Gene trap, gene knockout, gene knock-in, and transgenics in vascular development.
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Animals↗
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More to learn from gene knockouts.
Gene targeting by homologous recombination in mouse embryonic stem cells is a powerful technique to determine the physiological function of any gene product in embryonic and postnatal development and in molecular pathogenesis. Although the technique is very demanding and still in its developing stage several knockout mice carrying disrupted genes, which were once thought important for the development or molecular pathogenesis of certain tissues, have given unexpected results. A gene/function redundancy or superfluous and on-functional theory has been advanced by many investigators to explain the unexpected results. These surprising results may teach us a new lesson and lead to a revision of the strongly held view that highly conserved and abundantly expressed genes have a prominent role and function in cell physiology and development. Additional, they may also support the notion that molecular cross-talk among the genes may play an important role in determining the minimal phenotype.
Animals↗
[Generation of Ayu17-449 gene knockout mice by gene trapping].
We report the generation of Ayu17-449 gene knockout mice in an effort to facilitate studies of its function during mouse development, A special gene trapping vector was transfected into mouse ES cells. The clone with the trapped gene trap was identified by 5' RACE and Southern blot analysis. Ayu17-449 trapped mice were then generated by using this ES clone. In addition, the expression of Ayu17-449 in this mutant line was analyzed with Northern blot. Results showed that in these mutant mice, the trapping vector was located upstream of the Ayu17-449 initiation site, and as a result, the transcription of Ayu17-449 gene was inhibited. Ayu17-449 trapped mice are a useful tool for the analysis of Ayu17-449 gene function in mouse development.
Animals↗
Hypertension associated with decreased testosterone levels in natriuretic peptide receptor-A gene-knockout and gene-duplicated mutant mouse models.
Mice lacking the gene (Npr1) encoding the natriuretic peptide receptor A (NPRA) have hypertension with elevated blood pressure and cardiac hypertrophy. In particular, Npr1 gene-deficient male mice exhibit lethal vascular events similar to those seen in untreated human hypertensive patients. Serum testosterone levels tend to be lower in hypertensive male humans than in normal males without hypertension, but the genetic basis for this tendency remains unknown. To determine whether Npr1 gene function affects the testosterone level, we measured serum testosterone in male hypertensive mice lacking a functional Npr1 gene, wild-type animals with two copies, and the gene-duplicated littermates expressing four copies of the gene. In the Npr1 gene-knockout (zero-copy) mice, the serum testosterone level was 62% lower than that in the two-copy control mice (80+/-10 ts. 120+/-14 ng/ml, respectively; P < 0.005). Serum testosterone in the four-copy mice was 144% (P < 0.005) of that in the two-copy wild-type control mice. To investigate the role of NPRA in testicular steroidogenesis, we analyzed atrial natriuretic peptide (ANP)-dependent guanylyl cyclase activation, accumulation of intracellular cGMP, and testosterone production in purified primary Leydig cells from animals with zero, two, or four copies of the Npr1 gene. Leydig cells lacking the Npr1 gene did not show ANP-stimulated guanylyl cyclase activation or cGMP accumulation and had no ANP-dependent testosterone production. ANP stimulation of Leydig cells from the four-copy males elicited a 2-fold greater production of cGMP compared to that in the two-copy wild-type counterparts (260+/-12 vs. 126+/-7 pmol/l x 10(6) cells; P < 0.001). Similarly, ANP-dependent testosterone production in Leydig cells was nearly twice as high in four-copy mice as in two-copy wild-type controls (561+/-18 vs. 325+/-11 ng/l x 10(6) cells; P < 0.001). ANP-dependent guanylyl cyclase activation and production of cGMP in Leydig cells increased progressively with the number of Npr1 gene copies. Our results establish the existence of an alternate mechanism for testicular steroidogenesis that is stimulated by NPRA-dependent cGMP signaling, in addition to that mediated by gonadotropins, via a cAMP pathway. These findings demonstrate the role of Npr1 gene function in the maintenance of serum testosterone levels and testicular steroidogenesis and provide a genetic link between hypertension associated with decreased NPRA and low testosterone levels.
