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Results for “Gene knockout”

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At least 181 records · Page 10Linked to original sources

PubMed2001

Discrimination of computer-graphic stimuli by mice: a method for the behavioral characterization of transgenic and gene-knockout models.

An automated method is described for the behavioral testing of mice in an apparatus that allows computer-graphic stimulus material to be presented. Mice responded to these stimuli by making a nose-poke toward a computer monitor that was equipped with a touchscreen attachment for detecting responses. It was found that C57BL/6 mice were able to solve single-pair visual discriminations as well as 3-pair concurrent visual discriminations. The finding that mice are capable of complex visual discriminations introduces the possibility of testing mice on nonspatial tasks that are similar to those used with rats, monkeys, and humans. Furthermore, the method seems particularly well suited to the comprehensive behavioral assessment of transgenic and gene-knockout models.

Animals↗

PubMed2004

Brain penetration of methadone (R)- and (S)-enantiomers is greatly increased by P-glycoprotein deficiency in the blood-brain barrier of Abcb1a gene knockout mice.

RATIONALE: Methadone maintenance treatment is complicated by the wide variability of efficacy among patients. The large interindividual variability of the plasma concentrations of methadone was previously thought to be responsible for the variable therapeutic efficacy. However, recent studies suggested that methadone may be a substrate of P-glycoprotein (P-gp). Therefore, the function of P-gp in blood-brain barrier (BBB) may affect the concentration of methadone at its site(s) of action in the central nervous system, thereby contributing to its therapeutic efficacy and/or adverse events. OBJECTIVE: To investigate the effect of P-gp on brain penetration of methadone (R)- and (S)-enantiomers and their major oxidative metabolite 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP). METHODS: We compared the tissue distribution of methadone (R)- and (S)-enantiomers and EDDP in the Abcb1a-/- gene knockout mice and the Abcb1a+/+ wild-type mice 1 h following intraperitoneal administration of 15 microg Rac-methadone/g mouse. RESULTS: Plasma concentrations of (R)- and (S)-methadone were similar between the two animal groups. However, the brain concentrations of (R)- and (S)-methadone in the Abcb1a-/- mice were markedly higher (15- and 23-fold, respectively, P<0.0001) than those of the Abcb1a+/+ wild-type mice. No statistically significant difference was found for other organs between the mutants and controls. No organ difference was found for EDDP between the mutants and controls. CONCLUSIONS: (R)- and (S)-methadone are substrates of P-gp. The P-gp in BBB greatly limits the brain entry of (R)- and (S)-methadone to their central nervous system acting sites. The interindividual variation in expression of P-gp in BBB may represent a source of variation for the access and effects of methadone in the brain.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2006

KCNJ11 gene knockout of the Kir6.2 KATP channel causes maladaptive remodeling and heart failure in hypertension.

Heart failure is a growing epidemic, with systemic hypertension a major risk factor for development of disease. However, the molecular determinants that prevent the transition from a state of hypertensive load to that of overt cardiac failure remain largely unknown. Here in experimental hypertension, knockout of the KCNJ11 gene, encoding the Kir6.2 pore-forming subunit of the sarcolemmal ATP-sensitive potassium (K(ATP)) channel, predisposed to heart failure and death. Defective decoding of hypertension-induced metabolic distress signals in the K(ATP) channel knockout set in motion pathological calcium overload and aggravated cardiac remodeling through a calcium/calcineurin-dependent cyclosporine-sensitive pathway. Rescue of the failing K(ATP) knockout phenotype was achieved by alternative control of myocardial calcium influx, bypassing uncoupled metabolic-electrical integration. The intact KCNJ11-encoded K(ATP) channel is thus a required safety element preventing hypertension-induced heart failure, with channel dysfunction a molecular substrate for stress-associated channelopathy in cardiovascular disease.

Animals↗

PubMed2005

Caveolin-1 gene knockout impairs nitrergic function in mouse small intestine.

