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PubMed2006

Regulation of gene expression in mouse embryos and its embryonic cells through RNAi.

The ability to direct cell differentiation of embryo-derived cells should have considerable practical applications. A prerequisite for success is the availability of robust tools that allows efficient knockdown of specific genes to elucidate gene function and direct differentiation. Loss-of-function studies in the mouse have traditionally been carried out using gene knockout animals to obtain mutant embryonic cells. Despite improvements in this technology, it still remains a laborious method. In addition, the use of knockout for studying preimplantation embryonic cells is limited due to maternal contributions. RNA interference (RNAi) is a powerful genetic approach for specific gene silencing of target genes in which 21-23 nt small interfering RNAs work as sequence-specific RNAi mediators. Using RNAi approaches, gene function has been eliminated in mouse embryonic stem cells and carcinoma cells to direct cell differentiation. In mouse oocytes and embryos, RNAi eliminated both maternal and zygotic transcripts and disturbed normal development.

Animals↗
PubMed1997

Adenovirus-mediated transfer of a functional GAD gene into nerve cells: potential for the treatment of neurological diseases.

The recent development of efficient virus-mediated gene transfer into nerve cells allows the prospect of new strategies for the treatment of drug-resistant neurological diseases. Some forms of epilepsy may be amenable to gene therapy. Although there is no obvious candidate gene, the consensual GABA hypothesis of epilepsy suggests that the GAD gene may be beneficial. GAD gene expression may be useful in supplying the inhibitory neurotransmitter GABA to particular critical brain territories. We show herein that a nonreplicative recombinant adenovirus carrying the GAD67 gene under the control of Rous sarcoma virus long terminal repeat promoter is able to express the transgene in primary cultures of neurons and glial cells. Expression of the GAD67 gene was assessed by immunoblotting and immunohistochemical analysis. We demonstrated the functionality of the transgene, the expression of which resulted in production of large amounts of GABA in neuronal and glial cell cultures. Substantial production of the enzyme was also detected for several weeks in infected organotypic slices cultured from new-born rat hippocampal tissues. The virally encoded GAD67 was also expressed in vivo in various brain areas involved in various neurological disorders and thus may be of value for the development of gene therapies.

Adenoviridae↗
PubMed2005

Wax constituents on the inflorescence stems of double eceriferum mutants in Arabidopsis reveal complex gene interactions.

To shed new light on gene involvement in plant cuticular-wax production, 11 eceriferum (cer) mutants of Arabidopsis having dramatic alterations in wax composition of inflorescence stems were used to create 14 double cer mutants each with two homozygous recessive cer loci. A comprehensive analysis of stem waxes on these double mutants revealed unexpected CER gene interactions and new ideas about individual CER gene functions. Five of the 14 double cer mutants produced significantly more total wax than one of their respective cer parents, indicating from a genetic standpoint a partial bypassing (or complementation) of one cer mutation by the other. Eight of the 14 double cer mutants had alkane amounts lower than both respective cer parents, suggesting that most of these CER gene products play a major additive role in alkane synthesis. Other results suggested that some CER genes function in more than one step of the wax pathway, including those associated with sequential steps in acyl-CoA elongation. Surprisingly, complete epistasis was not observed for any of the cer gene combinations tested. Significant overlap or redundancy of genetic operations thus appears to be a central feature of wax metabolism. Future studies of CER gene product function, as well as the utilization of CER genes for crop improvement, must now account for the complex gene interactions described here.

Arabidopsis↗
PubMed2001

Efficient gene transfer into the embryonic mouse brain using in vivo electroporation.

Mouse genetic manipulation has provided an excellent system to characterize gene function in numerous contexts. A number of mutants have been produced by using transgenic, gene knockout, and mutagenesis techniques. Nevertheless, one limitation is that it is difficult to express a gene in vivo in a restricted manner (i.e., spatially and temporally), because the number of available enhancers and promoters which can confine gene expression is limited. We have developed a novel method to introduce DNA into in/exo utero embryonic mouse brains at various stages by using electroporation. More than 90% of operated embryos survived, and more than 65% of these expressed the introduced genes in restricted regions of the brain. Expression was maintained even after birth, 6 weeks after electroporation. The use of fluorescent protein genes clearly visualized neuronal morphologies in the brain. Moreover, it was possible to transfect three different DNA vectors into the same cells. Thus, this method will be a powerful tool to characterize gene function in various settings due to its high efficiency and localized gene expression.

