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PubMed1994

Efficient expression of drug-selectable genes in retroviral vectors under control of an internal ribosome entry site.

We describe a new retroviral vector system pSXLC/pHa that utilizes a putative internal ribosome entry site (IRES) from encephalomyocarditis virus downstream from a multicloning site to co-express drug-selectable markers with a second non-selectable cDNA in a eukaryotic expression vector. The positive drug-selectable marker, MDR1, and the positive-negative marker, herpes simplex virus thymidine kinase (HSV-TK), were successfully introduced and expressed in the pSXLC/pHa system. The pSXLC-MDR and pSXLC-TK vectors contain the drug-selectable genes under translational control of the IRES and multiple cloning sites upstream for insertion of second cDNAs which can be co-expressed in this system. The inserts of these pSXLC plasmids were designed for easy transfer to the pHa retrovirus vector which has a strong promoter from Harvey murine sarcoma virus. The IRES-MDR-carrying retroviral vector, pHa-MCS-IRES-MDR, conferred resistance to vincristine and adriamycin. The IRES-TK-containing vector, pHa-MCS-IRES-TK conferred HAT-resistance in TK-deficient cells and the transfectants showed hypersensitivity to ganciclovir. These "flexible" vectors should be useful for co-expression of genes for selectable gene transfer and for positive-negative (suicide) selections in vitro and in vivo.

3T3 Cells↗
PubMed1985

A cosmid for selecting genes by complementation in Aspergillus nidulans: Selection of the developmentally regulated yA locus.

We constructed a 9.9-kilobase cloning vector, designated pKBY2, for isolating genes by complementation of mutations in Aspergillus nidulans. pKBY2 contains the bacteriophage lambda cos site, to permit in vitro assembly of phage particles; a bacterial origin of replication and genes for resistance to ampicillin and chloramphenicol, to permit propagation in Escherichia coli; the A. nidulans trpC(+) gene, to permit selection in Aspergillus; and a unique BamHI restriction site, to permit insertion of DNA fragments produced by digestion with restriction endonucleases BamHI, BglII, Mbo I, or Sau3A. We used this cosmid to form a quasirandom recombinant DNA library containing 35- to 40-kilobase DNA fragments from a wild-type strain of A. nidulans. DNA from this library transformed a yellow-spored (yA(-)) pabaA(-)trpC(-)Aspergillus strain (FGSC237) to trpC(+) at frequencies of approximately 10 transformants per mug of DNA. Three of approximately 1000 trpC(+)pabaA(-) colonies obtained were putative yA(+) transformants, because they produced wild-type (green) spores. DNA from each of the green-spored transformants contained pKBY2 sequences and DNA from two transformants transduced E. coli to ampicillin resistance following treatment in vitro with a lambda packaging extract. The cosmids recovered in E. coli had similar restriction patterns and both yielded trpC(+) transformants of A. nidulans FGSC237, 85% of which produced green spores. Several lines of evidence indicate that the recovered cosmids contain a wild-type copy of the yA gene.

Journal Article↗
PubMed2003

A mathematical programming approach for gene selection and tissue classification.

MOTIVATION: Extracting useful information from expression levels of thousands of genes generated with microarray technology needs a variety of analytical techniques. Mathematical programming approaches for classification analysis outperform parametric methods when the data depart from assumptions underlying these methods. Therefore, a mathematical programming approach is developed for gene selection and tissue classification using gene expression profiles. RESULTS: A new mixed integer programming model is formulated for this purpose. The mixed integer programming model simultaneously selects genes and constructs a classification model to classify two groups of tissue samples as accurately as possible. Very encouraging results were obtained with two data sets from the literature as examples. These results show that the mathematical programming approach can rival or outperform traditional classification methods.

Algorithms↗
PubMed1992

Mutational spectrometry: a general approach for hot-spot point mutations in selectable genes.

