Inactivation of G-protein genes: double knockout in cell lines.
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Animals↗
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Results for “Gene knockout”
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New players in the 5-HT receptor field: genes and knockouts.
In recent years, cloning has revealed the existence of no less than 14 mammalian 5-HT receptors, as well as evidence that isoforms of the 5-HT4 and 5-HT7 receptors are generated by alternative splicing. In addition, molecular biology techniques have provided new tools with which to study the function of 5-HT receptors. For example, new technologies are emerging that will allow the generation of either inducible or tissue-specific knockouts (or both). In this review José Lucas and René Hen focus on the characteristics of the most recently cloned receptors and the contribution of molecular biology to the understanding of 5-HT receptor function.
Animals↗
Gene discovery and gene function assignment in filamentous fungi.
Filamentous fungi are a large group of diverse and economically important microorganisms. Large-scale gene disruption strategies developed in budding yeast are not applicable to these organisms because of their larger genomes and lower rate of targeted integration (TI) during transformation. We developed transposon-arrayed gene knockouts (TAGKO) to discover genes and simultaneously create gene disruption cassettes for subsequent transformation and mutant analysis. Transposons carrying a bacterial and fungal drug resistance marker are used to mutagenize individual cosmids or entire libraries in vitro. Cosmids are annotated by DNA sequence analysis at the transposon insertion sites, and cosmid inserts are liberated to direct insertional mutagenesis events in the genome. Based on saturation analysis of a cosmid insert and insertions in a fungal cosmid library, we show that TAGKO can be used to rapidly identify and mutate genes. We further show that insertions can create alterations in gene expression, and we have used this approach to investigate an amino acid oxidation pathway in two important fungal phytopathogens.
Alleles↗
Observations regarding the effect of targeted gene deletions (knockouts) on graft rejection.
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Animals↗
Involvement of the arginine repressor in lysine biosynthesis of Thermus thermophilus.
Lysine biosynthesis of Thermus thermophilus proceeds in a similar way to arginine biosynthesis, and some lysine biosynthetic enzymes from T. thermophilus so far investigated have the potential to function in arginine biosynthesis. These observations suggest that arginine might regulate the expression of genes for lysine biosynthesis. To test this hypothesis, the argR gene encoding the regulator of arginine biosynthesis was cloned from T. thermophilus and its function in lysine biosynthesis was analysed. The addition of arginine to the culture medium inhibited the growth of an arginase gene knockout mutant of T. thermophilus, which presumably accumulates arginine inside the cells. Arginine-dependent growth inhibition was not alleviated by the addition of ornithine, which is a biosynthetic intermediate of arginine and serves as a peptidoglycan component of the cell wall in T. thermophilus. However, the growth inhibition was cancelled either by the simultaneous addition of lysine and ornithine or by a knockout of the argR gene, suggesting the involvement of argR in regulation of lysine biosynthesis in T. thermophilus. Electrophoretic mobility shift assay and DNase I footprinting revealed that the ArgR protein specifically binds to the promoter region of the major lysine biosynthetic gene cluster. Furthermore, an alpha-galactosidase reporter assay for this promoter indicated that arginine repressed the promoter in an argR-dependent manner. These results indicate that lysine biosynthesis is regulated by arginine in T. thermophilus.
Arginase↗
Inhibition of L-deleted foot-and-mouth disease virus replication by alpha/beta interferon involves double-stranded RNA-dependent protein kinase.
We previously demonstrated that the ability of foot-and-mouth disease virus (FMDV) to form plaques in cell culture is associated with the suppression of alpha/beta interferon (IFN-alpha/beta). In the present study, we used Escherichia coli-expressed porcine and bovine IFN-alpha or -beta individually to demonstrate that each was equally effective in inhibiting FMDV replication. The block in FMDV replication appeared to be at the level of protein translation, suggesting a role for double-stranded RNA-dependent protein kinase (PKR). In support of these findings, treatment of porcine and bovine cells with 2-aminopurine, an inhibitor of PKR, increased the yield of virus 8.8- and 11.2-fold, respectively, compared to that in untreated infected cells. In addition, results of FMDV infection in mouse embryonic fibroblast cells derived from gene knockout mice lacking the gene for RNase L(-/-) or PKR(-/-) or both indicated an important role for PKR in the inhibition of FMDV replication.
Animals↗
Cytokines score a knockout. Harnessing gene targeting to gain insight into the pathogenesis of myocarditis.
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Animals↗
Oestrogen at the neonatal stage is critical for the reproductive ability of male mice as revealed by supplementation with 17beta-oestradiol to aromatase gene (Cyp19) knockout mice.
