RESUMEN
Single-stranded oligonucleotides (SSOs) mediate gene repair of punctual chromosomal mutations at a low frequency. We hypothesized that enhancement of DNA binding affinity of SSOs by intercalating agents may increase the number of corrected cells. Several biochemical modifications of SSOs were tested for their capability to correct a chromosomally integrated and mutated GFP reporter gene in human 293 cells. SSOs of 25 nucleotide length conjugated with acridine at their 5' end increased the efficiency of gene correction up to 10-fold compared to nonmodified SSOs. Acridine and psoralen conjugates were both evaluated, and acridine-modified SSOs were the most effective. Conjugation with acridine at the 3' end of the SSO inhibited gene correction, whereas flanking the SSO by acridine on both sides provided an intermediate level of correction. These results suggest that increasing the stability of hybridization between SSO and its target without hampering a 3' extension improves gene targeting, in agreement with the "annealing-integration" model of DNA repair.
Asunto(s)
Acridinas/metabolismo , Sustancias Intercalantes/metabolismo , Oligonucleótidos/genética , Oligonucleótidos/metabolismo , Reparación del Gen Blanco/métodos , Ficusina/metabolismo , Terapia Genética , Humanos , Oligonucleótidos Antisentido/genética , Oligonucleótidos Antisentido/metabolismo , TransfecciónRESUMEN
BACKGROUND: Gene targeting is a potential tool for gene therapy but is limited by the low rate of homologous recombination. Using highly homologous linear DNA improves gene targeting frequency but requires microinjection into nuclear cells to be effective. Because transfection of circular DNA is more efficient than transfection of linear DNA and adaptable to viral vectors, we developed a system for the intracellular release of linear fragments from circular plasmids. METHODS: Only one cutting site inside the "donor" DNA was not convenient because it led to integration of exogenous sequences into the target. So we constructed several "donor" plasmids containing the homologous sequences flanked by two I-Sce I recognition sites. Expression of I-Sce I allowed intracellular delivery of "ends-out" (replacement) vectors. We compared the efficiency of different constructions to correct a mutated gfp target. RESULTS: Co-transfection of "donor" plasmids and an I-Sce I expression vector into CHO cells enhanced the correction of an extrachromosomal mutated gfp target by at least 10 times. Maximum correction was observed with the greatest homology size and maximum effect of I-Sce I was obtained when the long hemi-sites of the duplicated I-Sce I sites were contiguous to the homologous sequence. Unexpectedly, the reverse orientation of I-Sce I sites provided little or no effect, probably due to the asymmetrical activity of the I-Sce I meganuclease. CONCLUSIONS: Releasing homologous DNA fragments with I-Sce I enhances gene replacement. This work provides the basis for the future design of viral vectors for gene replacement.
Asunto(s)
ADN Circular/genética , Desoxirribonucleasas de Localización Especificada Tipo II/farmacología , Vectores Genéticos/análisis , Proteínas Fluorescentes Verdes/genética , Recombinación Genética , Animales , Células CHO , Cricetinae , Marcación de Gen/métodos , Vectores Genéticos/genética , Proteínas Fluorescentes Verdes/análisis , Proteínas de Saccharomyces cerevisiae , TransfecciónRESUMEN
Directed mutagenesis in mammalian cells has been the focus of intense research because of its promising application for gene correction and engineering. Both natural and modified oligonucleotides (ODN), RNA-DNA chimeric oligonucleotide (RDO) and small fragment DNA (SFHR), as well as vector DNA were used for promoting homologous replacement with varying success. It was recently shown that a triple helix-forming oligonucleotide (TFO) tethered to an oligonucleotide (donor DNA) can enhance mutagenesis by homologous recombination in cells. The basic idea is to accelerate homology search by oligonucleotide-directed triple helix formation in the vicinity of the target site for donor DNA. Here we describe a new method named GOREC (guided homologous recombination) which shares similar gene targeting, but has notable difference in the concept with the previous method. It is made of a homing device (TFO) and a donor DNA for effecting distinct functions. They are linked together by non-covalent or covalent interaction. This modular concept allows guidance of either an oligonucleotide (ODN, RDO) or a small DNA fragment to the target site for homologous replacement. Therefore, the triple helix site can be hundreds of base pairs away from the target site. An episomal assay for proof-of-principle study will be presented and discussed.
