RESUMO
A series of dye-labeled oligonucleotide probes containing base and sugar modifications were tested for the ability to detect telomeric repeat sequences in FISH assays. These modified oligonucleotides, all 18 nt in length, were complementary to either the cytidine-rich (C(3)TA(2))(n)or guanosine-rich (T(2)AG(3))(n)telomere target sequences. Oligonucleotides were modified to either increase target affinity by enhancing duplex stability [2'-OMe ribose sugars and 5-(1-propynyl)pyrimidine residues] or inhibit the formation of inter- or intramolecular structures (7-deazaguanosine and 6-thioguanosine residues), which might interfere with binding to the target. Several dye-labeled oligonucleotide probes were found that could effectively stain the telomeric repeat sequences of either cytidine- or guanosine-rich strands in a specific manner. Such probes could be used as an alternative to peptide nucleic acids for investigating the dynamics of telomere length and maintenance. In principle, these relatively inexpensive and readily synthesized modified oligonucleotides could be used for other FISH-related assays.
Assuntos
Hibridização in Situ Fluorescente , Oligodesoxirribonucleotídeos/síntese química , Sequências Repetitivas de Ácido Nucleico , Telômero/química , Sequência de Bases , Sítios de Ligação , Modelos Químicos , Dados de Sequência MolecularRESUMO
Cl(-)-sensitive fluorescent indicators have been used extensively in cell culture systems to measure the Cl(-)-transporting function of the cystic fibrosis transmembrane conductance regulator protein CFTR. These indicators have been used in establishing a surrogate end point to assess the efficacy of CFTR cDNA delivery in human gene therapy trials. The ability to measure Cl- transport with high sensitivity in small and heterogeneous tissue samples makes the use of Cl- indicators potentially attractive in gene delivery studies. In this review article, the important technical aspects of Cl- transport measurements by fluorescent indicators such as SPQ are described, applications of Cl- indicators to assay CFTR function are critically evaluated, and new methodological developments are discussed. The available Cl- indicators have been effective in quantifying Cl- transport rates in cell culture models and in vitro systems such as isolated membrane vesicles and liposomes. However, the imperfect photophysical properties of existing Cl- indicators limit their utility in performing measurements in airway tissues, where gene transfer vectors are delivered in CF gene therapy trials. The low efficiency of gene transfer and the cellular heterogeneity in airway samples pose substantial obstacles to functional measurements of CFTR expression. Significant new developments in generating long-wavelength and dual-wavelength halide indicators are described, and recommendations are proposed for the use of the indicators in gene therapy trials.
Assuntos
Cloretos/química , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/terapia , DNA Complementar/administração & dosagem , Corantes Fluorescentes/química , Membrana Celular/metabolismo , Ensaios Clínicos como Assunto , Terapia Genética , Humanos , Transporte de ÍonsRESUMO
The anterior cruciate ligament of twenty-five adult dogs was replaced using fresh or deep-frozen patellar-tendon allografts. The morphology of these transplanted allografts was then evaluated using routine histological studies and a vascular-injection (Spalteholz) technique at various intervals from two weeks to one year postoperatively. The fresh patellar-tendon allografts incited a marked inflammatory and rejection response which was characterized by perivascular cuffing and lymphocyte invasion. Deep-frozen patellar-tendon allografts appeared to be benign within the joint and underwent alterations that were comparable with those observed in autogenous patellar-tendon grafts. These included avascular necrosis followed by revascularization and cellular proliferation. At one year, the gross and histological appearance of the patellar tendon allograft resembled that of a normal anterior cruciate ligament.
Assuntos
Articulação do Joelho/cirurgia , Ligamentos Articulares/cirurgia , Patela , Tendões/transplante , Animais , Cães , Congelamento , Rejeição de Enxerto , Sobrevivência de Enxerto , Tendões/patologia , Fatores de Tempo , Preservação de Tecido/métodos , Transplante HomólogoRESUMO
The DNA elements that account for the highly regulated expression of the cystic fibrosis transmembrane conductance regulator gene (CFTR) are poorly understood. The goal of this study was to assess the feasibility of using a yeast artificial chromosome (YAC)-based reporter gene construct to define these elements further. An approximately 350-kb YAC (y5'luc) was constructed by replacing CFTR with a luciferase reporter gene (luc). A second YAC (y5'lucI) was similarly constructed but included a putative positive regulatory element from CFTR intron 1. Stable Chinese hamster ovary (CHO-K1) cell clones were derived using each YAC to assess the role that luc copy number and the presence of intron 1 played in luc expression. The CHO-K1 clonal cell lines demonstrated a wide range of luciferase activity. On average, this activity was significantly higher in clones derived from y5'lucI. After correcting for luc copy number, the presence of intron 1 was still associated with an increase in luciferase activity (P < 0.05), despite the fact that luciferase activity did not correlate with luc copy number in y5'luc-derived clones (r = -0.12). In contrast, the luciferase activity correlated well with luc copy number in the clones derived from y5'luc (r = 0. 75). These data are consistent with a positive role for intron 1 in regulating CFTR expression, but suggest that copy number is not the only factor that determines expression levels, particularly when this element is present. This YAC-based reporter system will provide a unique strategy for further assessment of the cis-acting elements that control CFTR expression.