To observe point mutational spectra with a high degree of precision, independent large cultures of human lymphoblastoid cells were treated with a mutagen, benzo[a]pyrene diol epoxide, and mutants at the HPRT gene were selected en masse by 6-thioguanine resistance. An average of 1.6 x 10(4) 6-thioguanine-resistant mutants were created per experiment and the kinds, positions, and numbers of the most frequent mutations were examined in exon 3 of the HPRT gene by using a high-fidelity polymerase chain reaction and denaturing gradient gel electrophoresis. Sixteen exon 3-specific mutations were found to be predominantly G----T transversions and corresponded to an average of 3500 induced mutants per experiment. Of these mutations, 6 occurred within a run of 6 guanines and 5 occurred in the sequence 5'-GAAGAG-3'. The variation among independent experiments is consistent with the numerical expectation that all 16 mutations fulfill reasonable statistical criteria for mutational hot spots. The agreement with data from various systems using clone-by-clone analysis shows that the protocol reported herein can be a useful tool to study hot-spot point mutational spectra for DNA sequences for which phenotypic selection systems exist.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed2006

Ovulation-selective genes: the generation and characterization of an ovulatory-selective cDNA library.

Ovulation-selective/specific genes, that is, genes preferentially or exclusively expressed during the ovulatory process, have been the subject of growing interest. We report herein studies on the use of suppression subtractive hybridization (SSH) to construct a 'forward' ovulation-selective/specific cDNA library. In toto, 485 clones were sequenced and analyzed for homology to known genes with the basic local alignment tool (BLAST). Of those, 252 were determined to be nonredundant. Of these 252 nonredundant clones, 98 were analyzed by probing mouse preovulatory and postovulatory ovarian cDNA. Twenty-five clones (26%) failed to show any signal, and 43 cDNAs tested thus far display a true ovulation-selective/specific expression pattern. In this communication, we focus on one such ovulation-selective gene, the fatty acid elongase 1 (FAE-1) homolog, found to be localized to the inner periantral granulosa and to the cumulus granulosa cells of antral follicles. The FAE-1 gene is a beta-ketoacyl-CoA synthase belonging to the fatty acid elongase (ELO) family, which catalyzes the initial step of very long-chain fatty acid synthesis. All in all, the present study accomplished systematic identification of those hormonally regulated genes that are expressed in the ovary in an ovulation-selective/specific manner. These ovulation-selective/specific genes may have significant implications for the understanding of ovarian function in molecular terms and for the development of innovative strategies for both the promotion of fertility and its control.

Acetyltransferases↗
PubMed2003

Selective gene expression in brain microglia mediated via adeno-associated virus type 2 and type 5 vectors.

Microglia represent a crucial cell population in the central nervous system, participating in the regulation and surveillance of physiological processes as well as playing key roles in the etiologies of several major brain disorders. The ability to target gene transfer vehicles selectively to microglia would provide a powerful new approach to investigations of mechanisms regulating brain pathologies, as well as enable the development of novel therapeutic strategies. In this study, we evaluate the feasibility of specifically and efficiently targeting microglia relative to other brain cells, using vectors based on two different serotypes of adeno-associated virus (AAV) carrying cell-type-specific transcriptional elements to regulate gene expression. Among a set of promoter choices examined, an element derived from the gene for the murine macrophage marker F4/80 was the most discriminating for microglia. Gene expression from vectors controlled by this element was highly selective for microglia, both in vitro and in vivo. To our knowledge, this is the first demonstration of selective expression of transferred genes in microglia using AAV-derived vectors, as well as the first utilization of recombinant AAV-5 vectors in any macrophage lineage. These results provide strong encouragement for the application of these vectors and this approach for delivering therapeutic and other genes selectively to microglia.

Animals↗
PubMed2006

Assessing stability of gene selection in microarray data analysis.

BACKGROUND: The number of genes declared differentially expressed is a random variable and its variability can be assessed by resampling techniques. Another important stability indicator is the frequency with which a given gene is selected across subsamples. We have conducted studies to assess stability and some other properties of several gene selection procedures with biological and simulated data. RESULTS: Using resampling techniques we have found that some genes are selected much less frequently (across sub-samples) than other genes with the same adjusted p-values. The extent to which this type of instability manifests itself can be assessed by a method introduced in this paper. The effect of correlation between gene expression levels on the performance of multiple testing procedures is studied by computer simulations. CONCLUSION: Resampling represents a tool for reducing the set of initially selected genes to those with a sufficiently high selection frequency. Using resampling techniques it is also possible to assess variability of different performance indicators. Stability properties of several multiple testing procedures are described at length in the present paper.

Biomarkers, Tumor↗
PubMed2001

Development and evaluation of functional gene arrays for detection of selected genes in the environment.

