Analysis of Array-CGH Data Using the R and Bioconductor Software Suite

Background. Array-based comparative genomic hybridization (array-CGH) is an emerging high-resolution and high-throughput molecular genetic technique that allows genome-wide screening for chromosome alterations. DNA copy number alterations (CNAs) are a hallmark of somatic mutations in tumor genomes and congenital abnormalities that lead to diseases such as mental retardation. However, accurate identification of amplified or deleted regions requires a sequence of different computational analysis steps of the microarray data. Results. We have developed a user-friendly and versatile tool for the normalization, visualization, breakpoint detection, and comparative analysis of array-CGH data which allows the accurate and sensitive detection of CNAs. Conclusion. The implemented option for the determination of minimal altered regions (MARs) from a series of tumor samples is a step forward in the identification of new tumor suppressor genes or oncogenes

Use of cultivated osteoprogenitor cells to increase bone formation in segmental mandibular defects: an experimental pilot study in sheep

The hypothesis of the present experimental pilot study was that autogeneous cultivated osteoprogenitor cells in porous calcium phosphate scaffolds can increase bone formation in segmental defects of the mandible. The autogenous osteoprogenitor cells of eight sheep were cultivated from bone biopsies from the iliac crest and seeded into cylindrical scaffolds of pyrolized bovine bone of an overall length of 35 mm and 13 mm in diameter, Segmental defects of 35 mm length were created unilaterally in the mandibles of the animals. Reconstruction was performed using cylinders with cultivated osteoprogenitor cells in four animals and empty scaffolds in the remaining four sheep, which served as controls. After 5 months, the mandibles were retrieved and the reconstructed areas were analyzed by qualitative and quantitative histology in serial undecalcified thick-section specimens. There was significantly more bone formation in the group that had received scaffolds with cultivated bone cells (P=0.028). Bone formation was present in 34.4% of the evaluated cross-sectional units in the seeded scaffolds. while it was found in 10.4% in the control group, Although the spatial distribution of bone formation was significantly different across the scaffold in both groups, osteoprogenitor cells appeared to have increased bone formation, particularly in the centre of the defect when compared to the control group. It is concluded that the repair of segmental defects of the mandible can be enhanced by the transplantation of autogenous osteoprogenitor cells in a porous calcium phosphate scaffold

Improved bi-allelic modification of a transcriptionally silent locus in patient-derived iPSC by Cas9 nickase

Homology directed repair (HDR)-based genome editing via selectable long flanking arm donors can be hampered by local transgene silencing at transcriptionally silent loci. Here, we report efficient bi-allelic modification of a silent locus in patient-derived hiPSC by using Cas9 nickase and a silencing-resistant donor construct that contains an excisable selection/counter-selection cassette. To identify the most active single guide RNA (sgRNA)/nickase combinations, we employed a lentiviral vector-based reporter assay to determine the HDR efficiencies in cella. Next, we used the most efficient pair of sgRNAs for targeted integration of an improved, silencing-resistant plasmid donor harboring a piggyBac-flanked puro Delta tk cassette. Moreover, we took advantage of a dual-fluorescence selection strategy for bi-allelic targeting and achieved 100% counter-selection efficiency after bi-allelic excision of the selection/counter-selection cassette. Together, we present an improved system for efficient bi-allelic modification of transcriptionally silent loci in human pluripotent stem cells