Assuntos
Cromossomos Artificiais de Levedura/genética , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Íntrons/fisiologia , Luciferases/genética , Regiões Promotoras Genéticas/fisiologia , Animais , Southern Blotting , Células CHO , Células Clonais , Cricetinae , Dosagem de Genes , Regulação da Expressão Gênica , Genes Reporter/genética , Íntrons/genética , Luciferases/biossíntese , Luciferases/metabolismo , Transfecção , Regulação para CimaRESUMO
Gene isolation methods used during positional cloning rely on physical contigs consisting of bacterial artificial chromosomes, P1, or cosmid clones. However, in most instances, the initial framework for physical mapping consists of contigs of yeast artificial chromosome (YACs), large vectors that are suboptimal substrates for gene isolation. Here we report a strategy to identify gene sequences contained within a YAC by using cDNA representational difference analysis (RDA) to directly isolate transcripts expressed from the YAC in mammalian cells. The RDA tester cDNAs were generated from a previously reported hamster cell line derived by stable transfer of a 590-kb YAC (911D5) that expressed NPC1, the human gene responsible for Niemann-Pick type C (NP-C). The driver cDNAs were generated from a control hamster cell line that did not contain the YAC that expressed NPC1. Among the gene fragments obtained by RDA, NPC1 was the most abundant product. In addition, two non-NPC1 fragments were isolated that were mapped to and expressed from 911D5. One of these RDA gene fragments (7-R) spans more than one exon and has 98% sequence identity with a human cDNA clone reported previously as an expressed sequence tag (EST), but not mapped to a chromosomal region. The other fragment (2-R) that had no significant sequence similarities with known mammalian genes or ESTs, was further localized to the region of overlap between YACs 911D5 and 844E3. The latter YAC is part of a contig across the NP-C candidate region, but does not contain NPC1. This two-part approach in which stable YAC transfer is followed by cDNA RDA should be a useful adjunct strategy to expedite the cloning of human genes when a YAC contig is available across a candidate interval.
Assuntos
Cromossomos Artificiais de Levedura/genética , DNA Complementar/análise , Expressão Gênica/genética , Transcrição Gênica , Animais , Southern Blotting/métodos , Cricetinae , Humanos , Dados de Sequência Molecular , Hibridização de Ácido Nucleico/métodos , RNA Mensageiro/isolamento & purificação , Análise de Sequência de DNARESUMO
The introduction of high molecular weight DNA into mammalian cells is useful for gene expression studies. However, current transfection strategies are inefficient, necessitating propagation of stable DNA transformants prior to analysis of gene expression. Here we demonstrate that transient lipid-mediated DNA transfection can be used to assess gene expression from yeast artificial chromosomes (YACs) containing the 230 kb cystic fibrosis transmembrane conductance regulator gene ( CFTR ) and Escherichia coli lacZ . We also show that psoralen-UV inactivated adenovirus significantly enhances transfection efficiency. The ability to deliver high molecular weight DNA using lipid-mediated transfection should expedite the analysis of large human genes contained within artificial chromosome vectors.
Assuntos
Adenoviridae/genética , Cromossomos Artificiais de Levedura/genética , Expressão Gênica , Transfecção/métodos , Animais , Resinas de Troca de Cátion , Linhagem Celular , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Escherichia coli/genética , Ficusina , Vetores Genéticos/genética , Humanos , Óperon Lac/genética , Lipídeos , Fármacos Fotossensibilizantes , RNA Mensageiro/análise , Raios UltravioletaRESUMO
The transfer of high molecular weight (HMW) DNA into mammalian cells is an important strategy for assessing human gene expression and chromosome structure and function. However, using current methods, it is difficult to dependably prepare intact HMW DNA because of the susceptibility of the DNA to degradation and physical shearing. Here we describe a strategy whereby intact artificial chromosome DNA (as large as 1 Mb) can be routinely prepared from yeast. Strict adherence to this protocol has resulted in: (i) >90% of liquid DNA preparations containing largely intact DNA; (ii) transfection efficiencies for the development of stable human clonal cell lines ranging from 5 x 10(-7) to 8.8 x 10(-5); and (iii) the presence of markers from both YAC arms in 30-42% of the human fibrosarcoma cell HT1080 clones and 100% of the CF lung epithelial cell lines IB3-1 and CFT1 clones, suggesting that the HMW DNA is potentially intact in a substantial proportion of clones. Using this protocol for DNA preparation, successful transfection of functional 1 Mb human artificial chromosome DNA into human cells has also been achieved. This methodology should prove useful to those interested in using HMW human DNA for gene expression and functional analysis or for linear artificial chromosome construction, since integrity is absolutely critical for the success of these studies.