Caveolin-1 is a plasma membrane-associated protein that is responsible for caveolae formation. It plays an important role in the regulation of the function of different signaling molecules, among which are the different isoforms of nitric oxide synthase (NOS). Nitric oxide (NO) is known to be an important inhibitory mediator in the mouse gut. Caveolin-1 knockout mice (Cav1(-/-)) were used to examine the effect of caveolin-1 absence on the NO function in the mouse small intestine (ileum and jejunum) compared to their genetic controls and BALB/c controls. Immunohistochemical staining showed loss of caveolin-1 and NOS in the jejunal smooth muscles and myenteric plexus interstitial cells of Cajal (ICC) of Cav1(-/-) mice; however, nNOS immunoreactive nerves were still present in myenteric ganglia. Under nonadrenergic noncholinergic (NANC) conditions, small intestinal tissues from Cav1(-/-) mice relaxed to electrical field stimulation (EFS), as did tissues from control mice. Relaxation of tissues from control mice was markedly reduced by N-omega-nitro-L-arginine (10(-4) M), but relaxation of Cav1(-/-) animals was affected much less. Also, Cav1(-/-) mice tissues showed reduced relaxation responses to sodium nitroprusside (100 microM) compared to controls; yet there were no significant differences in the relaxation responses to 8-bromoguanosine-3': 5'-cyclic monophosphate (100 microM). Apamin (10(-6) M) significantly reduced relaxations to EFS in NANC conditions in Cav1(-/-) mice, but not in controls. The data from this study suggest that caveolin-1 gene knockout causes alterations in the smooth muscles and the ICC, leading to an impaired NO function in the mouse small intestine that could possibly be compensated by apamin-sensitive inhibitory mediators.

Animals↗

PubMed2006

Gene knockout of the KCNJ8-encoded Kir6.1 K(ATP) channel imparts fatal susceptibility to endotoxemia.

Sepsis, the systemic inflammatory response to infection, imposes a high demand for bodily adaptation, with the cardiovascular response a key determinant of outcome. The homeostatic elements that secure cardiac tolerance in the setting of the sepsis syndrome are poorly understood. Here, in a model of acute septic shock induced by endotoxin challenge with Escherichia coli lipopolysaccharide (LPS), knockout of the KCNJ8 gene encoding the vascular Kir6.1 K(ATP) channel pore predisposed to an early and profound survival disadvantage. The exaggerated susceptibility provoked by disruption of this stress-responsive sensor of cellular metabolism was linked to progressive deterioration in cardiac activity, ischemic myocardial damage, and contractile dysfunction. Deletion of KCNJ8 blunted the responsiveness of coronary vessels to cytokine- or metabolic-mediated vasodilation necessary to support myocardial perfusion in the wild-type (WT), creating a deficit in adaptive response in the Kir6.1 knockout. Application of a K(ATP) channel opener drug improved survival in the endotoxic WT but had no effect in the Kir6.1 knockout. Restoration of the dilatory capacity of coronary vessels was required to rescue the Kir6.1 knockout phenotype and reverse survival disadvantage in lethal endotoxemia. Thus, the Kir6.1-containing K(ATP) channel, by coupling vasoreactivity with metabolic demand, provides a vital feedback element for cardiovascular tolerance in endotoxic shock.

Animals↗

PubMed2004

Cloning of L-lactate dehydrogenase and elimination of lactic acid production via gene knockout in Thermoanaerobacterium saccharolyticum JW/SL-YS485.