Aromatase P450 (CYP19) is an enzyme responsible for the conversion of androgens to oestrogens. We generated CYP19 knockout (ArKO) mice by targeted disruption of Cyp19 and studied the role of oestrogens in male reproductive ability. Approximately 85% of ArKO males were unable to sire offspring. However, no obvious difference was found in testicular and epididymal weights, numbers of sperm in the epididymis or the ability of sperm to fertilize eggs in vitro between wild-type and ArKO males. An examination of mating behaviour demonstrated that ArKO males showed an impairment in mounting behaviour against sexually mature females. The inability of more than 90% of ArKO males to sire offspring was reversed by repeated subcutaneous injections of 17beta-oestradiol when initiated on the day of birth. The effects of 17beta-oestradiol on reproduction were concentration dependent and evident when supplementation was initiated on day 7, but not on day 15 after birth. These findings suggest that oestrogens acting during neonatal life are required for normal mating behaviour in adulthood.
Age Factors↗
Antisense oligonucleotide drug design.
Maneuvering single gene expression is not only an optimal way to study gene function but also an ambitious goal, which will lead to the treatment of a variety of human diseases whose main pathogenetic event is a genetic alteration. The recent efforts focusing on the genome project have led to array based, high throughput, gene expression analysis techniques that allow the study of complex molecular networks. Combining these powerful new technologies with modulation of gene expressions is making it possible to unravel complex molecular networks or, vice versa, to find new gene products responsible for pathological conditions on which exogenous modulation can be productive. Efficient and specific modulation of gene expression can be obtained either by producing transgenic or gene knockout organisms or cells (gene targeting), or by treating organisms or cells with short synthetic nucleic acid segments in antisense orientation with respect to the targeted mRNAs (mRNA targeting by antisense strategy). While genome manipulation is a time consuming and expensive approach, requiring invasive intervention, the "antisense strategy" is characterized by high flexibility resulting from safeness, specificity, reversibility, modulability, and low cost. The rationale of the antisense strategy is that, once one gene sequence is known, its expression can be silenced by application of synthetic single-strand nucleic acid segments (oligonucleotides) whose sequence is in antisense orientation compared to the targeted mRNA. Recently, this "informational" strategy has been boosted by the discovery of the RNA interference: a natural mechanism by which cells are thought to fight detrimental exogenous viruses and endogenous transposons. Despite promising futures, antisense-based therapeutics are far from being an established reality. This review analyses the recent improvements in antisense-based gene expression modulation, focuses on the treatment of diseases in the light of the past, and provides our personal findings on this topic.
Animals↗
Molecular characterization of the operon comprising the spoIV gene of Bacillus megaterium DSM319 and generation of a deletion mutant.
According to sequence analysis, the spoIV-locus of Bacillus megaterium DSM 319 is 1,185 bp long; it is the second gene of a sporulation operon, which altogether contains three open reading frames. The ORF preceding spoIV encodes a putative polypeptide with 94 amino acids; the 3rd ORF of the operon has 972 bp corresponding to 324 amino acids. The operon is flanked on both sides by palindromic sequences, probably representing Rho-independent terminators. A primer extension analysis revealed that mRNA synthesis starts immediately downstream of a promoter, which is similar to the consensus sequence of Bacillus subtilis sigma(E) dependent promoters. Both the -35 and the -10 region are within the terminator region of the preceding operon. Gene knockout experiments and reporter gene assays with a newly developed system based on the heterologous Paenibacillus macerans glucanase gene (bgl) confirmed sigma(E)-dependent transcription. Two open reading frames of a further upstream operon were also identified. Northern analysis revealed that transcription of these ORFs comes about in late sporulation phases. The genetic organization of the spoIV comprising operon and adjacent loci clearly resembles that of the B. subtilis yqfa-phoH gene cluster. Thus our findings are of general significance for endospore-forming bacteria.
Journal Article↗
Uridine phosphorylase (-/-) murine embryonic stem cells clarify the key role of this enzyme in the regulation of the pyrimidine salvage pathway and in the activation of fluoropyrimidines.
