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PubMed2007

A novel conditional knockout strategy applied to serotonin receptors.

Here we demonstrate the feasibility of a doubly regulatable transgenic mouse design that allows for gene manipulation by both Cre-recombinase and the tetracycline inducible system. Using a knock-in strategy to insert both elements of the tetracycline inducible system and a neomycin (neo) cassette flanked by loxP sequences (floxed) into the wild-type locus, we generated mice that express the 5-HT(1B) receptor in a conditional manner. In the presence of a floxed neo-cassette, receptor expression was silenced. Removal of this cassette by Cre-mediated recombination led to 5-HT(1B) receptor expression, which was highly regulatable when doxycycline, a derivative of tetracycline, was administered to the mice. This system allowed for a determination of an in vivo time course of receptor half-life and recovery. Physiological studies also demonstrated that rescued 5-HT(1B) receptors were functional, and that this functionality was reversible upon treatment with doxycycline. Crossing mice where the 5-HT(1B), or the 5-HT(1A), receptors were silenced by the neo-cassette, with mice expressing either Cre-recombinase or the tetracycline transactivator (tTA) under the control of tissue-specific promoters, led to tissue-specific re-expression of these receptors. Our studies thus demonstrate the potential of this strategy for achieving both a classic knockout, as well as subsequent tissue-specific and/or inducible knockouts.

Animals↗

PubMed2003

The effect of pfl gene knockout on the metabolism for optically pure D-lactate production by Escherichia coli.

The effect of gene knockout on metabolism in the pflA-, pflB-, pflC-, and pflD- mutants of Escherichia coli was investigated. Batch cultivations of the pfl- mutants and their parent strain were conducted using glucose as a carbon source. It was found that pflA- and pflB- mutants, but not pflC- and pflD- mutants, produced large amounts of D-lactate from glucose under the microaerobic condition, and the maximum yield was 73%. In order to investigate the metabolic regulation mechanism, we measured enzyme activities for the following eight enzymes: glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), pyruvate kinase, lactate dehydrogenase (LDH), phosphoenolpyruvate carboxylase, acetate kinase, and alcohol dehydrogenase. Intracellular metabolite concentrations of glucose 6-phosphate, fructose 1,6-bisphosphate, phosphoenolpyruvate, pyruvate, acetyl coenzyme A as well as ATP, ADP, AMP, NADH, and NAD+ were also measured. It was shown that the GAPDH and LDH activities were considerably higher in pflA- and pflB- mutants, which implies coupling between NADH production and consumption between the two corresponding reactions. The urgent energy requirement was shown by the lower ATP/AMP level due to both oxygen limitation and pfl gene knockout, which promoted significant stepping-up of glycolysis when using glucose as a carbon source. It was shown that the demand for energy is more important than intracellular redox balance, thus excess NADH produced through GAPDH resulted in a significantly higher intracellular NADH/NAD+ ratio in pfl- mutants. Consequently, the homolactate production was achieved to meet the requirements of the redox balance and the energy production through glycolysis. The effect of using different carbon sources such as gluconate, pyruvate, fructose, and glycerol was investigated.

Acetate Kinase↗

PubMed1999

DEAD-box protein HEL64 from Trypanosoma brucei: subcellular localization and gene knockout analysis.

RNA helicases are molecules that play a central role in the control of ribonucleic acid metabolism. Only two putative RNA helicase genes of the DEAD-box family have been identified in the protozoan parasite Trypanosoma brucei brucei. One of these genes is HEL64, a single-copy gene of unknown function. In this study we conducted targeted gene-disruption experiments of the HEL64 locus with the aim of identifying a phenotype that would suggest a function for the encoded protein. It is likely that HEL64 is an essential gene in insect-stage trypanosomes, since all attempts to create a HEL64 double-allele knockout cell line failed. Instead, we obtained a mutant derived from an unusual recombination event that nonetheless contained a functional copy of the gene. One allele of HEL64 is sufficient for the survival of the parasite. Single-allele knockout mutants showed no gross change in cell morphology and multiplied with a cell-doubling time identical to that of wild-type trypanosomes. Though HEL64 has high sequence homology with the nuclear DEAD-box protein p68, it is localized in the cytosol of trypanosomes and, thus, cannot be a homologue of p68 as previously suggested. Since the protein did not cross-hybridize with an anti-eIF-4A antibody, we excluded the possibility that HEL64 might be a homologue of the translation initiation factor eIF-4A. Although the function of HEL64 remains unknown, the present data indicate that the encoded DEAD-box protein plays an important role during the insect life-cycle stage of the parasite.