Animals↗
PubMed2002

Expressed sequence tag (EST) analysis of a Schistosoma japonicum cercariae cDNA library.

Expressed sequence tags (ESTs) constitute a rapid and informative strategy for studying gene-expression profiles of specific stages of schistosomes. To date, only approximately equal 2000 ESTs of Schistosoma japonicum have been deposited in databases. This is insufficient to understand the biology and development of this species. In this report, a cDNA library constructed from S. japonicum cercariae RNA was used to generate ESTs. Cercariae are the larval forms of Schistosoma responsible for infection of the vertebrate host and one of the main objectives of this research was to discover and characterize new and unique genes from this stage. The expression products of those stage-specific genes can potentially be useful as new drugs or vaccine targets applicable for controlling Asian schistosomiasis. In our study, 101 cDNA clones were sequenced either from 5' or 3' end of the cDNAs. Some 42 ESTs (42%) matched known genes, while 59 ESTs did not match with any known genes. Among the 42 former ESTs, 29 (informative ESTs) matched to functional genes and 13 matched with ribosomal proteins or RNA genes. Among the latter 59 ESTs, 21 matched with published ESTs of S. japonicum or S. mansoni, two matched with human ESTs and the other 18 did not match with any published sequences. The informative ESTs could be grouped into nine categories: regulatory and signaling proteins (24.1%), transcription and translation machinery proteins (13.8%), RNA binding proteins (6.9%), structural and cytoskeletal proteins (6.9%), DNA binding proteins (3.4%), DNA scaffold proteins (3.4%), transporter proteins (3.4%) and others (31%). Some functional genes relevant to the physiology of cercariae are discussed.

Animals↗
PubMed1995

Calcium transport proteins in the nonfailing and failing heart: gene expression and function.

In heart failure alterations of intracellular Ca2+ handling are thought to be a major reason for impaired contraction and relaxation. Peak Ca2+ transients are reduced, resting Ca2+ levels elevated, and the time course of diastolic Ca2+ decline is markedly prolonged in failing hearts. The proteins of the sarcoplasmic reticulum and the sarcolemmal Na+/Ca2+ exchanger are the most important tools for Ca2+ homeostasis in the cardiomyocyte, and their molecular cloning has allowed prediction of structure/function analysis. The investigation of function and gene expression of these proteins in failing myocardium has been an area of intensive research in recent years in order to provide a more detailed understanding of the pathophysiology of heart failure. Quantitative changes in expression of the sarcoplasmic reticulum Ca(2+)-ATPase, the ryanodine receptor, and the Na+/Ca2+ exchanger with correlations to functional alterations have been reported both in experimental animal models and in the human failing heart. However, in human heart failure these findings are currently the subject of a lively discussion because observations have apparently been in part contradictory. This review discusses the proteins involved in myocardial Ca2+ handling and describes the current state of research on expressional and functional alterations and their potential implication in the pathomechanism of heart failure.

Animals↗
PubMed2006

Directing RNA interference specifically to differentiated muscle cells.

A common approach for mediating RNA interference (RNAi) is to introduce DNA that encodes short hairpin RNA (shRNA), which is often contained in a plasmid that can express a shRNA in a wide variety of cell types. Muscle cells and certain other cell types grown in culture can exist in both a dividing state and in a post-mitotic, differentiated state, and it is sometimes useful to induce RNAi selectively in terminally differentiated cells to study the function of a gene, particularly when the gene is also required for propagation of dividing cells. We describe two methods for studying gene function by RNAi specifically in terminally differentiated skeletal muscle cells in culture. We developed a shRNA expression vector, based on myosin light chain 1f gene regulatory sequences, which is designed to induce shRNA expression specifically after differentiation has been initiated. We show that this vector can mediate RNAi and is only active in differentiated muscle cells. Also, we developed an adenoviral vector that is designed to be able to deliver shRNAs directly to post-mitotic muscle cells. We show that adenoviruses produced using this vector mediate RNAi in differentiated muscle cells. These methods add to the repertoire of RNAi tools that can be used for identifying genes involved in any event of interest that occurs in differentiated muscle cells.

Animals↗
PubMed2005

Functional genomics using high-throughput RNA interference.