We have reported the elevation of uridine phosphorylase (UPase) in many solid tumors and the presence of a variant phosphorolytic activity in breast cancer tissues (M. Liu et al., Cancer Res., 58: 5418-5424, 1998). To better understand the biological and pharmacological significance of these findings, we have developed an UPase gene knockout embryonic stem (ES) cell model by specific gene targeting techniques. In this cellular model, we establish the critical role of UPase as an important anabolic enzyme in 5-fluorouracil (5-FU) activation and pyrimidine salvage pathway regulation. It has long been known that UPase regulates the plasma concentration of uridine; however, little is known of the role of UPase in the activation and metabolism of 5-FU and its derivatives, mainly because of the lack of an appropriate model system. The experimental data indicate that the disruption of UPase activity in murine ES cells leads to a 10-fold increase in 5-FU IC(50) and a 2-3-fold reduction in its incorporation into nucleic acids, whereas no differences in toxicity is seen with other pyrimidine nucleoside analogues such as 5-fluorouridine, 2'-deoxy-5-fluorouridine, and 1-beta-D-arabinofuranosylcytosine compared with WT (wild-type) ES cells. Benzylacyclouridine can specifically prevent the WT ES cells from the sensitivity of 5-FU. Our data also shows the effect of UPase on the cytotoxicity of 5'-deoxy-5-fluorouridine (5'DFUR), a 5-FU prodrug. The IC(50) is increased almost 16-fold in the knockout cells compared with the wild type cells, demonstrating the role of UPase in catalyzing the conversion of 5'DFUR to 5-FU. These findings additionally elucidate the tumor-specific selectivity of capecitabine, the oral fluoropyrimidine prodrug approved for the treatment of metastatic breast and colorectal cancers. Not only do the knockout cells present a decreased incorporation of 5-FU into nucleic acids but also an increased reliance on the pyrimidine salvage pathway. The reduced dependence of UPase knockout cells on the pyrimidine de novo synthesis is reflected in the apparent resistance to phosphonacetyl-L-aspartic acid, a specific inhibitor of pyrimidine pathway, with a 5-fold elevation in its IC(50) in UPase-nullified cells compared with WT. In summary, we have successfully generated an UPase gene knockout cell model that presents reduced sensitivity to 5-FU, 5'DFUR, and phosphonacetyl-L-aspartic acid, although it does not affect the basic cellular physiology under normal tissue culture conditions. Considering the role of UPase in 5-FU metabolism and the elevated expression of this protein in cancer cells compared with paired normal tissues, additional investigation should be warranted to firmly establish the clinical role of UPase in the tumor selective activation of 5-FU and capecitabine.
Animals↗
Lipopolysaccharide aggravates cerebral pathology in B10.PL-derived CD1-/-, beta2m-/-, TCRalpha-/-, and TCRdelta-/- knockout mice.
Adult B10.PL-derived immunological genes knockout mice injected with 100 microg lipopolysaccharide (LPS) showed severe hydrocephalus and meningitis. A consequence of the hydrocephalus is pineal hyperplasia, sponginess of periventricular parenchyma, gliosis and, at the last stage of hydrocephalus formation, disappearance of the ependymal layer and the Gomori-positive subependymal astrocytes. Possible mechanisms for the aggravation of cerebral pathology induced by LPS are discussed.
Animals↗
An evaluation of what the mouse knockout experiments are telling us about mammalian behaviour.
The early gene knockout studies with a neurobiological focus were directed at fairly obvious target genes and added very little to our knowledge of behavioural neuroscience. On the contrary, since the behavioural consequences were often predictable, this helped confirm that the technology was working. However, a substantial number of knockouts of genes expressed in the brain have been without obvious behavioural consequences, supporting the concept of genetic canalisation and redundancy. Others have produced a behavioural deficit for which there is no obvious explanation. Many cells of different tissue types have a capacity for memory, and in the brain, cells of the hippocampus are important for spatial learning and memory. Deleting genes that are expressed in the hippocampus has received considerable attention in this behavioural context. Although the initial studies experienced problems of interpretation, considerable advances have since been made. Knockout mice are now subject to tests of different forms of learning, multicellular hippocampal recordings, and restricted gene deletion specific to cells of component regions. This multi-level approach is proving more informative. Nevertheless, there is still a need to recognise that behavioural expression is several steps removed from gene expression, and that the relationship between genes and behaviour can be reciprocal.
Animals↗
Gene targeting in mosquito cells: a demonstration of 'knockout' technology in extrachromosomal gene arrays.
BACKGROUND: Gene targeting would offer a number of advantages over current transposon-based strategies for insect transformation. These include freedom from both position effects associated with quasi-random integration and concerns over transgene instability mediated by endogenous transposases, independence from phylogenetic restrictions on transposon mobility and the ability to generate gene knockouts. RESULTS: We describe here our initial investigations of gene targeting in the mosquito. The target site was a hygromycin resistance gene, stably maintained as part of an extrachromosomal array. Using a promoter-trap strategy to enrich for targeted events, a neomycin resistance gene was integrated into the target site. This resulted in knockout of hygromycin resistance concurrent with the expression of high levels of neomycin resistance from the resident promoter. PCR amplification of the targeted site generated a product that was specific to the targeted cell line and consistent with precise integration of the neomycin resistance gene into the 5' end of the hygromycin resistance gene. Sequencing of the PCR product and Southern analysis of cellular DNA subsequently confirmed this molecular structure. CONCLUSIONS: These experiments provide the first demonstration of gene targeting in mosquito tissue and show that mosquito cells possess the necessary machinery to bring about precise integration of exogenous sequences through homologous recombination. Further development of these procedures and their extension to chromosomally located targets hold much promise for the exploitation of gene targeting in a wide range of medically and economically important insect species.