Animals↗
SelectRepair Knockout: Efficient PTC-Free Gene Knockout Through Selectable Homology-Directed DNA Repair.
Generating nonessential gene knockouts using CRISPR/Cas9 technology is becoming increasingly common in biological research. In a typical workflow, the Cas9 endonuclease is used to induce a DNA double-strand break that relies on nonhomologous end-joining (NHEJ) to introduce a premature termination codon (PTC) in the target gene. The goal is to isolate clones in which the gene produces PTC-containing mRNA transcripts that are degraded via nonsense-mediated mRNA decay (NMD) to cause loss of gene function. Unfortunately, this approach is laborious, and not all PTCs trigger NMD. More importantly, mounting evidence suggest that PTC mutations can also result in a transcriptional adaptation response that can mask the effects of a PTC-mediated gene knockout. In this chapter, we present a PTC-free gene knockout strategy that implements homology-directed DNA repair (HDR) with selectable markers to substantially reduce the complexity of the screening and validation of genome edits in cells containing more than one gene copy as in the case of the commonly used hypotriploid HEK293 cell line. We describe how to obtain a complete knockout of the Ligase IV protein (LIG4) and provide considerations for the application of this SelectRepair Knockout method to other genes.
Humans↗
Gene knockout reveals a novel gene cluster for the synthesis of a class of cell wall lipids unique to pathogenic mycobacteria.
Surface-exposed unusual lipids containing phthiocerol and phenolphthiocerol are found only in the cell wall of slow-growing pathogenic mycobacteria and are thought to play important roles in host-pathogen interaction. The enzymology and molecular genetics of biosynthesis of phthiocerol and phenolphthiocerol are unknown. We postulate the domain organization of a set of multifunctional enzymes and a cluster of genes (pps) that would encode these enzymes for the biosynthesis of phthiocerol and phenolphthiocerol. A cosmid containing the postulated pps gene cluster was identified by screening a genomic library of Mycobacterium bovis BCG with the postulated homologous domains from mycocerosic acid synthase and fatty acid synthase genes as probes. Homologous cosmids were also identified in the genomic libraries of Mycobacterium tuberculosis and Mycobacterium leprae. M. bovis BCG was transformed with a pps disruption construct, made from the BCG cosmid by introducing the hygromycin resistance gene as the positive-selectable marker and the sacB gene as the counter-selectable marker. Gene disruption by homologous recombination with double crossover was confirmed by polymerase chain reaction and Southern hybridization. Chromatographic analysis showed that the phenolphthiocerol derivative, mycoside B, and phthiocerol dimycocerosates were not produced by the gene knockout mutants. This result confirms the identity of the pps genes. With the identification of the pps gene clusters in both M. tuberculosis and M. leprae, it should be possible to test the postulated roles of these unique lipids in tuberculosis and leprosy.
Acyl Coenzyme A↗
Arabidopsis gene knockout: phenotypes wanted.
Gene knockout is considered to be a major component of the functional genomics toolbox, and is aimed at revealing the function of genes discovered through large-scale sequencing programs. In the past few years, several Arabidopsis populations mutagenized with insertion elements, such as the T-DNA of Agrobacterium or transposons, have been produced. These large populations are routinely screened for insertions into specific genes, allowing mass-isolation of knockout lines. Although many Arabidopsis knockouts have already been obtained, few of them have been reported to present informative phenotypes that provide a direct clue to gene function. Although functional redundancy explains the lack of phenotypical alterations in some cases, it also appears that many mutations are conditional and/or do not alter plant morphology even in the presence of severe physiological defects. Consequently, gene knockout per se is not sufficient to assess gene function and must be integrated into a more global approach for determining biological functions.
Arabidopsis↗
New functions of histamine found in histidine decarboxylase gene knockout mice.