Assuntos
DNA/genética , Transfecção , Linhagem Celular , Cromossomos Artificiais de Levedura , Regulador de Condutância Transmembrana em Fibrose Cística/genética , HumanosRESUMO
Saccharomyces cerevisiae contains an irregular telomere sequence (TG1-3)n, which differs from the regular repeat (TTAGGG)n found at the telomeres of higher organisms including humans. We have modified the entire 16-nt template region of the S. cerevisiae telomerase RNA gene (TLC1) to produce (TTAGGG)n repeats, the human telomere sequence. Haploid yeast strains with the tlc1-human allele are viable with no growth retardation and express the humanized gene at a level comparable to wild type. Southern hybridization demonstrates that (TTAGGG)n repeats are added onto the yeast chromosome ends in haploid strains with the tlc1-human allele, and sequencing of rescued yeast artificial chromosome ends has verified the addition of human telomeric repeats at the molecular level. These data suggest that the irregularity of the yeast telomere sequence is because of the template sequence of the yeast telomerase RNA. Haploid strains with the tlc1-human allele will provide an important tool for studying the function of telomerase and its regulation by telomere-binding proteins, and these strains will serve as good hosts for human artificial chromosome assembly and propagation.
Assuntos
Telomerase/genética , Southern Blotting , Cromossomos Artificiais de Levedura/genética , DNA/química , Proteínas de Ligação a DNA/metabolismo , Humanos , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , RNA Fúngico/química , Sequências Repetitivas de Ácido Nucleico , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Telomerase/metabolismo , Telômero/genética , Moldes GenéticosRESUMO
A human artificial chromosome (HAC) vector was constructed from a 1-Mb yeast artificial chromosome (YAC) that was selected based on its size from among several YACs identified by screening a randomly chosen subset of the Centre d'Etude du Polymorphisme Humain (CEPH) (Paris) YAC library with a degenerate alpha satellite probe. This YAC, which also included non-alpha satellite DNA, was modified to contain human telomeric DNA and a putative origin of replication from the human beta-globin locus. The resultant HAC vector was introduced into human cells by lipid-mediated DNA transfection, and HACs were identified that bound the active kinetochore protein CENP-E and were mitotically stable in the absence of selection for at least 100 generations. Microdissected HACs used as fluorescence in situ hybridization probes localized to the HAC itself and not to the arms of any endogenous human chromosomes, suggesting that the HAC was not formed by telomere fragmentation. Our ability to manipulate the HAC vector by recombinant genetic methods should allow us to further define the elements necessary for mammalian chromosome function.
Assuntos
Cromossomos Artificiais de Levedura/genética , DNA Satélite/genética , Dosagem de Genes , Linhagem Celular , Cromossomos/genética , Células Clonais/metabolismo , Fragmentação do DNA/genética , Sondas de DNA/genética , Vetores Genéticos/genética , Globinas/genética , Humanos , Hibridização in Situ Fluorescente , Mitose/genética , Origem de Replicação/genética , Telômero/genética , Transfecção/genéticaRESUMO
A major obstacle in positional cloning is identifying the specific mutated gene from within a large physical contig. Here we describe the application of DNA microarray technology to a defined genomic region (physical map) to identify: (i) exons without a priori sequence data and (ii) the disease gene based on differential gene expression in a recessive disorder. The feasibility was tested using resources from the positional cloning of the Neimann-Pick Type C (NP-C) disease gene, NPC1. To identify NPC1 exons and optimize the technology, an array was generated from genomic fragments of the 110-kb bacterial artificial chromosome, 108N2, which encodes NPC1. First, as a test case for blindly identifying exons, fluorescently labeled NPC1 cDNA identified 108N2 fragments that contained NPC1 exons, many of which also contained intronic sequences and could be used to determine part of the NPC1 genomic structure. Second, to demonstrate that the NPC1 disease gene could be identified based upon differential gene expression, subarrays of 108N2 fragments were hybridized with fluorescently labeled cDNA probes generated from total RNA from hamster cell lines differentially expressing NPC1. A probe derived from the NP-C cell line CT60 did not detect NPC1 exons or other genomic fragments from 108N2. In contrast, several NPC1 exons were detected by a probe generated from the non-NP-C cell line 911D5A13, which was derived from CT60, and expressed NPC1 as a consequence of stable transduction with a YAC that contains NPC1 and encompasses 108N2. Thus, the array technology identified NPC1 as a candidate gene based on a physical contig and differential NPC1 expression between NP-C and non-NP-C cells. This technique should facilitate gene identification when a physical contig exists for a region of interest and mutations result in changes in the mRNA level of the disease gene or portions thereof.