore, eNOS mutant mice still show a protective effect of female gender, indicating that the mechanism of this protection cannot be limited to upregulation of eNOS expression. The eNOS mutant mice also prove that eNOS modulates the cardiac contractile response to ss-adrenergic agonists and baseline diastolic relaxation. Atrial natriuretic peptide, upregulated in the hearts of eNOS mutant mice, normalizes cGMP levels and restores normal diastolic relaxation.

Animals↗
PubMed2004

Effect of monoamine oxidase gene knockout on dopamine metabolism in mouse brain structures.

Experiments were performed on knockout Tg8 mice lacking monoamine oxidase A gene that plays a major role in dopamine catabolism. The study by the method of high-performance liquid chromatography revealed considerable regional differences in the contents of dopamine and its metabolite dihydroxyphenylacetic acid in brain structures of these animals. Tg8 mice differed from the parent C3H/HeJ strain by low level of dihydroxyphenylacetic acid in the striatum, midbrain, hypothalamus, and hippocampus and high concentration of dopamine in the striatum. No differences were revealed in the contents of dopamine and dihydroxyphenylacetic acid in the frontal cortex and amygdala. The 2.4-4.8-fold decrease in the content of dihydroxyphenylacetic acid in various brain structures was not accompanied by changes in dopamine concentration. These data reflect the effective compensation for deficiency of dopamine metabolism. Our results suggest that monoamine oxidases A and B and catechol-O-methyltransferase play different roles in dopamine metabolism in various brain structures.

3,4-Dihydroxyphenylacetic Acid↗
PubMed2003

IL-2 gene knockout affects T lymphocyte trafficking and the microglial response to regenerating facial motor neurons.

Following facial nerve axotomy in mice, T cells cross the intact blood-brain barrier (BBB), home to nerve cell bodies in the facial motor nucleus (FMN), and augment neuroregenerative processes. The pivotal T cell immunoregulatory cytokine, IL-2, appears to have bidirectional effects on neuronal and microglial cell function, suggesting rival hypotheses that IL-2 could either enhance or disrupt processes associated with regeneration of axotomized facial motor neurons. We tested these competing hypotheses by comparing the effect of facial nerve axotomy on C57BL/6-IL-2(-/-) knockout and C57BL/6-IL-2(+/+) wild-type littermates. Since IL-2 may also be produced endogenously in the brain, we also sought to determine whether differences between the knockout and wild-type mice were attributable to loss of IL-2 gene expression in the CNS, loss of peripheral sources of IL-2 and the associated effects on T cell function, or a combination of these factors. To address this question, we bred novel congenic mice with the SCID mutation (mice lacking T cell derived IL-2) that were homozygous for either the IL-2 knockout or wild-type gene alleles (C57BL/6scid-IL-2(-/-) and C57BL/6scid-IL-2(+/+) littermates, respectively). Groups were assessed for differences in (1) T lymphocytes entering the axotomized FMN; (2) perineuronal CD11b(+) microglial phagocytic clusters, a measure of motor neuron death; and (3) activated microglial cells as measured by MHC-II positivity. C57BL/6-IL-2(-/-) knockout mice had significantly higher numbers of T cells and lower numbers of activated MHC-II-positive microglial cells in the regenerating FMN than wild-type littermates, although the number of CD11b(+) phagocytic microglia clusters did not differ. Thus, despite the significant impairment of T cell function known to be associated with loss of peripheral IL-2, the increased number of T cells entering the axotomized FMN appears to have sufficient activity to support neuroregenerative processes. Congenic C57BL/6scid-IL-2(-/-) knockout mice had lower numbers of CD11b(+) microglial phagocytic clusters than congenic C57BL/6scid-IL-2(+/+) wild-type littermates, suggesting that loss of the IL-2 gene in the CNS (and possibly the loss of other unknown sources of the gene) enhanced neuronal regeneration. Further study of IL-2's complex actions in neuronal injury may provide greater understanding of key variables that determine whether or not immunological processes in the brain are proregenerative.

Animals↗
PubMed2003

An improved and highly standardised transformation procedure allows efficient production of single and multiple targeted gene-knockouts in a moss, Physcomitrella patens.

The moss Physcomitrella patens is the only land plant known to date with highly efficient homologous recombination in its nuclear DNA, making it a unique model for plant functional genomics approaches. For high-throughput production of knockout plants, a robust transformation system based on polyethylene glycol-mediated transfection of protoplasts was developed and optimised. Both the DNA conformation and pre-culture of plants used for protoplast isolation significantly affected transformation efficiencies. Employing a newly developed PCR high-throughput method, the gene-targeting efficiency in more than 1000 plants transformed with different cDNA-based knockout constructs was determined and analysed with regard to the length and intron/exon structure of the homologous gene locus. Different targeting constructs, each containing an identical selectable marker gene, were applied as batch DNA in a single transformation experiment and resulted in double-knockout plants. Thus, the fast and efficient generation of multiple targeted gene-knockouts is now feasible in Physcomitrella.

Amino Acid Sequence↗
PubMed1999

Use of gene knockouts in cultured cells to study apoptosis.

The avian DT40 cell system represents a novel method to generate loss of function mutations in vertebrate cells. These chicken B lymphoma cells undergo homologous recombination at very high frequencies and can thus be used to "knock out" genes believed to function in apoptotic processes. The knockout cells can then be used to determine how the cell death process is modulated after induction of apoptosis and to order components in cell death pathways. The system can be further modified, using tetracycline-responsive promoters, to allow expression of wild-type cDNAs to rescue "knockout cells" if the gene of interest is essential. Alternatively, cDNA expression constructs containing mutations or deletions in the cDNA encoding the absent protein can be used to delineate functional domains. cDNA expression libraries or known proteins believed to function downstream of the target in a signal transduction pathway could also be transfected into the knockout cell line, and the resultant cells could be assayed for complementation and/or rescue of the apoptotic alteration/defect. Finally, the system has recently been adapted to allow disruption of human genes in DT40/human hybrid cell lines thereby potentially extending this system for use in studying human genes.

Animals↗
PubMed2006

Effects of methylmalonyl-CoA mutase gene knockouts on erythromycin production in carbohydrate-based and oil-based fermentations of Saccharopolyspora erythraea.

In carbohydrate-based fermentations of Saccharopolyspora erythraea, a polar knockout of the methylmalonyl-CoA mutase (MCM) gene, mutB, improved erythromycin production an average of 126% (within the range of 102-153% for a 0.95 confidence interval). In oil-based fermentations, where erythromycin production by the wild-type strain averages 184% higher (141-236%, 0.95 CI) than in carbohydrate-based fermentations, the same polar knockout in mutB surprisingly reduced erythromycin production by 66% (53-76%, 0.95 CI). A metabolic model is proposed where in carbohydrate-based fermentations MCM acts as a drain on the methylmalonyl-CoA metabolite pool, and in oil-based fermentations, MCM acts in the reverse direction to fill the methylmalonyl-CoA pool. Therefore, the model explains, in part, how the well-known oil-based process improvement for erythromycin production operates at the biochemical level; furthermore, it illustrates how the mutB erythromycin strain improvement mutation operates at the genetic level in carbohydrate-based fermentations.

Bacterial Proteins↗