Gene targeting techniques have revolutionized the investigation of the effects of bioactive substances in pathological and physiological conditions. Histamine synthesis is uniquely catalyzed by L-histidine decarboxylase. The knockout mice of this gene express no histamine-producing activity and lack histamine. These mice have been used to examine the mechanisms of histamine in several known phenotypes, e.g., gastric acid secretion, contraction of smooth muscles, vascular permeability, and awakening, and have also been used to explore unreported effects of histamine in the whole body. First, we will review the former mechanisms and then move to the latter, new effects. Especially, in the latter mechanisms, we focus on several important roles of histamine in angiogenesis, neutrophil and eosinophil recruitment, bacterial infection, and systemic anaphylaxis in this review. Moreover, to our surprise, the morphology of mast cells in the knockout mice was severely affected by the absence of histamine in terms of their granules.
Anaphylaxis↗
Muscle growth after postdevelopmental myostatin gene knockout.
Constitutive myostatin gene knockout in mice causes excessive muscle growth during development. To examine the effect of knocking out the myostatin gene after muscle has matured, we generated mice in which myostatin exon 3 was flanked by loxP sequences (Mstn[f/f]) and crossed them with mice bearing a tamoxifen-inducible, ubiquitously expressed Cre recombinase transgene. At 4 mo of age, Mstn[f/f]/Cre+ mice that had not received tamoxifen had a 50-90% reduction in myostatin expression due to basal Cre activity but were not hypermuscular relative to Mstn[w/w]/Cre+ mice (homozygous for wild-type myostatin gene). Three months after tamoxifen treatment (initiated at 4 mo of age), muscle mass had not changed from the pretreatment level in Mstn[w/w]/Cre+ control mice. Tamoxifen administration to 4-mo-old Mstn[f/f]/Cre+ mice reduced myostatin mRNA expression to less than 1% of normal, which increased muscle mass approximately 25% over the following 3 mo in both male and female mice (P<0.005 vs. control). Fiber hypertrophy appeared to be sufficient to explain the increase in muscle mass. The pattern of expression of genes encoding the various myosin heavy-chain isoforms was unaffected by postdevelopmental myostatin knockout. We conclude that, even after developmental muscle growth has ceased, knockout of the myostatin gene induces a significant increase in muscle mass.
Animals↗
Acute renal injury in the interferon-gamma gene knockout mouse: effect on cytokine gene expression.
We studied major histocompatibility complex (MHC) and cytokine mRNA induction after renal injury in the absence of interferon-gamma (IFN-gamma) using IFN-gamma gene knockout (GKO) mice. The left renal pedicle of normal (wild-type) and GKO BALB/c mice was clamped for 60 minutes; cytokine and MHC mRNA expression were monitored in the injured kidney and compared to the contralateral control kidney. After a single episode of ischemic injury, the expression of mRNA for MHC class I and II, interleukin-2, interleukin-10, granulocyte-macrophage colony-stimulating factor, tumor necrosis factor-alpha, and transforming growth factor-beta 1 was increased in wild-type and GKO mice, whereas preproepidermal growth factor (ppEGF) was reduced. IFN-gamma expression was induced in wild-type mice but absent in the GKO mice. Therefore, local injury was equally effective in both wild-type and GKO mice with equivalent cytokine and MHC mRNA induction, proving that local tissue injury can induce MHC expression by non-IFN-gamma factors.
Animals↗
Cell surface oxygen consumption by mitochondrial gene knockout cells.
Mitochondrial gene knockout (rho(0)) cells that depend on glycolysis for their energy requirements show an increased ability to reduce cell-impermeable tetrazolium dyes by electron transport across the plasma membrane. In this report, we show for the first time, that oxygen functions as a terminal electron acceptor for trans-plasma membrane electron transport (tPMET) in HL60rho(0) cells, and that this cell surface oxygen consumption is associated with oxygen-dependent cell growth in the absence of mitochondrial electron transport function. Non-mitochondrial oxygen consumption by HL60rho(0) cells was extensively inhibited by extracellular NADH and NADPH, but not by NAD(+), localizing this process at the cell surface. Mitochondrial electron transport inhibitors and the uncoupler, FCCP, did not affect oxygen consumption by HL60rho(0) cells. Inhibitors of glucose uptake and glycolysis, the ubiquinone redox cycle inhibitors, capsaicin and resiniferatoxin, the flavin centre inhibitor, diphenyleneiodonium, and the NQO1 inhibitor, dicoumarol, all inhibited oxygen consumption by HL60rho(0) cells. Similarities in inhibition profiles between non-mitochondrial oxygen consumption and reduction of the cell-impermeable tetrazolium dye, WST-1, suggest that both systems may share a common tPMET pathway. This is supported by the finding that terminal electron acceptors from both pathways compete for electrons from intracellular NADH.