Asunto(s)
Marcación de Gen/métodos , Terapia Genética/métodos , Mutagénesis Sitio-Dirigida , Recombinación Genética , Animales , Células CHO , Cricetinae , ADN/administración & dosificación , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/genética , Oligonucleótidos/genética , Trasplante HomólogoRESUMEN
BACKGROUND: The combination of physiologically and pharmacologically controlled elements may provide a means to ensure both the regulation and the safety of transgene expression--two major goals in gene therapy. METHODS: A two-gene modulation system was developed that uses the following three levels of control: (i) the hypoxia-responsive element directing the transcription of the tetracycline-controlled transactivator (tTA); (ii) part of the oxygen-degradation domain limiting the production of tTA in normoxia; and (iii) the tetracycline switch of the transactivator activity (the tet-off system). RESULTS: This triple-control system allowed high expression of the gene of interest (luciferase or erythropoietin) by transfected cells upon hypoxia and low expression under normoxia or in the presence of tetracycline. This control of transgene expression was also obtained in mouse tumors. CONCLUSIONS: This multiple-control system is of interest for spatially restricting transgene expression into hypoxic tumors, and for finely adjusting the expression level of a therapeutic protein to the oxygen supply in medical applications such as neoangiogenesis or the erythropoietin-mediated treatment of anemia.
Asunto(s)
Regulación de la Expresión Génica/efectos de los fármacos , Terapia Genética , Oxígeno/metabolismo , Transgenes/genética , Adenoviridae/efectos de los fármacos , Adenoviridae/genética , Adenoviridae/metabolismo , Animales , Carcinoma Pulmonar de Lewis/metabolismo , Cartilla de ADN/química , Eritropoyetina/genética , Vectores Genéticos , Humanos , Hipoxia/genética , Luciferasas/genética , Neoplasias Pulmonares/metabolismo , Ratones , Tetraciclina/farmacología , Transcripción Genética/efectos de los fármacos , TransfecciónRESUMEN
Transcription factor p45 NF-E2 is highly expressed in the erythroid and megakaryocytic lineages. Although p45 recognizes regulatory regions of several erythroid genes, mice deficient for this protein display only mild dyserythropoiesis but have abnormal megakaryocytes and lack circulating platelets. A number of megakaryocytic marker genes have been extensively studied, but none of them is regulated by NF-E2. To find target genes for p45 NF-E2 in megakaryopoiesis, we used an in vivo immunoselection assay: genomic fragments bound to p45 NF-E2 in the chromatin of a megakaryocytic cell line were immunoprecipitated with an anti-p45 antiserum and cloned. One of these fragments belongs to the second intron of the thromboxane synthase gene (TXS). We demonstrate that the TXS gene, which is mainly expressed in megakaryocytes, is indeed directly regulated by p45 NF-E2. First, its promoter contains a functional NF-E2 binding site; second, the intronic NF-E2 binding site is located within a chromatin-dependent enhancer element; third, p45-null murine megakaryocytes do not express detectable TXS mRNA, although TXS expression can be detected in other cells. These data, and the structure of the TXS promoter and enhancer, suggest that TXS belongs to a distinct subgroup of genes involved in platelet formation and function.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Regulación Enzimológica de la Expresión Génica , Megacariocitos/enzimología , Tromboxano-A Sintasa/biosíntesis , Tromboxano-A Sintasa/genética , Factores de Transcripción/metabolismo , Animales , Secuencia de Bases , Línea Celular , Cromatina/fisiología , Clonación Molecular , Cartilla de ADN , Elementos de Facilitación Genéticos , Factores de Unión al ADN Específico de las Células Eritroides , Hematopoyesis , Humanos , Intrones , Ratones , Datos de Secuencia Molecular , Factor de Transcripción NF-E2 , Subunidad p45 del Factor de Transcripción NF-E2 , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , Proteínas Recombinantes/biosíntesis , Secuencias Reguladoras de Ácidos Nucleicos , Transfección , Dedos de ZincRESUMEN
Mucosal lymphocytes are increasingly activated in Crohn's disease. In order to investigate this phenomenon we tested the response of cultured colonic mucosal T lymphocytes to CD3 activation. These lymphocytes were obtained by biopsy from control subjects and Crohn's disease patients. T cells from mucosa involved in Crohn's disease showed a higher CD3-induced proliferative response compared to control T lymphocytes (P < 0.01). T cells from uninvolved areas showed a wide range of response, ranging from normal to very high proliferative indices. Our results suggest that the exaggerated response of mucosal T lymphocytes, due to the impairment of the downregulation of the CD3-dependent signaling process, may contribute to the pathogenesis of Crohn's disease.