To determine the potential of DNA array technology for assessing functional gene diversity and distribution, a prototype microarray was constructed with genes involved in nitrogen cycling: nitrite reductase (nirS and nirK) genes, ammonia mono-oxygenase (amoA) genes, and methane mono-oxygenase (pmoA) genes from pure cultures and those cloned from marine sediments. In experiments using glass slide microarrays, genes possessing less than 80 to 85% sequence identity were differentiated under hybridization conditions of high stringency (65 degrees C). The detection limit for nirS genes was approximately 1 ng of pure genomic DNA and 25 ng of soil community DNA using our optimized protocol. A linear quantitative relationship (r(2) = 0.89 to 0.94) was observed between signal intensity and target DNA concentration over a range of 1 to 100 ng for genomic DNA (or genomic DNA equivalent) from both pure cultures and mixed communities. However, the quantitative capacity of microarrays for measuring the relative abundance of targeted genes in complex environmental samples is less clear due to divergent target sequences. Sequence divergence and probe length affected hybridization signal intensity within a certain range of sequence identity and size, respectively. This prototype functional gene array did reveal differences in the apparent distribution of nir and amoA and pmoA gene families in sediment and soil samples. Our results indicate that glass-based microarray hybridization has potential as a tool for revealing functional gene composition in natural microbial communities; however, more work is needed to improve sensitivity and quantitation and to understand the associated issue of specificity.

Bacteria↗
PubMed2005

The MODY1 gene HNF-4alpha regulates selected genes involved in insulin secretion.

Mutations in the gene encoding hepatocyte nuclear factor-4alpha (HNF-4alpha) result in maturity-onset diabetes of the young (MODY). To determine the contribution of HNF-4alpha to the maintenance of glucose homeostasis by the beta cell in vivo, we derived a conditional knockout of HNF-4alpha using the Cre-loxP system. Surprisingly, deletion of HNF-4alpha in beta cells resulted in hyperinsulinemia in fasted and fed mice but paradoxically also in impaired glucose tolerance. Islet perifusion and calcium-imaging studies showed abnormal responses of the mutant beta cells to stimulation by glucose and sulfonylureas. These phenotypes can be explained in part by a 60% reduction in expression of the potassium channel subunit Kir6.2. We demonstrate using cotransfection assays that the Kir6.2 gene is a transcriptional target of HNF-4alpha. Our data provide genetic evidence that HNF-4alpha is required in the pancreatic beta cell for regulation of the pathway of insulin secretion dependent on the ATP-dependent potassium channel.

Animals↗
PubMed1995

Identification of a novel gene, selectively up-regulated in human carcinomas, using the differential display technique.

Using the differential display technique, selecting for genes up-regulated in renal cell carcinoma compared with normal renal parenchyma, we isolated a novel gene, designated DD96. As determined by in situ and Northern blot hybridization studies, DD96 is expressed only in rare normal epithelial cell populations, such as the proximal tubular epithelial cells of the kidney. However, it is expressed diffusely in malignant epithelial cells of the wide majority of renal cell carcinomas. In addition, DD96 is overexpressed markedly in various human carcinomas originating from the colon, breast, and lung, as well as in a number of cell lines derived from tumors of these organs compared with normal epithelial cell populations. Furthermore, the expression of DD96 is induced in immortalized breast ductal epithelial cell lines compared with normal breast ductal epithelial cells, and, in vivo, in premalignant conditions, such as adenoma of the colon and ductal carcinoma in situ of the breast. Sequence analysis of a complete cDNA clone isolated from a human kidney cDNA library revealed that DD96 encodes for a protein of approximately Mr 13,500. These results suggest that DD96 may play a role in the early events associated with malignant transformation; however, its function remains to be determined.

Amino Acid Sequence↗
PubMed1997

Two vectors for the insertion of mammalian selectable genes into yeast artificial chromosome cloned DNA.

The introduction of cloned DNA into mammalian cells allows functional testing of genes contained on the fragments. In many cases, the exogenous DNA introduced into mammalian cells requires selectable genes that mark the presence of input DNA. Two new vectors, carrying mammalian selectable markers encoding for either neomycin-resistance (neo) or histidinol-resistance (hol), have been constructed for targeted integration to specific single-copy sites within yeast artificial chromosome (YAC) insert DNA. The integration cassettes comprise a single selectable yeast gene adjacent to a mammalian selectable gene, either LEU2 with neo or HIS3 with hol. Modification of the YAC occurs in yeast by transfection with linear DNA containing YAC-specific, unique, recombinogenic ends, thereby ensuring co-integration of the markers. Analysis of modified YACs confirms that both vectors correctly integrate into the targeted unique sites. The precise localization of selectable marker genes in the cloned DNA ensures the integrity of the genomic fragments during functional testing. Placement of mammalian selectable markers within the YAC insert DNA should allow for YAC-based gene targeting experiments in a variety of mammalian cell lines.