Aerobiosis↗
[Functional analysis of drug metabolizing enzymes using gene knockout animals].
Several gene knockout mice have been widely used to analyze the role of drug-metabolizing enzymes in pharmacologic and physiologic responses. The metabolic shift of endogenous and exogenous compounds causes pharmacologic and physiologic alterations. Microsomal epoxide hydrolase (mEH)-null mice are less susceptible to the skin tumorigenesis, splenic immunotoxicity, and embryonic toxicity of 7,12-dimethylbenz[a] anthracene (DMBA). The production of DMBA-3,4-diol is detected in the target organs of wild-type mice, but not in those of mEH-null mice. Soluble epoxide hydrolase (sEH)-null mice exhibit markedly reduced rates of epoxyeicosatrienoic acid conversion to dihydroxyei-cosatrienoic acid in the liver and kidney. Furthermore, sEH-null male mice have a lower blood pressure phenotype compared with male wild-type mice, suggesting the importance of sEH in blood pressure regulation. Nuclear bile acid receptor, farnesoid X receptor (FXR)-null mice are distinguished from wild-type mice by elevated bile acid levels in the liver and serum. However, hepatic lithocholic acid (LCA) levels are lower in LCA-fed FXR-null female mice compared to those in wild-type female mice. Furthermore, FXR-null female mice are less susceptible to liver damage by LCA compared with female wild-type mice. Marked increases in hepattic LCA-sulfating activity and hepatic hydroxysteroid sulfotransferase and biliary sulfated bile acid levels are detected in FXR-null female mice, suggesting the protective role of hydroxysteroid sulfotransferase in LCA-induced liver damage. These and other studies indicate that mice null for drug-metabolizing enzymes and nuclear receptors are of great value in the study of the role of drug-metabolizing enzymes in pharmacologic and physiologic responses.
9,10-Dimethyl-1,2-benzanthracene↗
Methylnitrosourea-induced tumorigenesis in MGMT gene knockout mice.
Gene targeting was used to obtain mice defective in the MGMT gene, encoding O6-methylguanine-DNA methyltransferase [Tsuzuki et al., Carcinogenesis (Lond.), 17: 1215-1220, 1996]. These MGMT-/- mice were most sensitive to the alkylating carcinogen, methylnitrosourea; when varied doses of methylnitrosourea were administered to 6-week-old mice and survivals at the 30th day were determined, LD50s of MGMT-/- and MGMT+/+ mice were 20 and 240 mg/kg of body weight, respectively. MGMT+/- mice were as resistant as MGMT+/+ mice, but some difference in survival time was noted when the two genotypes of mice were exposed to a relatively high dose of methylnitrosourea. A large number of thymic lymphomas, as well as lung adenomas, occurred in MGMT-/- mice exposed to methylnitrosourea at a dose of 2.5 mg/kg of body weight. In case of exposure to the same dose of drug, no or few tumors occurred in the MGMT+/+ and MGMT+/- mice. It appears that the DNA repair methyltransferase protein protected these mice from methylnitrosourea-induced tumorigenesis.
Animals↗
Global metabolic response of Escherichia coli to gnd or zwf gene-knockout, based on 13C-labeling experiments and the measurement of enzyme activities.
