RESUMEN
Retroviral vectors are used widely in research and are also being designed for use in gene therapy trials. In practice, these vectors usually contain a marker gene, which is often a drug selection gene. In this report, a novel retroviral vector has been constructed which contains a gene that allows selection for infected cells by a magnet. This gene is a single-chain antibody (sFv) to a specific hapten molecule 4-ethoxymethylene-2-phenyl-2-oxazolin-5-one (phOx). sFv specific for phOx is displayed on the surface of infected cells. This feature allows binding to phOx-BSA coated magnetic beads which are used to isolate the infected cells.
Asunto(s)
Vectores Genéticos , Separación Inmunomagnética/métodos , Retroviridae , Línea Celular , Proteínas Fluorescentes Verdes , Haptenos/inmunología , Humanos , Fragmentos de Inmunoglobulinas/genética , Región Variable de Inmunoglobulina/genética , Proteínas Luminiscentes/genética , Magnetismo , Oxazoles/inmunologíaRESUMEN
Retroviral RNA molecules are plus, or sense in polarity, equivalent to mRNA. During reverse transcription, the first strand of the DNA molecule synthesized is minus-strand DNA. After the minus strand is polymerized, the plus-strand DNA is synthesized using the minus-strand DNA as the template. In this study, a helper cell line that contains two proviruses with two different mutated gfp genes was constructed. Recombination between the two frameshift mutant genes resulted in a functional gfp. If recombination occurs during minus-strand DNA synthesis, the plus-strand DNA will also contain the functional sequence. After the cell divides, all of its offspring will be green. However, if recombination occurs during plus-strand DNA synthesis, then only the plus-strand DNA will contain the wild-type gfp sequence and the minus-strand DNA will still carry the frameshift mutation. The double-stranded DNA containing this mismatch was subsequently integrated into the host chromosomal DNA of D17 cells, which were unable to repair the majority of mismatches within the retroviral double-strand DNA. After the cell divided, one daughter cell contained the wild-type gfp sequence and the other daughter cell contained the frameshift mutation in the gfp sequence. Under fluorescence microscopy, half the cells in the offspring were green and the other half of the cells were colorless or clear. Thus, we demonstrated that more than 98%, if not all, retroviral recombinations occurred during minus-strand DNA synthesis.
Asunto(s)
ADN Viral/genética , Provirus/genética , Recombinación Genética , Retroviridae/genética , Línea Celular , ADN Viral/metabolismo , Vectores Genéticos , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/genética , Mutación , Retroviridae/aislamiento & purificación , Transformación Genética , Replicación ViralRESUMEN
Retroviral vectors usually contain drug resistance genes, which are used to select for infected cells and to determine the viral titers. The viral titer is referred to as colony-forming units (CFUs). Color reporter genes, such as the lacZ gene and the green fluorescent protein gene (gfp), have been widely used as markers in retroviral vectors. In this report, a simple and rapid method for the determination of retroviral titers has been developed. The number of viral particles capable of forming individual green cells per unit volume is defined as marker-forming units (MFUs). The MFUs determined by using gfp as a marker were found to be proportional to the CFUs obtained by using drug selection for five different drug resistance genes. In addition, after adjusting the time factor, the MFUs are higher than CFUs in viruses released from 30 stable helper cell lines. The lower titers determined by CFUs are likely due to the toxicity on transduced cells.
Asunto(s)
Farmacorresistencia Microbiana/genética , Genes Reporteros/genética , Proteínas Luminiscentes/metabolismo , Retroviridae/crecimiento & desarrollo , Replicación Viral , Animales , Antibacterianos/farmacología , Recuento de Células/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Línea Celular , Color , Perros , Relación Dosis-Respuesta a Droga , Farmacorresistencia Microbiana/fisiología , Marcadores Genéticos/genética , Vectores Genéticos/genética , Proteínas Fluorescentes Verdes , Humanos , Proteínas Luminiscentes/genética , Microscopía Fluorescente , Retroviridae/genética , Factores de Tiempo , TransfecciónRESUMEN
As a consequence of being diploid viruses, members of the Retroviridae have a high recombination rate. To measure recombination between two identical sequences within the same RNA molecule per round of retroviral replication cycle, a murine leukemia virus based vector (JZ442 + 3' Hyg) has been constructed. It carries a drug resistance gene, hyg, and a 290-bp repeat sequence of the 3' hyg gene inserted into the 3' untranslated region of the green fluorescent protein gene (gfp). Under fluorescence microscopy, Hygr cells containing the recombinant proviruses were clear, while a green color was observed in the drug-resistant cells carrying the parental proviruses. The rate of recombination was determined by the ratio of the number of clear colonies to the total number of Hygr colonies (green and clear colonies). The rate of recombination was found to be 62% by this method. The intermolecular recombination rate between an infectious virus bearing two copies of the 290-bp segment and a noninfectious chimeric RNA virus containing only a single copy of this sequence was also measured.