The gene encoding L-lactate dehydrogenase from Thermoanaerobacterium saccharolyticum JW/SL-YS485 was cloned, sequenced, and used to obtain an L-ldh deletion mutant strain (TD1) following a site-specific double-crossover event as confirmed by PCR and Southern blot. Growth rates and final cell densities were similar for strain TD1 and the wild-type grown on glucose and xylose. Lactic acid was below the limit of detection (0.3 mM) for strain TD1 on both glucose and xylose at all times tested, but was readily detected for the wild-type strain, with average final concentrations of 8.1 and 1.8 mM on glucose and xylose, respectively. Elimination of lactic acid as a fermentation product was accompanied by a proportional increase in the yields of acetic acid and ethanol. The results reported here represent a step toward using metabolic engineering to develop strains of thermophilic anaerobic bacteria that do not produce organic acids, and support the methodological feasibility of this goal.

Acetic Acid↗

PubMed2026

Targeted multiplex gene knockouts in Lemna minor using CRISPR/Cas9.

Lemna minor (commonly known as duckweed) is a fast-growing aquatic plant recognized as a promising green bioreactor for recombinant protein production. Its rapid proliferation, high protein yield, environmental adaptability, and edibility make it highly attractive for biotechnological applications. It is essential to develop and expand genetic tools tailored to this species to maximize these advantages and further unlock its biotechnological potential. A key strategy for achieving this goal is the implementation of advanced genome editing technologies, such as the CRISPR/Cas9 system. Although multiplex CRISPR/Cas9 gene editing has previously been successfully applied in Lemna aequinoctialis, the capability of the endogenous plant tRNA processing system for multiplex editing in L. minor using the polycistronic tRNA-sgRNA (PTG)/Cas9 system has not yet been explored. In this study, a PTG construct was engineered to include four sgRNAs designed to simultaneously target two plant-specific glycosyltransferase genes: α-1,3-fucosyltransferase (FucT) and β-1,2-xylosyltransferase (XylT). As anticipated, the PTG-Cas9 system successfully induced frameshift mutations, characterized by insertions and deletions (indels), in regenerated L. minor plants derived from transformed calli. Validation via PCR and RT-PCR analysis, followed by sequencing of the target loci, confirmed the presence of indels at the target sites. Furthermore, western blot analyses utilizing antibodies specific to XylT and FucT in two homozygous lines (lines 44 and 217) revealed truncated XylT proteins in both lines. Moreover, an in-frame FucT protein was detected in line 217, whereas FucT expression was absent in line 44. This study marked the first successful demonstration of PTG-Cas9 system for multiplex genome editing in L. minor, paving the way for advanced genetic engineering in this species.

CRISPR-Cas Systems↗

PubMed2005

Effect of lpdA gene knockout on the metabolism in Escherichia coli based on enzyme activities, intracellular metabolite concentrations and metabolic flux analysis by 13C-labeling experiments.

The lipoamide dehydrogenase (LPD) encoded by lpdA gene is a component of the pyruvate dehydrogenase complex (PDHc), alpha-ketoglutarate dehydrogenase (AKGDH) and the glycine cleavage multi-enzyme (GCV) systems. In the present study, cell growth characteristics, enzyme activities and intracellular metabolite concentrations were compared between the parent strain Escherichia coli BW25113 and its lpdA knockout mutant in batch and continuous cultures. The lpdA knockout mutant produced significantly more pyruvate and L-glutamate under aerobiosis. Some D-lactate and succinate also accumulated in the culture broth. Based on the investigation of enzyme activities and intracellular metabolite concentrations, acetyl-CoA was considered to be formed by the combined reactions through pyruvate oxidase (PoxB), acetyl-CoA synthetase (Acs) and acetate kinase (Ack)-phosphoacetyltransferase (Pta) in the lpdA mutant. The effect of the lpdA gene knockout on the intracellular metabolic flux distributions was investigated based on 1H-13C NMR spectra and GC-MS signals obtained from 13C-labeling experiment using the mixture of [U-13C] glucose, [1-13C] glucose, and naturally labeled glucose. Flux analysis of the lpdA mutant indicated that the Entner-Doudoroff (ED) pathway and the glyoxylate shunt were activated. The fluxes through glycolysis and oxidative pentose phosphate (PP) pathway (except for the flux through glucose-6-phosphate dehydrogenase) were slightly downregulated. The TCA cycle was also downregulated in the mutant strain. On the other hand, the fluxes through the anaplerotic reactions of PEP carboxylase, PEP carboxykinase and malic enzyme were upregulated, which were consistent with the results of enzyme activities. Furthermore, the influence of the poxB gene knockout on the growth of E. coli was also studied because of its similar function to PDHc which connects the glycolysis to the TCA cycle. Under aerobiosis, a comparison of lpdA mutant and poxB mutant indicated that PDHc is the main enzyme which catalyzes the reaction from pyruvate to acetyl-CoA in the parent strain, while PoxB plays a very important role in the PDHc-deficient strain.