An integrated study on cell growth, enzyme activities and carbon flux redistribution was made to investigate how the central metabolism of Escherichia coli changes with the knockout of genes in the oxidative pentose phosphate pathway (PPP). Mutants deficient in glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were constructed by disrupting the zwf and gnd genes and were grown in minimal media with two different carbon sources, such as glucose or pyruvate. It was shown that the knockout of either gnd or zwf gene did not affect the cell growth rate significantly, but the cellular metabolism was changed. While the specific substrate uptake rate and the specific carbon dioxide evolution rate for either mutant grown on glucose were higher than those obtained for the parent strain, these two rates were markedly decreased in mutants grown on pyruvate. The measurement of enzyme activities implied a significant change in metabolism, when alternative pathways such as the Entner-Doudoroff pathway (EDP) and the malic enzyme pathway were activated in the gnd mutant grown on glucose. As compared with the parent strain, the activities of phosphoglucose isomerase were increased in mutants grown on glucose but decreased in mutants grown on pyruvate. The metabolic flux redistribution obtained based on 13C-labeling experiments further indicated that the direction of the flux through the non-oxidative PPP was reversed in response to the gene knockout. Moreover, the knockout of genes caused an increased flux through the tricarboxlic acid cycle in mutants grown on glucose but caused a decrease in the case of using pyruvate. There was also a negative correlation between the fluxes through malic enzyme and isocitrate dehydrogenase in the mutants; and a positive correlation was found between the fluxes through malic enzyme and phosphoenolpyruvate carboxylase.
Carbohydrate Metabolism↗
Construction of lycopene-overproducing E. coli strains by combining systematic and combinatorial gene knockout targets.
Identification of genes that affect the product accumulation phenotype of recombinant strains is an important problem in industrial strain construction and a central tenet of metabolic engineering. We have used systematic (model-based) and combinatorial (transposon-based) methods to identify gene knockout targets that increase lycopene biosynthesis in strains of Escherichia coli. We show that these two search strategies yield two distinct gene sets, which affect product synthesis either through an increase in precursor availability or through (largely unknown) kinetic or regulatory mechanisms, respectively. Exhaustive exploration of all possible combinations of the above gene sets yielded a unique set of 64 knockout strains spanning the metabolic landscape of systematic and combinatorial gene knockout targets. This included a global maximum strain exhibiting an 8.5-fold product increase over recombinant K12 wild type and a twofold increase over the engineered parental strain. These results were further validated in controlled culture conditions.
Carotenoids↗
Glutathione peroxidase-1 deficiency leads to increased nuclear light scattering, membrane damage, and cataract formation in gene-knockout mice.
PURPOSE: Previous in vitro studies with transgenic and gene-knockout mice have shown that lenses with elevated levels of glutathione peroxidase (GPX)-1 activity are able to resist the cytotoxic effect of H(2)O(2), compared with normal lenses and lenses from GPX-1-deficient animals. The purpose of this study was to investigate the functional role of this enzyme in antioxidant mechanisms of lens in vivo by comparing lens changes of gene-knockout mice with age-matched control animals. METHODS: In vivo lens changes were monitored by slit lamp biomicroscopy, and enucleated lenses were examined under a stereomicroscope in gene-knockout animals and age-matched control animals ranging in age from 3 weeks to 18 months. Transmission (TEM) and confocal microscopy were performed on different regions of lenses after the mice were killed at various times. RESULTS: Slit lamp images showed an increase in nuclear light scattering (NLS) in gene-knockout mice compared with control animals. TEM revealed changes in the nucleus as early as 3 weeks of age by the appearance of waviness of fiber membranes. With increasing age, there was greater distortion of fiber membranes and distension of interfiber space at the apex of fiber cells compared with control mice. The changes in nuclear fiber membranes were even more dramatic, as observed by confocal microscopy, which was performed on thicker sections. In contrast to the changes in the lens nucleus, the morphology of the epithelium and superficial cortex remained unchanged in knockout animals during the same experimental period, consistent with slit lamp observations. Stereomicroscopy of ex vivo lenses demonstrated a significant increase in opacification in gene-knockout mice relative to control animals of the same age. This effect became evident in mice aged 5 to 9.9 months and persisted thereafter in older animals, resulting in mature cataracts after 15 months. CONCLUSIONS: The results demonstrate the critical role of GPX-1 in antioxidant defense mechanisms of the lens nucleus. The increased NLS appears to be associated with damage to fiber membranes in the nucleus, which is particularly susceptible to oxidative challenge because of the deficiency of GPX-1. It is suggested that the lens membrane changes in the knockout animals may be due to the formation of lipid peroxides, which serve as substrates for GPX-1. Cataract development in gene-knockout mice appeared to progress from focal opacities, apparent at an earlier age, to lamellar cataracts between 6 and 10 months, and finally to complete opacification in animals older than 15 months. This is the first reported phenotype in GPX-1-knockout mice.