Alleles↗

PubMed2006

Protein-carbonyl accumulation in the non-replicative senescence of the methionine sulfoxide reductase A (msrA) knockout yeast strain.

The major enzyme of the methionine sulfoxide reductase (Msr) system is MsrA. Senescing msrA knockout mother yeast cells accumulated significant amounts of protein-carbonyl both at 5 generation-old (young) and 21 generation-old (old) cultures, while the control mother cells showed significant levels of protein-carbonyl mainly in the old culture. The Msr activities of both yeast strains declined with age and exposure of cells to H(2)O(2) caused an accumulation of protein-carbonyl especially in the msrA knockout strain. It is suggested that a compromised MsrA activity may serve as a marker for non-replicative aging.

Aging↗

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↗

PubMed2005

Identification of low frequency knockout mutants in Dictyostelium discoideum created by single or double homologous recombination.

Generation and characterization of knockout clones is a widely used approach to evaluate the specific function of a gene product in Dictyostelium discoideum. The mutant clones are generally obtained by double homologous recombination in the target gene. A frequent limitation to obtaining mutants is the low frequency of homologous recombination. Here we present an easy method to identify rare mutants, based on PCR analysis of pools of clones. This method also allows the isolation of functional knockout mutants created by a single homologous recombination event, which can be more frequent than a double recombination event.

Animals↗

PubMed2006

Suicide vectors for antibiotic marker exchange and rapid generation of multiple knockout mutants by allelic exchange in Gram-negative bacteria.

Allelic exchange is frequently used in bacteria to generate knockout mutants in genes of interest, to carry out phenotypic analysis and learn about their function. Frequently, understanding of gene function in complex processes such as pathogenesis requires the generation of multiple mutant strains. In Pseudomonads and other non-Enterobacteriaceae, this is a time-consuming and laborious process based on the use of suicide vectors and allelic exchange of the appropriate mutant version of each gene, disrupted by a different antibiotic marker. This often implies the generation of a series of mutants for each gene, each disrupted by a different antibiotic marker, in order to obtain all possible double or multiple mutant combinations. In this work, we have modified this method by developing a set of 3 plasmid derivatives from the previously described suicide vector for allelic exchange, pKAS32, to make antibiotic marker exchange easier and thus accelerate the entire process. Briefly, the construction of each single gene knockout mutant is carried out by allelic exchange of the chromosomal gene with a mutant allele disrupted by the insertion of a kanamycin resistance cassette. When a double mutant strain is required, antibiotic marker exchange is performed in either one of the single mutants, using any of the three plasmid derivatives that carry the kanamycin resistance gene disrupted by either a chloramphenicol, gentamycin, or streptomycin resistance cassette. The single mutant strain, carrying now an antibiotic resistance marker other than kanamycin, can be used to introduce a second mutation using the original plasmid constructs, to generate a double mutant. The process can be repeated sequentially to generate multiple mutants. We have validated this method by generating strains carrying different combinations of mutations in genes encoding different transcriptional regulators of the Hrp type III secretion system in Pseudomonas syringae. We have also tested the genetic organisation and stability of the resulting mutant strains during growth in laboratory conditions as well as in planta.

Anti-Bacterial Agents↗

PubMed2002

Adenovirus-mediated G(alpha)(q)-protein antisense transfer in neurons replicates G(alpha)(q) gene knockout strategies.

Antisense approaches are increasingly used to dissect signaling pathways linking cell surface receptors to intracellular effectors. Here we used a recombinant adenovirus to deliver G-protein alpha(q) antisense into rat superior cervical ganglion (SCG) neurons and neuronal cell lines to dissect G(alpha)(q)-mediated signaling pathways in these cells. This approach was compared with other G(alpha)(q) gene knockdown strategies, namely, antisense plasmid and knockout mice. Infection with adenovirus expressing G(alpha)(q) antisense (G(alpha)(q)AS AdV) selectively decreased immunoreactivity for the G(alpha)(q) protein. Expression of other G(alpha) protein subunits, such as G(alpha)(oA/B,) was unaltered. Consistent with this, modulation of Ca(2+) currents by the G(alpha)(q)-coupled M(1) muscarinic receptor was severely impaired in neurons infected with G(alpha)(q)AS AdV whereas modulation via the G(alpha)(oA)-coupled M(4) muscarinic receptor was unchanged. In agreement, activation of phospholipase C and consequent mobilization of intracellular Ca(2+) by UTP receptors was lost in NG108-15 cells infected with G(alpha)(q)AS AdV but not in cells infected with the control GFP-expressing adenovirus. Results obtained with this recombinant AdV strategy qualitatively and quantitatively replicated results obtained using SCG neurons microinjected with G(alpha)(q) antisense plasmids or SCG neurons from G(alpha)(q) knockout mice. This combined antisense/recombinant adenoviral approach can therefore be useful for dissecting signal transduction mechanisms in SCG and other neurons.

