Towards Development of the 4C-Based Method Detecting Interactions of Plasmid DNA with Host Genome.

Chromosome conformation capture techniques have revolutionized our understanding of chromatin architecture and dynamics at the genome-wide scale. In recent years, these methods have been applied to a diverse array of species, revealing fundamental principles of chromosomal organization. However, structural organization of the extrachromosomal entities, like viral genomes or plasmids, and their interactions with the host genome, remain relatively underexplored.

Modification of the Hi-C Technology for Molecular Genetic Analysis of Formalin-Fixed Paraffin-Embedded Sections of Tumor Tissues.

Molecular genetic analysis of tumor tissues is the most important step towards understanding the mechanisms of cancer development; it is also necessary for the choice of targeted therapy. The Hi-C (high-throughput chromatin conformation capture) technology can be used to detect various types of genomic variants, including balanced chromosomal rearrangements, such as inversions and translocations. We propose a modification of the Hi-C method for the analysis of chromatin contacts in formalin-fixed paraffin-embedded (FFPE) sections of tumor tissues.

Method for the Isolation of "RNA-seq-Quality" RNA from Human Intervertebral Discs after Mortar and Pestle Homogenization.

The problem of isolating high-quality total RNA from intervertebral discs has no recognized solution yet. This is due to the extremely low content of live cells in the samples and the voluminous intercellular matrix. A variety of published protocols focused on isolating RNA from articular cartilage have recommended the use of expensive equipment, enzymatic matrix cleavage, or cell culture.

Differential DNA Methylation of the IMMP2L Gene in Families with Maternally Inherited 7q31.1 Microdeletions is Associated with Intellectual Disability and Developmental Delay.

Most copy number variations (CNVs) in the human genome display incomplete penetrance with unknown underlying mechanisms. One such mechanism may be epigenetic modification, particularly DNA methylation. The IMMP2L gene is located in a critical region for autism susceptibility on chromosome 7q (AUTS1).

Postsynthetic On-Column 2' Functionalization of RNA by Convenient Versatile Method.

We report a universal straightforward strategy for the chemical synthesis of modified oligoribonucleotides containing functional groups of different structures at the 2' position of ribose. The on-column synthetic concept is based on the incorporation of two types of commercial nucleotide phosphoramidites containing orthogonal 2'--protecting groups, namely 2'--thiomorpholine-carbothioate (TC, as "permanent") and 2'---butyl(dimethyl)silyl (BDMS, as "temporary"), to RNA during solid-phase synthesis. Subsequently, the support-bound RNA undergoes selective deprotection and follows postsynthetic 2' functionalization of the naked hydroxyl group.

Targeted genomic integration of EGFP under tubulin beta 3 class III promoter and mEos2 under tryptophan hydroxylase 2 promoter does not produce sufficient levels of reporter gene expression.

Neuronal tracing is a modern technology that is based on the expression of fluorescent proteins under the control of cell type-specific promoters. However, random genomic integration of the reporter construct often leads to incorrect spatial and temporal expression of the marker protein. Targeted integration (or knock-in) of the reporter coding sequence is supposed to provide better expression control by exploiting endogenous regulatory elements.

A mosaic intragenic microduplication of LAMA1 and a constitutional 18p11.32 microduplication in a patient with keratosis pilaris and intellectual disability.

The application of array-based comparative genomic hybridization and next-generation sequencing has identified many chromosomal microdeletions and microduplications in patients with different pathological phenotypes. Different copy number variations are described within the short arm of chromosome 18 in patients with skin diseases. In particular, full or partial monosomy 18p has also been associated with keratosis pilaris.

Allele-Specific Biased Expression of the CNTN6 Gene in iPS Cell-Derived Neurons from a Patient with Intellectual Disability and 3p26.3 Microduplication Involving the CNTN6 Gene.

Copy number variations (CNVs) of the human CNTN6 gene caused by megabase-scale microdeletions or microduplications in the 3p26.3 region are often the cause of neurodevelopmental disorders, including intellectual disability and developmental delay. Surprisingly, patients with different copy numbers of this gene display notable overlapping of neuropsychiatric symptoms.

Alternative dominance of the parental genomes in hybrid cells generated through the fusion of mouse embryonic stem cells with fibroblasts.

For the first time, two types of hybrid cells with embryonic stem (ES) cell-like and fibroblast-like phenotypes were produced through the fusion of mouse ES cells with fibroblasts. Transcriptome analysis of 2,848 genes differentially expressed in the parental cells demonstrated that 34-43% of these genes are expressed in hybrid cells, consistent with their phenotypes; 25-29% of these genes display intermediate levels of expression, and 12-16% of these genes maintained expression at the parental cell level, inconsistent with the phenotype of the hybrid cell. Approximately 20% of the analyzed genes displayed unexpected expression patterns that differ from both parents.

Deterministic versus stochastic model of reprogramming: new evidence from cellular barcoding technique.

Factor-mediated reprogramming of somatic cells towards pluripotency is a low-efficiency process during which only small subsets of cells are successfully reprogrammed. Previous analyses of the determinants of the reprogramming potential are based on average measurements across a large population of cells or on monitoring a relatively small number of single cells with live imaging. Here, we applied lentiviral genetic barcoding, a powerful tool enabling the identification of familiar relationships in thousands of cells.

Comparison of the three-dimensional organization of sperm and fibroblast genomes using the Hi-C approach.

Background: The three-dimensional organization of the genome is tightly connected to its biological function. The Hi-C approach was recently introduced as a method that can be used to identify higher-order chromatin interactions genome-wide. The aim of this study was to determine genome-wide chromatin interaction frequencies using the Hi-C approach in mouse sperm cells and embryonic fibroblasts.

Allelic expression and DNA methylation profiles of promoters at the parental Oct4 and Nanog genes in Mus musculus ES cell/Mus caroli splenocyte hybrid cells.

Expression of the parental Oct4 and Nanog alleles and DNA methylation of their promoters were studied in a set of Mus musculus embryonic stem (ES) cell/M. caroli splenocyte hybrid cells containing a variable ratio of parental chromosomes 6 and 17. The transcripts of the reactivated splenocyte Oct4 and Nanog genes were revealed in all hybrid cell clones positive for M.