RNA interference (RNAi) describes the post-transcriptional silencing of gene expression that occurs in response to the introduction of double-stranded RNA into cells. Application of RNAi in experimental systems has provided a great leap forward in the elucidation of gene function. To facilitate large-scale functional genomics studies using RNAi, several high throughput approaches have been developed based on microarray or microwell assays. Recent establishment of large libraries of RNAi reagents combined with a variety of detection assays further opens the door for genome-wide screens of gene function in mammalian cells.

Animals↗
PubMed2002

Gene targeting of tissue factor, factor X, and factor VII in mice: their involvement in embryonic development.

Inactivation of specific genes in mammals by gene targeting has accelerated our ability to determine gene function. Nearly all genes involved in the blood coagulation system have been knocked out in mice. Tissue factor (TF) is the main initiator of the coagulation system and functions as a cell surface receptor for coagulation factor VII (FVII). Knockout studies have shown that TF deficiency results in lethality around embryonic day (E) 8.5-10.5. The results suggest a role for TF in embryonic blood vessel development and maintenance of vascular integrity in the yolk sac. In addition, TF may be involved in the maintenance of the placental labyrinth. Factor X (FX) deficiency causes partial embryonic lethality between E11.5-12.5. FX-/- mice that were born died from fatal neonatal bleeding. In contrast, FVII deficiency is not embryonic lethal, but FVII-/- neonates died from hemorrhage within the first days after birth. The various lethal phenotypes of deficiencies of the different coagulation factors suggest involvement in processes beyond hemostasis. Both TF/FVIIa and FXa can trigger intracellular signaling events in certain cell types. Signaling by coagulation proteases and protease-activated receptors (PARs) may have important roles in embryonic development.

Animals↗
PubMed2003

Chemically regulated gene expression in plants.

Chemically inducible systems that activate or inactivate gene expression have many potential applications in the determination of gene function and in plant biotechnology. The precise timing and control of gene expression are important aspects of chemically inducible systems. Several systems have been developed and used to analyze gene function, marker-free plant transformation, site-specific DNA excision, activation tagging, conditional genetic complementation, and restoration of male fertility. Chemicals that are used to regulate transgene expression include the antibiotic tetracycline, the steroids dexamethasone and estradiol, copper, ethanol, the inducer of pathogen-related proteins benzothiadiazol, herbicide safeners, and the insecticide methoxyfenozide. Systems that are suitable for field application are particularly useful for experimental systems and have potential applications in biotechnology.

Copper↗
PubMed2005

RNA interference of dual oxidase in the plant nematode Meloidogyne incognita.

RNA interference (RNAi) is a powerful tool for the analysis of gene function in model organisms such as the nematode Caenorhabditis elegans. Recent demonstrations of RNAi in plant parasitic nematodes provide a stimulus to explore the potential of using RNAi to investigate disruption of gene function in Meloidogyne incognita, one of the most important nematode pests of global agriculture. We have used RNAi to examine the importance of dual oxidases (peroxidase and NADPH oxidase), a class of enzyme associated with extracellular matrix cross-linking in C. elegans. RNAi uptake by M. incognita juveniles is highly efficient. In planta infection data show that a single 4-h preinfection treatment with double-stranded RNA derived from the peroxidase region of a dual oxidase gene has effects on gene expression that are phenotypically observable 35 days postinfection. This RNAi effect results in a reduction in egg numbers at 35 days of up to 70%. The in vitro feeding strategy provides a powerful tool for identifying functionally important genes, including those that are potential targets for the development of new agrochemicals or transgenic resistance strategies.

Amino Acid Sequence↗
PubMed2005

Modulation of gene expression by RNAi.

RNA interference (RNAi) is a form of posttranscriptional gene silencing in which the presence within the cell of double-stranded RNA (dsRNA) leads to the specific degradation of mRNA with a complimentary sequence. RNAi is a natural phenomenon that can be exploited as a powerful tool to study gene function by generating gene "knockdowns" in various cell types. RNAi is mediated by short interfering RNAs (siRNAs), which are generated within cells from long dsRNAs. To avoid generalized toxic effects, mammalian cells are transfected directly with 21-23-bp-long siRNAs generated either by chemical synthesis or obtained by a series of enzymatic reactions. The present chapter deals with siRNA design, synthesis, transfection, and readout of efficiency in a mammalian cell culture system. The general principle is illustrated by the functional knockdown of p97/VCP (valosin-containing protein) in HeLa cells using five different siRNA sequences.

Animals↗
PubMed2006

Linking floral symmetry genes to breeding system evolution.