Biological Transport↗

PubMed2000

Increased blood pressure in alpha-calcitonin gene-related peptide/calcitonin gene knockout mice.

Nerves that contain calcitonin gene-related peptide (CGRP) are components of the sensory nervous system. Although these afferent nerves have traditionally been thought to sense stimuli in the periphery and transmit the information centrally, they also have an efferent vasodilator function. Acute administration of a CGRP receptor antagonist increases the blood pressure (BP) in several models of hypertension, which indicates that this potent vasodilator plays a counterregulatory role to attenuate the BP increase in these settings. To determine the role of this peptide in the long-term regulation of cardiovascular function, including hypertension, we obtained mice that have a deletion of the alpha-calcitonin gene-related peptide (alpha-CGRP) gene. Although the beta-calcitonin gene-related peptide (beta-CGRP) gene is intact in these mice, alpha-CGRP is by far the predominant species of CGRP produced in dorsal root ganglia (DRG) sensory neurons. Initially, we examined the effect of deletion of the alpha-CGRP on baseline BP and beta-CGRP and substance P mRNA expression. Systolic BP was significantly higher in the knockout mice (n=7) compared with wild-type in both male (160+/-6.1 vs 125+/-4.8 mm Hg) and female (163+/-4.8 vs 135+/-33 mm Hg) mice. Next, groups (n=7) of knockout and wild-type mice had catheters surgically placed in the right carotid artery for mean arterial pressure recording. With the animals fully awake and unrestrained, the knockout mice displayed an elevated mean arterial pressure compared with wild-type in both male (139+/-4.9 vs 118+/-4.9 mm Hg) and female (121+/-3.4 vs 107+/-3.1 mm Hg) mice. Northern blot analysis of DRG RNA samples confirmed the absence of alpha-CGRP mRNA in the knockout mice. Substance P mRNA content in DRG was unchanged between the 2 groups; however, beta-CGRP mRNA levels were reduced 2-fold in the knockout mice. These results indicate for the first time that alpha-CGRP may be involved in the long-term regulation of resting BP and suggest that these mice are particularly sensitive to challenges to BP homeostasis because of the loss of a compensatory vasodilator mechanism.

Animals↗

PubMed2002

Quantitative autoradiographic mapping of opioid receptors in the brain of delta-opioid receptor gene knockout mice.

