RESUMEN
Recombinant adeno-associated virus (rAAV) is the leading platform of gene delivery for its long-lasting gene transformation and low immunogenicity. Characterization of the integrity and purity of the rAAV genome is critical to ensure clinical potency and safety. However, current rAAV genome characterization methods that can provide size assessment are either time-consuming or not easily accessible to general labs. Additionally, there is a lack of right reference standard for analyzing long single-stranded DNA (ssDNA) fragments. Here, we have developed an ssDNA assay on a microfluidic capillary electrophoresis platform using ssDNA reference standard. This assay provides size calling for ssDNA fragment, a detection sensitivity at â¼89 pg/µL (3 × 1010 GC/mL AAV) for 5.1 kb ssDNA fragment, and a turnaround time at â¼100 s per sample with a high throughput sample analyzing capability. Moreover, we have observed that the annealing of AAV ssDNA subsequent to its release from the capsid might introduce an additional double-stranded DNA (dsDNA) peak. This phenomenon is dependent on the sample processing workflow. To avoid the risk of mischaracterization, we recommend the use of dual-reference standards in combination with other orthogonal methods to have a comprehensive understanding of the rAAV genome size and integrity.
RESUMEN
Biological maintenance of cells under variable conditions should affect gene expression of only certain genes while leaving the rest unchanged. The latter, termed "housekeeping genes," by definition must reflect no change in their expression levels during cell development, treatment, or disease state anomalies. However, deviations from this rule have been observed. Using DNA microarray technology, we report here variations in expression levels of certain housekeeping genes in prostate cancer and a colorectal cancer gene therapy model system. To highlight, differential expression was observed for ribosomal protein genes in the prostate cancer cells and beta-actin in treated colorectal cells. High-throughput differential gene expression analysis via microarray technology and quantitative PCR has become a common platform for classifying variations in similar types of cancers, response to chemotherapy, identifying disease markers, etc. Therefore, normalization of the system based on housekeeping genes, such as those reported here in cancer, must be approached with caution.
Asunto(s)
Algoritmos , Biomarcadores de Tumor/metabolismo , Neoplasias Colorrectales/metabolismo , Perfilación de la Expresión Génica/métodos , Proteínas de Neoplasias/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Neoplasias de la Próstata/metabolismo , Biomarcadores de Tumor/genética , Calibración , Línea Celular Tumoral , Neoplasias Colorrectales/genética , Perfilación de la Expresión Génica/normas , Humanos , Células Jurkat , Masculino , Proteínas de Neoplasias/genética , Análisis de Secuencia por Matrices de Oligonucleótidos/normas , Neoplasias de la Próstata/genética , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
The env gene of three simian immunodeficiency virus (SIV) variants developed convergent mutations during disease progression in six rhesus macaques. The monkeys had been inoculated with supercoiled plasmids encoding infectious proviruses of SIVmac239 (a pathogenic, wild-type strain), SIVdelta3 (the live attenuated vaccine strain derived from SIVmac239), or SIVdelta3+ (a pathogenic progeny virus that had evolved from SIVdelta3). All six monkeys developed immunodeficiency and progressed to fatal disease. Although many divergent mutations arose in env among the different hosts, three regions consistently mutated in all monkeys studied; these similar mutations developed independently even though the animals had received only a single infectious molecular clone rather than standard viral inocula that contain viral quasispecies. Together, these data indicate that the env genes of SIVmac239, SIVdelta3, and SIVdelta3+, in the context of different proviral backbones, evolve similarly in different hosts during disease progression.
Asunto(s)
ADN Viral/genética , Evolución Molecular , Productos del Gen env/genética , Macaca mulatta/virología , Provirus/genética , Provirus/fisiología , Virus de la Inmunodeficiencia de los Simios/genética , Secuencia de Aminoácidos , Animales , Productos del Gen env/química , Datos de Secuencia Molecular , Mutación/genética , Filogenia , Síndrome de Inmunodeficiencia Adquirida del Simio , ViremiaRESUMEN
mda-7 is a novel tumor suppressor with cytokine properties. Adenoviral mda-7 (Ad-mda7) induces apoptosis and cell death selectively in tumor cells. The molecular mechanisms underlying the anti-tumor activity of Ad-mda7 in breast and lung cancer lines were investigated. Microarray analyses implicated both the beta-catenin and the PI3K signaling pathways. Ad-mda7 treatment increased protein expression from tumor suppressor genes, including E-cadherin, APC, GSK-3beta, and PTEN, and decreased expression of proto-oncogenes involved in beta-catenin and PI3K signaling. Ad-mda7 caused a redistribution of cellular beta-catenin from the nucleus to the plasma membrane, resulting in reduced TCF/LEF transcriptional activity, and upregulated the E-cadherin-beta-catenin adhesion complex in a tumor cell-specific manner. Expression of the PI3K pathway members (p85 PI3K, FAK, ILK-1, Akt, and PLC-gamma) was downregulated and expression of the PI3K antagonist PTEN was increased. Consistent with this result, pharmacological inhibition of PI3K by wortmannin did not abrogate killing by Ad-mda7. Killing of breast cancer cells by Ad-mda7 required both MAPK and MEK1/2 signaling pathways, whereas these pathways were not essential for MDA-7-mediated killing in lung cancer cells. Thus, in breast and lung tumor cells MDA-7 protein expression modulates cell-cell adhesion and intracellular signaling via coordinate regulation of the beta-catenin and PI3K pathways.