RESUMEN
Our group and others have recently demonstrated the ability of recombinant baculoviruses to transduce mammalian cells at high frequency. To further characterize the use of baculovirus as a mammalian gene delivery system, we examined the status of transduced DNA stably maintained in Chinese hamster ovary (CHO) cells. Four independent clones carrying two introduced markers, the genes for neomycin resistance (Neo) and green fluorescent protein (GFP), were selected. PCR analysis, Southern blotting, and DNA sequencing showed that discrete portions of the 148-kb baculovirus DNA were present as single-copy fragments ranging in size from 5 to 18 kb. Integration into the CHO cell genome was confirmed by fluorescent in situ hybridization (FISH) analysis. For one clone, the left and right viral/chromosomal junctions were determined by DNA sequencing of inverse PCR products. Similarly, for a different clone, the left viral/chromosomal junction was determined; however, the right junction sequence revealed the joining to another viral fragment by a short homology (microhomology), a hallmark of illegitimate recombination. The random viral breakpoints and the lack of homology between the virus and flanking chromosomal sequences are also suggestive of an illegitimate integration mechanism. To examine the long-term stability of reporter gene expression, all four clones were grown continuously for 36 passages in either the presence or absence of selection for Neo. Periodic assays over a 5-month period showed no loss of GFP expression for at least two of the clones. This report represents the first detailed analysis of baculovirus integrants within mammalian cells. The potential advantages of the baculovirus system for the stable integration of genetic material into mammalian genomes are discussed.
Asunto(s)
Baculoviridae/genética , ADN Recombinante/genética , Vectores Genéticos , Transducción Genética , Integración Viral , Animales , Secuencia de Bases , Southern Blotting , Células CHO , Cricetinae , Genes Reporteros , Proteínas Fluorescentes Verdes , Hibridación Fluorescente in Situ , Proteínas Luminiscentes/biosíntesis , Proteínas Luminiscentes/genética , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa/métodos , Proteínas Recombinantes/biosíntesis , Análisis de Secuencia de ADNRESUMEN
Previous work in our laboratory using functional assays for tumorigenicity identified a tumor suppressor element on human chromosome 11q for the cutaneous squamous cell carcinoma cell line A388.6TG.c2. In this report, we screened a variety of agents for differential effects on A388.6TG.c2 compared to a growth-suppressed chromosome 11 microcell hybrid of A388.6TG.c2. One of the agents, 1, 25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3); calcitriol), exerted a growth-altering effect on A388.6TG.c2, which formed rounded cell clusters across the surface of the raft by Day 6 of treatment. In contrast, full-length chromosome 11 hybrids of A388.6TG.c2, as well as two other squamous cell carcinoma cell lines (FaDu and A431), when treated with 1,25(OH)(2)D(3), failed to demonstrate this cell-clumping phenotype. To pursue the hypothesis that the growth suppressor element is involved in altering the response to 1, 25(OH)(2)D(3), we tested microcell hybrids carrying t(X;11) chromosomes lacking large portions of 11q. Although these hybrids, like the parent A388.6TG.c2 cells, demonstrated extensive growth in organotypic cultures, they failed to form cell clusters with 1, 25(OH)(2)D(3) treatment. These results suggest that the chromosome 11 element that alters the response to 1,25(OH)(2)D(3) is distinct from the growth-suppressing element. An examination of differentiation marker expression revealed identical patterns of basal and suprabasal markers for A388.6TG.c2 and for a chromosome 11 hybrid with or without treatment with 1,25(OH)(2)D(3). Finally, characterization of candidate tumor suppressor gene PPP2R1B, which encodes for a subunit of protein phosphatase 2A (PP2A), showed seemingly insignificant alterations by cDNA sequence analysis. Collectively, the data suggest that human chromosome 11 contains two different tumor suppressor elements that may account for the two areas of loss of heterozygosity observed on the long arm of this chromosome.