Using quantitative receptor autoradiography we have determined if deletion of the delta-opioid receptor gene (Oprd1) results in compensatory changes in the expression of other opioid receptors. Gene targeting was used to delete exon 1 of the mouse delta-opioid receptor gene and autoradiography was carried out on brains from wild-type, heterozygous and homozygous knockout mice. Delta-opioid receptors were labeled with [(3)H]deltorphin I (7 nM), mu- with [(3)H]DAMGO (4 nM), and kappa- with [(3)H]CI-977 (2.5 nM) or [(3)H]bremazocine (2 nM in the presence of DPDPE and DAMGO) and non-specific binding determined with naloxone. [(3)H]Deltorphin I binding was reduced by approximately 50% in heterozygous animals. In homozygous animals specific binding could only be detected after long-term film exposure (12 weeks). Regions exhibiting this residual [(3)H]deltorphin I binding correlated significantly with those demonstrating high levels of the mu-receptor and were abolished in the presence of the mu-agonist DAMGO. Autoradiographic mapping showed significant overall reductions in [(3)H]DAMGO and [(3)H]CI-977 binding throughout the brain following loss of both copies of the Oprd1 gene. In contrast, overall levels of [(3)H]bremazocine binding were higher in brains from -/- than +/+ mice. Our findings suggest that residual [(3)H]deltorphin I binding in the brain of delta-receptor gene knockout mice is the result of cross-reactivity with mu-sites and that there are no delta-receptor subtypes derived from a different gene. Changes in mu- and kappa-receptor labeling suggest compensatory changes in these subtypes in response to the absence of the delta-receptor. The differences in [(3)H]CI-977 and [(3)H]bremazocine binding indicate these ligands show differential recognition of the kappa-receptor.

Animals↗

PubMed2000

Role for matrix metalloproteinase 9 after focal cerebral ischemia: effects of gene knockout and enzyme inhibition with BB-94.

It has been shown recently that matrix metalloproteinases (MMPs) are elevated after cerebral ischemia. In the current study, we investigated the pathophysiologic role for MMP-9 (gelatinase B, EC.3.4.24.35) in a mouse model of permanent focal cerebral ischemia, using a combination of genetic and pharmacologic approaches. Zymography and Western blot analysis demonstrated that MMP-9 protein levels were rapidly up-regulated in brain after ischemic onset. Reverse transcription polymerase chain reaction showed increased transcription of MMP-9. There were no differences in systemic hemodynamic parameters and gross cerebrovascular anatomy between wild type mice and mutant mice with a targeted knockout of the MMP-9 gene. After induction of focal ischemia, similar reductions in cerebral blood flow were obtained. In the MMP-9 knockout mice, ischemic lesion volumes were significantly reduced compared with wild type littermates in male and female mice. In normal wild type mice, the broad spectrum MMP inhibitor BB-94 (batimastat) also significantly reduced ischemic lesion size. However, BB-94 had no detectable protective effect when administered to MMP-9 knockout mice subjected to focal cerebral ischemia. These data demonstrate that MMP-9 plays a deleterious role in the development of brain injury after focal ischemia.

Animals↗

PubMed2000

Refinement of the ipsilateral retinocollicular projection is disrupted in double endothelial and neuronal nitric oxide synthase gene knockout mice.

Development of retinal connections to the superior colliculus (SC) requires an activity dependent refinement process in which axons gradually become restricted to appropriate retinotopic locations. Nitric oxide has been implicated in this process. We tested this possibility by studying the refinement of the ipsilateral retinocollicular projections (IRP) in normal C57-BL/6 mice and in double knockout mice in which the genes for the edothelial and neuronal isoforms of nitric oxide synthase (e, nNOS) were disrupted. Mice aged between P19 and adulthood were perfused 44-48 h after anterograde injections of WGA-HRP into one eye in order to measure the distribution of the labeled IRP. In normal mice, segregation of the IRP was complete at P21, with the ipsilateral projection restricted to the rostro-medial SC. By contrast, the ipsilateral projection was spread over much more of the SC in double e, nNOS knockouts at P21 with patches of label distributed across the entire medio-lateral axis of the rostral 700 microm. Although the distribution of the ipsilateral projection became more restricted in knockout animals at later ages, it was still more extensive than that of normal mice of the same age at P28 and P42. In the adult, the distribution of axons was similar in both normal and double knockout animals. These results show that refinement of the IRP is delayed when expression of eNOS and nNOS is disrupted, presumably to axons with uncorrelated activity because nitric oxide serves as a repellant molecule during normal development.

Animals↗

PubMed2025

Genetic Engineering and Screening Using Base Editing and Inducible Gene Knockout.