Adenoviridae↗

PubMed1998

The vasopressin deficient Brattleboro rats: a natural knockout model used in the search for CNS effects of vasopressin.

Behavioral neuroscience is using more and more gene knockout techniques to produce animals with a specific deletion. These studies have their precedent in nature. A mutation may result in a limited genetic defect, as seen in the vasopressin (VP) deficiency in the Brattleboro rat. The mutation is in a single pair of autosomal loci, and the sequences of VP gene from wild-type and homozygous Brattleboro rats are identical except for a single nucleotide deletion in the second exon. The deletion results in the synthesis of an altered VP precursor that is unable to enter the secretory pathway. The genetic disturbance results in a central diabetes insipidus comparable to that found in humans. Starting with our work during the early 1970s we found that the genetic defect in the availability of VP causes deficits in central nervous system (CNS) functions. Behavioral processes from cognition to drug tolerance appeared to be disturbed by the absence of VP, but not all behaviors are affected. The specificity of the absence of VP in causing behavioral deficits is shown in many cases. However, certain deficits are due to genetic factors other than the deletion of the VP gene. The picture is further complicated by differences in testing conditions, the absence of proper controls, i.e. heterozygous and wild-type Brattleboro rats, sex, compensation phenomena, and the absence of neuropeptides co-localized with VP. Interestingly, an age dependent spontaneous shunt to a heterozygous phenotype in vasopressinergic neurons might also compensate for the disturbance. Accordingly, findings in knockout animals should be interpreted with caution. One should realize that brain functions are modulated by multiple neuropeptides and that neuropeptides possess multiple CNS effects.

Animals↗

PubMed2003

Gene expression patterns for metabolic pathway in pgi knockout Escherichia coli with and without phb genes based on RT-PCR.

Metabolic regulations were investigated from the viewpoint of gene expressions for Escherichia coli JM109 and pgi knockout E. coli with and without phb genes using RT-PCR. One of the main features of pgi knockout E. coli is the overproduction of NADPH produced in pentose phosphate (PP) pathway. NADPH overproduction in PP pathway in pgi mutant causes some reducing power imbalance that ultimately affects the cell growth. It was shown that this reducing power imbalance can be recovered to some extent by introducing NADPH absorbing pathway such as PHB synthetic pathway into pgi mutant E. coli. To get insight into the regulation mechanism of pgi mutant E. coli at the transcriptional level, 87 E. coli genes involved in central metabolic pathways and key regulatory mechanisms were investigated by semi-quantitative RT-PCR analysis. The analysis showed that pentose phosphate pathway genes and part of the glycolysis pathway genes were affected significantly by expression of phb genes in pgi mutant E. coli DF11/pAeKG1 as well as in pgi mutant E. coli DF11 as compared with those in E. coli JM109. In contrast, most of the TCA cycle genes except icdA were downregulated in both pgi mutants E. coli. The upregulation of icdA gene may be due to the positive regulation of fruR. Moreover, it was found that ack gene as well as aceA and aceB genes involved in the glyoxylate shunt were upregulated in pgi mutants while ppc gene was downregulated, indicating that pgi inactivation changes the anaplerotic pathway from ppc pathway to glyoxylate shunt. Enzyme activities of glk, zwf, tpiA, fbaA, ldhA, gltA, aceA, mdh and maeB were also measured and compared with the corresponding gene expressions. Most of them are well correlated except for aceA gene indicating that glyoxylate pathway is regulated on the protein level, not on the gene level.

Down-Regulation↗

PubMed2003

Analysis of metabolic and physiological responses to gnd knockout in Escherichia coli by using C-13 tracer experiment and enzyme activity measurement.