Understanding the genetic basis of ecologically important traits is a major focus of evolutionary research. Recent advances in molecular genetic techniques should significantly increase our understanding of how regulatory genes function. By contrast, our understanding of the broader macro-evolutionary implications of developmental gene function lags behind. Here we review published data on the floral symmetry gene network (FSGN), and conduct phylogenetic analyses that provide evidence of a link between floral symmetry and breeding systems in angiosperms via dichogamy. Our results suggest that known genes in the FSGN and those yet to be described underlie this association. We posit that the integration of floral symmetry and the roles of other regulatory genes in plant breeding system evolution will provide new insights about macro-evolutionary patterns and processes in flowering plants.

Biological Evolution↗
PubMed1989

Constitutive mutants in the yeast pheromone response: ordered function of the gene products.

The alpha factor pheromone inhibits the division of yeast a cells. A general method was developed for isolating mutants that exhibit constitutive activation of the pheromone response pathway. A dominant allele of the STE4 locus was recovered in addition to recessive mutations in the SCG1 gene. SCG1 and STE4 are known to encode G alpha and G beta homologs, respectively. Analysis of double mutants suggests that the STE4 gene product functions after the SCG1 product but before the STE5 product.

Crosses, Genetic↗
PubMed1994

Avian erythroblastosis virus E26: only one (myb) of two cell-derived coding regions is necessary for oncogenicity.

The oncogene hypothesis postulates that mutated cellular genes, termed proto-onc genes, function as cancer genes because they are related to retroviral onc genes. However, in contrast to retroviral onc genes, mutated proto-onc genes from cancers are not sufficient for carcinogenesis. Therefore, it has been proposed that mutated proto-onc genes depend on other proto-onc genes for carcinogenesis. Since the oncogene of the avian leukemia virus E26 includes coding regions derived from two cellular proto-onc genes, proto-myb and proto-ets, this hybrid gene has been proposed to be a model for two-gene-carcinogenesis. Here we set out to test this proposal. For this purpose myb and ets deletion mutants of cloned E26 provirus were prepared, and the corresponding viruses, produced by transfected primary chicken embryo cells, were tested for leukemogenicity in newborn chickens. It was found that an ets deletion mutant was just as leukemogenic as the wild-type virus and that a myb deletion mutant lacked leukemogenicity completely. To eliminate the possibility that our E26 myb deletion mutant failed to be leukemogenic because it failed to replicate, the virus was titered by a quantitative polymerase chain reaction (PCR) method. By this method, E26 from the plasma of infected chickens was first allowed to reverse-transcribe viral RNA to cDNA in vitro, and then the cDNA concentration was determined from the lowest dilution that gave a positive signal after amplification of E26 cDNA by the PCR method. Virus titers of about 10(5) per ml were found for wild type and for myb and ets deletion mutants of E26. It is concluded that the ets region is not essential for carcinogenesis, and E26 derives transforming function from overexpression of its proto-myb coding region via the retroviral promoter. Thus, E26 is a single-hit carcinogen and, like all other oncogenic retroviruses, is not a model for two-gene-carcinogenesis. Viral ets probably reflects a genetic accident that transduced sequences of proto-ets together with proto-myb in generating E26.

Alpharetrovirus↗
PubMed2006

Gene expression profiles of T lymphocytes are sensitive to the influence of heavy smoking: A pilot study.

Cigarette smoke components have a proven negative influence on human health. Adverse metabolic effects were observed in tissues and single cells. T lymphocytes get in contact with affected organs (e.g., lung) or cells (e.g., erythrocytes), as well as with smoke components and bioactive molecules, whose production is triggered by tobacco smoke. We therefore compared the gene expression profiles in these cells of the adaptive immune system of three male heavy smokers and three male nonsmokers using rapid T cell isolation and Affymetrix GeneChip HG U133A 2.0 microarray analysis. Eighty-eight genes were found to be significantly (t test) differentially expressed by a factor of 1.5-fold or larger between smokers and nonsmokers. Using the gene function groups of the gene ontology consortium to categorize the functions of the differentially expressed genes, the group termed "response to stimulus" was found to be most significantly affected by smoking. Our data indicate a prominent role of cytotoxic T lymphocytes in response to smoking. Several genes that are typically expressed in these cells were found regulated although the ratio of cytotoxic and helper T lymphocytes remained unchanged in smokers. Our data show that, in principle, it might be possible to identify health-related biomarkers in the transcriptome of T lymphocytes.

Adult↗

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