Aedes↗
Establishing insulin 1 and insulin 2 knockout congenic strains on NOD genetic background.
As insulin is a major autoantigen in autoimmune diabetes and because the insulin gene region locus in humans has been linked to diabetes risk, we have bred insulin gene knockouts onto the NOD mouse. Mice differ from humans in terms that they express two nonallelic genes of insulin. Insulin 2 is the murine homologue of the human insulin gene and is located on mouse chromosome 7. Insulin 1 is thought to have evolved by a gene duplication event, lacks the second intron of the insulin 2 gene, and is located on mouse chromosome 19. The differential thymic expression of the insulin gene may be important for central tolerance induction. Here, we present the initial establishment of congenic knockouts and characterization of the congenic intervals corresponding to insulin 1 and insulin 2 knockout genes on mouse chromosome 19 and 7, respectively.
Animals↗
Genetically modified animal models as tools for studying bone and mineral metabolism.
Genetic modification of mice is a powerful tool for the study of bone development and metabolism. This review discusses the advantages and disadvantages of various approaches used in bone-related research and the contributions these studies have made to bone biology. Genetic modification of mice is a powerful tool for the study of bone development and metabolism. This review discusses the advantages and disadvantages of various approaches used in bone-related research and the contributions these studies have made to bone biology. The approaches to genetic modification included in this review are (1) overexpression of genes, (2) global gene knockouts, (3) tissue-specific gene deletion, and (4) gene knock-in models. This review also highlights issues that should be considered when using genetically modified animal models, including the rigorous control of genetic background, use of appropriate control lines, and confirmation of tissue specificity of gene expression where appropriate. This technology provides a unique and powerful way to probe the function of genes and is already revolutionizing our approach to understanding the physiology of bone development and metabolism.
Animals↗
Conditional genome alteration in mice.
The recent ability to inactivate specific genes in mice has significantly accelerated our understanding of molecular, cellular, and even behavioral aspects of normal and disease processes. However, this ability has also demonstrated the extreme complexity of genetic determination in mammals, in particular, that genes in the same family or pathway can be functionally redundant and that a given gene often has multiple roles. Thus, inactivation of a gene often does not indicate its complete spectrum of functions. To circumvent this problem, many new tools and novel applications of classic techniques have been developed to place spatial and temporal restrictions on the genomic alterations. These approaches include chimera and mosaic studies, organ transplantation, complementation assays, dominant negative mutants, conditional gene knockouts, and lineage-specific gene rescue. Not only has this opened up more sophisticated ways to make genomic alterations, but it has provided the opportunity to create animal models for sporadic human genetic diseases.
Animals↗
Construction, detection and microarray analysis on the Shigella flexneri 2a sitC mutant.
In order to overcome the defects of difficult gene operations in low-copy suicide plasmid pCVD442, Gateway technology was applied in the construction process of recombinant plasmid for gene knockout in this study. With this improved knockout system, we inactivated sitC gene, which is associated with iron transport in Shigella flexneri 2a strain 301, to yield the mutant, MTS. The functional detection of the mutant was performed at the level of culture medium, cell and animal experiment, respectively. The gene expression profiles were compared with DNA microarray between the mutant and the wild type under iron-restricted conditions. The results showed that MTS grew obviously less well than the wild-type strains in L broth containing 150 micromol/L iron chelator DIP (2,2'-dipyridyl). Addition of iron or manganese to the cultures stimulated the growth of MTS to wild-type levels in rich culture medium. In either the experiment on the ability of intracellular multiplication and cell-to-cell spread in HeLa and U937 cell lines, or the experiment on keratoconjunctivitis in guinea pigs, MTS showed no obvious changes in virulence compared with the parental strain Sf301. When 65 micromol/L DIP was added to the cultured HeLa cells, the ability of intracellular multiplication of MTS reduced about 51.6% as compared with that of Sf301. The analysis of expression profiles under iron-limited condition showed that MTS was more sensitive for the change of iron deficiency than Sf301. There are 106 more up-regulated genes in MTS than in wild-type strains, which are involved in membrane transportation, amino acid metabolism and uncategorized function genes, while down-regulated genes are mainly involved in energy and carbohydrate metabolism. Under low iron conditions, the expression levels of known iron-transport associated genes generally increased. Additionally, the number of these genes and their increase amplitude in MTS are more than those in Sf301. Together, these results confirmed that Sit iron-transport system is important for the growth of Shigella.
ATP-Binding Cassette Transporters↗