3-Isopropylmalate Dehydrogenase↗
PubMed2001

Efficient and cancer-selective gene transfer to hepatocellular carcinoma in a rat using adenovirus vector with iodized oil esters.

Gene therapy for cancer requires efficient, selective gene transfer to cancer cells. In gene therapy for hepatocellular carcinoma (HCC), gene transfer is efficient for small tumors, but not for large tumors. The delivery of anticancer agents and of iodized oil esters as embolic agents through tumor-feeding arteries is known as transarterial embolization. We speculate that genes may be efficiently and selectively transferred for HCC using iodized oil esters because these esters may remain together with a genetic vector within HCC selectively. Hence, we have studied the effect of iodized oil esters on adenovirus vector-mediated gene transfer for HCC in vivo. A rat model of HCC induced with diethylnitrosamine and phenobarbital was injected with either AxCALacZ, which expresses the beta-galactosidase of Escherichia coli, or AxCALacZ and iodized oil esters into the hepatic artery. Histological comparisons revealed that the beta-galactosidase expression in the rats with HCC injected with AxCALacZ and iodized oil esters was greater (P<.0001) in small tumors (P=.0046) and large tumors (P=.0023), and more selective (P=.0229) than in only AxCALacZ-injected rats. These results suggest that iodized oil esters are injected into hepatic artery together with adenovirus vector, and that genes may be efficiently and cancer-selectively transferred to HCC.

Adenoviridae↗
PubMed2004

In silico gene selection strategy for custom microarray design.

Microarray technology has become an important tool for studying large-scale gene expression for a diversity of biological applications. However, there are a number of experimental settings for which commercial arrays are either unsuitable or unavailable despite the existence of sequence information. With the increasing availability of custom array manufacturing services, it is now feasible to design high-density arrays for any organism having sequence data. However, there have been relatively few reports discussing gene selection, an important first step in array design. Here we propose an in silico strategy for custom microarray gene selection that is applicable to a wide range of organisms, based on utilizing public domain microarray information to interrogate existing sequence data and to identify a set of homologous genes in any organism of interest. We demonstrate the utility of this approach by applying it to the selection of candidate genes for a custom Xenopus laevis microarray. A significant finding of this study is that 3%-4% of Xenopus expressed sequence tags (ESTs) are in an orientation contrary to that indicated in the public database entry (http://mssaha.people.wm.edu/suppMSS.html).

Algorithms↗
PubMed2004

Gene selection for sample classifications in microarray experiments.

DNA microarray technology provides useful tools for profiling global gene expression patterns in different cell/tissue samples. One major challenge is the large number of genes relative to the number of samples. The use of all genes can suppress or reduce the performance of a classification rule due to the noise of nondiscriminatory genes. Selection of an optimal subset from the original gene set becomes an important prestep in sample classification. In this study, we propose a family-wise error (FWE) rate approach to selection of discriminatory genes for two-sample or multiple-sample classification. The FWE approach controls the probability of the number of one or more false positives at a prespecified level. A public colon cancer data set is used to evaluate the performance of the proposed approach for the two classification methods: k nearest neighbors (k-NN) and support vector machine (SVM). The selected gene sets from the proposed procedure appears to perform better than or comparable to several results reported in the literature using the univariate analysis without performing multivariate search. In addition, we apply the FWE approach to a toxicogenomic data set with nine treatments (a control and eight metals, As, Cd, Ni, Cr, Sb, Pb, Cu, and AsV) for a total of 55 samples for a multisample classification. Two gene sets are considered: the gene set omegaF formed by the ANOVA F-test, and a gene set omegaT formed by the union of one-versus-all t-tests. The predicted accuracies are evaluated using the internal and external crossvalidation. Using the SVM classification, the overall accuracies to predict 55 samples into one of the nine treatments are above 80% for internal crossvalidation. OmegaF has slightly higher accuracy rates than omegaT. The overall predicted accuracies are above 70% for the external crossvalidation; the two gene sets omegaT and omegaF performed equally well.