Genetic engineering and screening in human cells are powerful techniques for the precise and comprehensive identification and analysis of gene and protein domain functions. Genome-wide knockout screens have been extensively utilized to discover essential genes, tumor suppressors, and genes that regulate responses to various chemicals, including antimitotic and therapeutic drugs. The advent of base editors, which facilitate the targeted mutation of single amino acids, has advanced the identification of critical and functional domains or motifs. In this context, we outline methods for creating efficient base editor and inducible knockout cell lines for targeted gene manipulation and conducting genetic screens to elucidate the roles of genes and their domains within a specific cell biological context.

Humans↗

PubMed2006

Effects of ergot alkaloids on food preference and satiety in rabbits, as assessed with gene-knockout endophytes in perennial ryegrass (Lolium perenne).

Neotyphodium species are fungal endophytes best known for their protection of grass hosts and production of bioactive metabolites including ergot alkaloids. Perennial ryegrass-Neotyphodium sp. Lp1 symbiota that have altered ergot alkaloid profiles (resulting from knockouts in two different endophyte genes) were fed, along with controls, to rabbits to test the effects of ergot alkaloids on food preference and satiety. Interestingly, rabbits dramatically preferred plants that were endophyte-infected but free of ergot alkaloids over endophyte-free plants (P = 0.01). Accumulation of ergot alkaloids of the clavine class counteracted the added appeal of endophyte-infected plants. In satiety tests, consumption of ergovaline (the ultimate ergot pathway product in wild-type endophyte), but not of several other ergot alkaloids, during an initial meal had a negative effect on subsequent rabbit chow consumption (P < 0.05). The data indicate that clavines were sufficient to reduce the appeal of endophyte-infected grasses, whereas only ergovaline reduced appetite.

Animals↗

PubMed2002

Heavy-ion-induced mutations in the gpt delta transgenic mouse: effect of p53 gene knockout.

The influence of the loss of p53 gene on heavy-ion-induced mutations was examined by constructing a new line of transgenic mice, p53 knockout (p53(-/-)) gpt delta. In this mouse model, deletions in lambda DNA integrated into the mouse genome are preferentially selected as Spi(-) phages, which can then be subjected to molecular analysis. Mice were exposed to 10 Gy of whole-body carbon-ion irradiation. The carbon ions were accelerated to 135 MeV/u by the RIKEN Ring Cyclotron. The p53 defect markedly enhanced the Spi(-) mutant frequency (MF) in the kidneys of mice exposed to C-ion irradiation: the Spi(-) MF increased 4.4- and 2.8-fold over the background level after irradiation in p53(-/-) and p53(+/+) mice, respectively. There was no significant difference in the background Spi(-) MF between p53(-/-) and p53(+/+) mice. Sequence analysis of the Spi(-) mutants indicated that the enhancement of kidney Spi(-) MF in p53(-/-) mice was primarily due to an increase in complex or rearranged-type deletions. In contrast to the kidney, the p53 defect had no effect on the Spi(-) MF in liver: Spi(-) MF increased 3.0- and 2.7-fold after the irradiation in p53(-/-) and p53(+/+) mice, respectively. Our results suggest that p53 suppresses deletion mutations induced by heavy-ion irradiation in an organ-specific manner.

Animals↗

PubMed2005

Histochemical characteristics of soleus muscle in angiotensin-converting enzyme gene knockout mice.