The physiological and metabolic responses to gnd knockout in Escherichia coli K-12 was quantitatively investigated by using the (13)C tracer experiment (GC-MS/NMR) together with the enzyme activity analysis. It was shown that the general response to the gene knockout was the local flux rerouting via Entner-Doudoroff pathway and the direction reversing via non-oxidative pentose phosphate pathway (PPP). The mutant was found to direct higher flux to phosphoglucose isomerase reaction as compared to the wild-type, but the respiratory metabolism was comparable in both strains. The anaplerotic pathway catalyzed by malic enzyme was identified in the mutant, which was accompanied with an up-regulation of phosphoenolpyruvate carboxylase and down-regulation of phosphoenolpyruvate carboxykinase. The presented results provide first evidence that compensatory mechanism existed in PPP and anaplerotic pathway in response to the gnd deletion.

Carbon Isotopes↗

PubMed2000

Hepatic fibrosis and cytochrome P450: experimental models of fibrosis compared to AHR knockout mice.

Hepatic fibrosis is characterized by abnormal collagen deposition resulting from increased collagen synthesis and decreased collagen degradation. Cytochrome P450 mediates major drug metabolizing enzyme activity in the liver and this activity is reduced in hepatic fibrosis. In this study we assess cytochrome P450 and CYP 1A mRNA in livers of animals that have been induced to hepatic fibrosis using the heterologous serum induced model of fibrosis in rats compared to controls. Fibrosis was confirmed by assessing the collagen in liver sections as quantified by Sirius red/Fast green staining, a quantitative measure of fibrosis as well as by visualization of the hepatic fibrosis. Collagen levels in the liver sections of heterologous serum induced fibrotic rats was increased by 33% compared to controls and a typical fibrotic pattern was seen. Messenger RNA was prepared from heterologous serum induced fibrotic rats and compared to controls. CYP 1A2 was assessed using a specific probe and the CYP 1A2 level was significantly reduced in the heterologous serum induced fibrotic rats compared to controls. These results further suggest that cytochrome P450 is reduced in the presence of hepatic fibrosis. Thus, in three well established experimental models of hepatic fibrosis which had clearly developed hepatic fibrosis (as shown by Sirius red/Fast green staining), cytochrome P450 mediated enzyme activity, or specifically, CYP 1A messenger RNA is decreased. We then investigated a transgenic mouse, deficient in the arylhydrocarbon hydroxylase receptor (AHR), which has undetectable levels of CYP 1A messenger RNA. We quantitated the collagen in liver sections obtained from AHR knockout mice compared to controls, as an indication of the presence of hepatic fibrosis. Collagen concentration was significantly increased by 53% (P<0.0005) in sections from Ahr-/- (knockout) mice compared to wild-type controls. Collagen in livers of the Ahr+/- heterozygous mice was not different from wild-type controls. The increase in collagen concentration in liver sections is an indication of fibrosis in Ahr-/- mice. Collagen protein deposition was also elevated in liver sections from bile duct ligated rats (by 44%) compared to sham operated controls, was elevated in liver sections from heterologous serum induced fibrosis in rats (33%) compared to controls, and was elevated in liver sections from yellow phosphorous induced hepatic fibrosis (74%) compared to vehicle treated controls. In conclusion, these results indicate that cytochrome P450 and specific subtypes of P450, the CYP 1A subgroup, are significantly reduced in three experimental models of hepatic fibrosis when there is evidence of increased collagen deposition in the livers. These results also indicate that mice that are deficient in CYP 1A have elevated levels of hepatic collagen protein, an indication of hepatic fibrosis.

Journal Article↗

PubMed1998

Knockout mice in neuropsychopharmacology: present and future.

The technique of targeted inactivation of individual genes in mice has undoubtedly revolutionized biomedicine. Applications for gene knockout mice in neuropsychopharmacology are manifold: Determination of targets for established treatments is eased, while development of novel drugs is facilitated for a given target. This review discusses advantages and limitations of gene knockout strategies and, in particular, their relevance for the development of novel diagnostic and therapeutic approaches in psychiatric disorders. In addition to the classical targeted disruption of one or several genes simultanously, it specifically emphasizes those novel techniques that allow the inactivation of a gene with spatio-temporal selectivity.

Journal Article↗

PubMed1998

The adenosine A(2A) receptor knockout mouse: a model for anxiety?