Asunto(s)
Calcitriol/farmacología , Agonistas de los Canales de Calcio/farmacología , Carcinoma de Células Escamosas , Cromosomas Humanos Par 11 , Neoplasias Cutáneas , Anciano , Anciano de 80 o más Años , Agregación Celular/efectos de los fármacos , Agregación Celular/genética , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Regulación Neoplásica de la Expresión Génica , Genes Supresores de Tumor/genética , Humanos , Células Híbridas/citología , Células Híbridas/efectos de los fármacos , Pérdida de Heterocigocidad , Masculino , Fenotipo , Fosfoproteínas Fosfatasas/genética , Proteína Fosfatasa 2 , Células Tumorales CultivadasRESUMEN
Several DNA sequence elements are thought to stimulate homologous recombination, illegitimate recombination, or both in mammalian cells. Some are implicated by their recurrence around rearrangement breakpoints, others by their effects on recombination of extrachromosomal plasmids. None of these sequences, however, has been tested on the chromosome in a defined context. In this paper we show how the adenine phosphoribosyltransferase locus in CHO cells can be used to study the recombinogenic potential of defined DNA sequences. As an example we have measured the effect on homologous recombination of a dinucleotide repeat, (GT)29, which has been shown to stimulate homologous recombination in extrachromosomal vectors 3-20 fold. On the chromosome at the adenine phosphoribosyltransferase locus, however, this sequence shows no capacity to stimulate recombination or to influence the distribution of recombination events.
Asunto(s)
Adenina Fosforribosiltransferasa/genética , ADN Satélite/genética , ADN/genética , Repeticiones de Microsatélite/genética , Recombinación Genética , Animales , Secuencia de Bases , Células CHO , Cricetinae , Datos de Secuencia Molecular , Análisis de SecuenciaRESUMEN
To examine the mechanisms of recombination governing the illegitimate integration of transfected DNA into a mammalian genome, we developed a cell system that selects for integration events in defined genomic regions. Cell lines with chromosomal copies of the 3' portion of the adenine phosphoribosyltransferase (APRT) gene (targets) were established. The 5' portion of the APRT gene, which has no homology to the integrated 3' portion, was then electroporated into the target cell lines, and selection for APRT gene function was applied. The reconstruction of the APRT gene was detected at frequencies ranging from less than 10(-7) to 10(-6) per electroporated cell. Twenty-seven junction sequences between the integrated 5' APRT and its chromosomal target were analyzed. They were found to be randomly distributed in a 2-kb region immediately in front of the 3' portion of the APRT gene. The junctions fell into two main classes: those with short homologies (microhomologies) and those with inserted DNA of uncertain origin. Three long inserts were shown to preexist elsewhere in the genome. Reconstructed cell lines were analyzed for rearrangements at the target site by Southern blotting; a variety of simple and complex rearrangements were detected. Similar analysis of individual clones of the parental cell lines revealed analogous types of rearrangement, indicating that the target sites are unstable. Given the high frequency of integration events at these sites, we speculate that transfected DNA may preferentially integrate at unstable mammalian loci. The results are discussed in relation to possible mechanisms of DNA integration.
Asunto(s)
ADN/genética , Recombinación Genética , Transfección , Adenina Fosforribosiltransferasa/genética , Animales , Secuencia de Bases , Células CHO , Cricetinae , Cartilla de ADN/genética , Elementos Transponibles de ADN , Reordenamiento Génico , Genoma , Modelos Genéticos , Datos de Secuencia Molecular , Reacción en Cadena de la PolimerasaRESUMEN
The FLP/FRT site-specific recombination system was established and characterized at the APRT gene in CHO cells. Targeting frequencies with FLP-stimulation were about 1 to 5 X 10(-5), which were 6-22-fold above gene targeting frequencies in the absence of FLP. Fifty two APRT+ cell lines were analyzed by Southern blotting: 56% were FLP-targeted integrants; 33% were APRT target convertants; 11% gave undefined patterns. In separate experiments we first enriched for integrants by screening for two additional markers carried on the targeting vector; 18 of 19 (95%) of the resulting cell lines were integrants. Intrachromosomal site-specific recombination was tested by reexposing integrants to FLP. Intrachromosomal popouts were stimulated over 200-fold, while homologous recombination in an adjacent interval was unchanged. The utility of this system was demonstrated by one-step FLP targeting to generate chromosomal substrates for homologous recombination, and by a two-step, FLP-and-run procedure to construct a chromosomal substrate for illegitimate recombination.