Animals↗
Development of a gene knockout system for the halophilic archaeon Haloferax volcanii by use of the pyrE gene.
So far, the extremely halophilic archaeon Haloferax volcanii has the best genetic tools among the archaea. However, the lack of an efficient gene knockout system for this organism has hampered further genetic studies. In this paper we describe the development of pyrE-based positive selection and counterselection systems to generate an efficient gene knockout system. The H. volacanii pyrE1 and pyrE2 genes were isolated, and the pyrE2 gene was shown to code for the physiological enzyme orotate phosphoribosyl transferase. A DeltapyrE2 strain was constructed and used to isolate deletion mutants by the following two steps: (i) integration of a nonreplicative plasmid carrying both the pyrE2 wild-type gene, as a selectable marker, and a cloned chromosomal DNA fragment containing a deletion in the desired gene; and (ii) excision of the integrated plasmid after selection with 5-fluoroorotic acid. Application of this gene knockout system is described.
Amino Acid Sequence↗
TGF-beta1-induced Smad 3 binding to the Smad 7 gene: knockout of Smad 7 gene transcription by sense phosphorothioate oligos, autoregulation, and effect on TGF-beta1 secretion: bleomycin acts through TGF-beta1.
Bleomycin produces its fibrogenic effect, at least in part, by TGF-beta1 secretion. Treatment of IMR-90 human embryonic lung fibroblasts with bleomycin at 0.5 microg/ml results in a 1.6-fold increase of TGF-beta1 as determined by a specific ELISA assay for TGF-beta1 after acidification of the conditioned media. This elevation of TGF-beta1 secretion is furthermore enhanced in vivo by TGF-beta1 autoinduction of the TGF-beta1 gene. To demonstrate TGF-beta1 autoinduction, the fibroblasts were pretreated with 12.5 ng/ml TGF-beta1, washed extensively to remove any residual TGF-beta1, and then allowed to incubate for 24 h in AIM V synthetic serum-free media. The media when assayed using the ELISA assay contained a 1.6-fold increase of TGF-beta1. The distal promoter of the human TGF-beta1 gene contains a Smad 3 element (CAGGACA), which is homologous to the Smad 3 binding element motif (CAGA). The nuclear extracts of human embryonic lung fibroblasts treated for either 15 min or 24 h with TGF-beta1 did not demonstrate specificity of binding of a protein(s) to the homologous Smad 3 element as determined by cold wild-type oligodeoxynucleotide competition experiments. However, specific Smad 3 binding to the Smad 3 element (GTCTAGAC) found in proximal promoter of the Smad 7 gene was observed by cold oligo competition and supershift assays using a goat polyclonal Smad 3 antibody in the presence and absence of an N-terminal Smad 3 peptide. To determine the functionality of this Smad 3 binding to the Smad 3 element in the proximal promoter of the Smad 7 inhibitory gene to TGF-beta1 secretion, fibroblasts were transiently pretransfected with double-stranded phosphorothioate oligo "decoys" containing the Smad 7/Smad 3 element in the presence of plasmin to convert latent TGF-beta1 to active TGF-beta1. Under these conditions, which simulate the in vivo situation of 2.2-fold increase of total active TGF-beta1 was observed. Fibroblasts were also pretransfected with these double-stranded oligo "decoys," washed, then treated with TGF-beta1, washed and incubated in AIM V for an additional 24 h. In this latter experiment, a superinduction of TGF-beta1 secretion was observed. We propose that these oligo "decoys" bind Smad 3 preventing this initiation factor from binding to the Smad 7/Smad 3 element thereby decreasing the transcription of the Smad 7 gene. The decrease of the inhibitory Smad 7 would result in less binding of this Smad inhibitor to the Type I TGF-beta receptor and less antagonism of active TGF-beta1, more autoinduction of the TGF-beta1 gene, and more of the fibrogenic effects of TGF-beta1.
Bleomycin↗