We examined the histochemical characteristics of soleus muscle in the angiotensin-converting enzyme (ACE) gene (Ace in mice, ACE in humans) knockout mice. Serial sections of soleus muscle of wild-type (Ace+/+, n=20) and heterozygous mutant (Ace+/-, n=24) mice were stained for myosin adenosine triphosphatase activity to identify different muscle fiber types. Capillaries were visualized by amylase-periodic acid-Schiff staining. ACE activity in the serum and gastrocnemius muscle was higher in male mice than in female mice. Female and male Ace+/- mice had markedly lower ACE activity in the serum and the gastrocnemius muscle than did female and male Ace+/+ mice, respectively. In both male and female mice, the composition of fiber types (type I and IIa) did not differ significantly between Ace+/+ and Ace+/- mice. There was no significant gender difference in capillary density. Ace+/- mice had significantly more capillaries around type IIa fibers (5.44 +/- 0.18 vs. 5.01 +/- 0.13, p<0.05) than Ace+/+ mice. The differences in the number of capillaries around type I fibers and in the number of capillaries around per fiber (capillary:fiber ratio) between Ace+/- and Ace+/+ mice were not significant (p<0.1). There was no significant difference in the mean cross-sectional area occupied by one capillary and the number of capillaries per fiber area between Ace+/+ and Ace+/- mice. In conclusion, knockout of the Ace gene in mice increased capillary density, as expressed by the mean number of capillaries around type IIa fibers. This finding suggests a possible mechanism for the cardioprotective effects of ACE inhibitors.

Angiotensin-Converting Enzyme Inhibitors↗

PubMed1996

Physiological roles for parathyroid hormone-related protein: lessons from gene knockout mice.

A null mutation in the PTHrP gene produces profound abnormalities in endochondral bone formation in vivo. The role of PTHrP as a modulator of the chondrocytic proliferation and differentiation program is evident in the alterations that occur in its absence in the temporal and spatial sequence of chondrocyte development and subsequent endochondral bone formation that is necessary for normal bone elongation. These actions of PTHrP are probably responsible for the delay in chondrocyte development seen in Jansen osteochondrodystrophy, a disease caused by ligand-independent activation of the PTH-PTHrP receptor (Schipani et al., 1995). Furthermore, these conclusions have been corroborated by the observation that chondrocyte-specific overexpression of PTHrP causes a profound delay in the developmental program of chondrocyte differentiation and endochondral ossification (Weir et al., 1995). The morphological abnormalities in the knockout mice were limited to the skeletal system, despite the widespread production of PTHrP during fetal development. At this point, one can only speculate about the limited tissue distribution of the abnormalities. It is possible, for example, that other gene products, such as PTH, can compensate for the loss of PTHrP in some tissues. Alternatively, possible abnormalities in proliferation and differentiation may be present but morphologically subtle. A molecular assessment of these possible actions may well reveal more widespread effects of PTHrP.

Animals↗

PubMed2000

Homologous gene knockout in the archaeon Halobacterium salinarum with ura3 as a counterselectable marker.

To facilitate the functional genomic analysis of an archaeon, we have developed a homologous gene replacement strategy for Halobacterium salinarum based on ura3, which encodes the pyrimidine biosynthetic enzyme orotidine-5'-monophosphate decarboxylase. H. salinarum was shown to be sensitive to 5-fluoroorotic acid (5-FOA), which can select for mutations in ura3. A spontaneous 5-FOA-resistant mutant was found to contain an insertion in ura3 and was a uracil auxotroph. Integration of ura3 at the bacterioopsin locus (bop ) of this mutant restored 5-FOA sensitivity and uracil prototrophy. Parallel results were obtained with a Deltaura3 strain constructed by gene replacement and with derivatives of this strain in which ura3 replaced bop. These results show that H. salinarum ura3 encodes functional orotidine-5'-monophosphate decarboxylase. To demonstrate ura3-based gene replacement, a Deltabop strain was constructed by transforming a Deltaura3 host with a bop deletion plasmid containing a mevinolin resistance marker. In one approach, the host contained intact ura3 at the chromosomal bop locus; in another, ura3 was included in the plasmid. Plasmid integrants selected with mevinolin were resolved with 5-FOA, yielding Deltabop recombinants at a frequency of > 10-2 in both approaches. These studies establish an efficient new genetic strategy towards the systematic knockout of genes in an archaeon.

Amino Acid Sequence↗