Colonic Neoplasms↗
PubMed2006

Regularization network-based gene selection for microarray data analysis.

Microarray data contains a large number of genes (usually more than 1000) and a relatively small number of samples (usually fewer than 100). This presents problems to discriminant analysis of microarray data. One way to alleviate the problem is to reduce dimensionality of data by selecting important genes to the discriminant problem. Gene selection can be cast as a feature selection problem in the context of pattern classification. Feature selection approaches are broadly grouped into filter methods and wrapper methods. The wrapper method outperforms the filter method but at the cost of more intensive computation. In the present study, we proposed a wrapper-like gene selection algorithm based on the Regularization Network. Compared with classical wrapper method, the computational costs in our gene selection algorithm is significantly reduced, because the evaluation criterion we proposed does not demand repeated training in the leave-one-out procedure.

Algorithms↗
PubMed2005

An entropy-based gene selection method for cancer classification using microarray data.

BACKGROUND: Accurate diagnosis of cancer subtypes remains a challenging problem. Building classifiers based on gene expression data is a promising approach; yet the selection of non-redundant but relevant genes is difficult. The selected gene set should be small enough to allow diagnosis even in regular clinical laboratories and ideally identify genes involved in cancer-specific regulatory pathways. Here an entropy-based method is proposed that selects genes related to the different cancer classes while at the same time reducing the redundancy among the genes. RESULTS: The present study identifies a subset of features by maximizing the relevance and minimizing the redundancy of the selected genes. A merit called normalized mutual information is employed to measure the relevance and the redundancy of the genes. In order to find a more representative subset of features, an iterative procedure is adopted that incorporates an initial clustering followed by data partitioning and the application of the algorithm to each of the partitions. A leave-one-out approach then selects the most commonly selected genes across all the different runs and the gene selection algorithm is applied again to pare down the list of selected genes until a minimal subset is obtained that gives a satisfactory accuracy of classification. The algorithm was applied to three different data sets and the results obtained were compared to work done by others using the same data sets. CONCLUSION: This study presents an entropy-based iterative algorithm for selecting genes from microarray data that are able to classify various cancer sub-types with high accuracy. In addition, the feature set obtained is very compact, that is, the redundancy between genes is reduced to a large extent. This implies that classifiers can be built with a smaller subset of genes.

Algorithms↗
PubMed1991

Gene transfer with subsequent removal of the selection gene from the host genome.

A general method of gene transfer that does not leave behind a selectable marker in the host genome is described. A luciferase gene was introduced into the tobacco genome by using the hygromycin phosphotransferase gene (hpt) as a linked selectable marker. Flanked by recombination sites from the bacteriophage P1 Cre/lox recombination system, the hpt gene was subsequently excised from the plant genome by the Cre recombinase. The Cre-catalyzed excision event in the plant genome was precise and conservative--i.e., without loss or alteration of nucleotides in the recombinant site. After removal of the Cre-encoding locus by genetic segregation, plants were obtained that had incorporated only the desired transgene. Gene transfer without the incorporation of antibiotic-resistance markers in the host genome should ease public concerns over the field release of transgenic organisms expressing such traits. Moreover, it would obviate the need for different selectable markers in subsequent rounds of gene transfer into the same host.

Agrobacterium tumefaciens↗
PubMed2004

Reduced inflammatory pain in mice deficient in the differential screening-selected gene aberrative in neuroblastoma.

Differential screening-selected gene aberrative in neuroblastoma (Dan) protein is produced in small neurons of dorsal root ganglia. Thermal and mechanical allodynia and Fos expression in the spinal dorsal horn evoked by inflammation and neuropathic pain were investigated using Dan-deficient mice. Mice showed pain reactions induced by the introduction of complete Freund's adjuvant (CFA) into their hind paw (inflammatory pain model) and after sciatic nerve ligation (neuropathic pain model). In the inflammatory pain model, thermal and mechanical pain thresholds in Dan-deficient mice were significantly higher than those of wild-type mice. The number of Fos-immunoreactive cells in the dorsal horn during the inflammatory period was significantly less in Dan-deficient mice. However, in the neuropathic pain model, no differences in thermal hypersensitivity, mechanical allodynia, or the number of Fos-immunoreactive cells in the dorsal horn were observed between the mice. These data suggest that Dan may be a neuromodulator in inflammatory pain.

Animals↗