The main behavioural features of the adenosine A(2A) receptor knockout mouse include anxiety, aggressiveness in males and a paradoxical response to caffeine. These behavioural characteristics caused by the lack of adenosine A(2A) receptor function in mice correspond to the effects of functional antagonism of adenosine A(2A) receptors in humans and rodents. Increased anxiety in patients with panic disorder and increased psychotic symptomatology in patients with schizophrenia have been observed after caffeine administration. Several hypotheses have been developed suggesting a reduced adenosine A(2A) receptor-mediated transmission as a contributing factor in the pathogenesis of these disorders. Recent genetic studies, in particular of panic disorder, suggest an involvement of adenosine A(2A) receptor gene variation. If future studies prove a pathogenetic role for a genetically determined loss of A(2A) receptor function in psychiatric disorders, the A(2A) receptor knockout mouse will be a valuable model to develop novel pharmacological therapies for these disorders.

Journal Article↗

PubMed1999

Knockout Corner.

Serotonin (5-hydroxytryptamine, 5-HT) is a neurotransmitter involved in a number of physiological functions including sleep, appetite, pain perception, and sexual activity. Several pathological states such as migraine, depression, and anxiety have been linked to the serotonergic system, and serotonergic drugs have been used to treat these disorders. To date, there are 14 known serotonin receptor subtypes through which serotonin exerts its multiple actions. The classic pharmacological approach to study how these individual receptor subtypes contribute to various behaviours has been to use selective drugs that either block or activate certain receptor subtypes, and then study the effects of these compounds on physiology and behaviour. A complementary genetic approach is the technique of gene targeting. Using this technology, we and others have begun to examine the contribution of several serotonin receptor subtypes to complex behaviours through the generation of knockout mice that lack the genes encoding these receptors. In this review, we will describe what we have learned about the serotonergic system and the function of the 5-HT(1B) receptor by the analysis of 5-HT(1B) receptor knockout mice. Furthermore, we will discuss the implications of these findings and our plans for future studies.

Journal Article↗

PubMed2003

Cyclin D-knockout uncouples developmental progression from sugar availability.

Multicellular organisms need to modulate proliferation and differentiation in response to external conditions. An important role in these processes plays the mitogen-stimulated induction of cyclin D (cycD) gene expression. D-type cyclins have been identified as the crucial intracellular sensors for cell-cycle regulation in all eukaryotes. However, cycD deletions have been found to cause specific phenotypic alterations in animals but not yet in plants. An insertional mutation of a so far uncharacterized Arabidopsis cycD gene did not alter the plant phenotype. To gain new insights into CycD function of land plants, we generated targeted cycD gene knockouts in the moss Physcomitrella patens and observed a surprisingly limited disruption phenotype. While wild-type plants reacted to exogenous glucose sources with prolonged growth of juvenile stages and retarded differentiation, cycD knockouts exhibited developmental progression independent of sugar supply. On the other hand, growth rate, cell sizes or plant size were not affected. Thus, we conclude that Physcomitrella CycD might not be essential for cell-cycle regulation but is important for coupling the developmental progression to nutrient availability.

Blotting, Southern↗

PubMed2003

Production of knockout rats using ENU mutagenesis and a yeast-based screening assay.

The rat is a widely used model in biomedical research and is often the preferred rodent model in many areas of physiological and pathobiological research. Although many genetic tools are available for the rat, methods to produce gene-disrupted knockout rats are greatly needed. In this study, we developed protocols for creating N-ethyl-N-nitrosourea (ENU)-induced germline mutations in several rat strains. F1 preweanling pups from mutagenized Sprague Dawley (SD) male rats were then screened for functional mutations in Brca1 and Brca2 using a yeast gap-repair, ADE2-reporter truncation assay. We produced knockout rats for each of these two breast cancer suppressor genes.

Animals↗

PubMed2006

Temporal and spatial delineation of mouse Otx2 functions by conditional self-knockout.

To identify the independent spatial and temporal activities of the essential developmental gene the Otx2, the germline mutation of which is lethal at embryonic day 8.5, we floxed one allele and substituted the other with an inducible CreER recombinase gene. This makes 'trans' self-knockout possible at any developmental stage. The transient action of tamoxifen pulses allows time-course mutation. We demonstrate efficient temporal knockout and demarcate spatio-temporal windows in which Otx2 controls the head, brain structures and body development.

Animals↗