Method For Human Cells Research Articles

Direct optical measurement of intramolecular distances with angstrom precision.

Optical investigations of nanometer distances between proteins, their subunits, or other biomolecules have been the exclusive prerogative of Förster resonance energy transfer (FRET) microscopy for decades. In this work, we show that MINFLUX fluorescence nanoscopy measures intramolecular distances down to 1 nanometer-and in planar projections down to 1 angstrom-directly, linearly, and with angstrom precision. Our method was validated by quantifying well-characterized 1- to 10-nanometer distances in polypeptides and proteins. Moreover, we visualized the orientations of immunoglobulin subunits, applied the method in human cells, and revealed specific configurations of a histidine kinase PAS domain dimer. Our results open the door for examining proximities and interactions by direct position measurements at the intramacromolecular scale.

Advantages of a 21-loci short tandem repeat method for detection of cross-contamination in human cell lines.

Cross-contamination of cell lines is a highly relevant and pervasive problem. The analysis of short tandem repeats (STR) is a simple and commercially available technique to authenticate cell lines for more than two decades. At present, STR multiple amplification kits have been developed up to 21 loci while the current STR databases only provide 9-loci STR profiles. Here, we compared the advantages of 21-loci STR methodology using the same algorithm as 9-loci method. The 21-loci method reduced the uncertainty ratio for authentications by 97.5% relative to the 9-loci method and exclude effectively false positive. We show that the additional 12 loci helped to greatly reduce sample-site marker specificity arising from genetic isolation and the occurrence of null alleles, suggesting that inclusion of additional loci in these databases will ultimately improve the efficiency and accuracy of authentication of cell lines. Taken together, we demonstrate the utility of a 21-loci method in human cells, providing a novel marker panel for use as a valuable alternative to 9-loci analyses to minimize cell line authentication errors and reduce costs due to erroneous experiments.

Centromere Chromosome Orientation Fluorescent in situ Hybridization (Cen-CO-FISH) Detects Sister Chromatid Exchange at the Centromere in Human Cells.

Human centromeres are composed of large tandem arrays of repetitive alpha satellite DNA, which are often sites of aberrant rearrangement in cancers ( Mitelman et al., 1997 ; Padilla- Nash et al., 2001 ). To date, annotation of the human centromere repetitive sequences remains incomplete, greatly hindering in-depth functional studies of these regions essential for chromosome segregation. In order to monitor sister chromatid exchange happening at the centromere (C-SCE) due to recombination and mutagenic events, I have applied the Chromosome-Orientation Fluorescence in situ Hybridization (CO-FISH) technique to centromeres (Cen-CO-FISH) in human cells. This hybridization-based method involves (1) the incorporation of nucleotide analogs through a single round of replication, (2) enzymatic digestion of the newly synthesized DNA strand and (3) subsequent hybridization of single-stranded probes, in absence of a denaturation step. The resulting signal allows to differentially label each sister chromatid based on the 5'-3' directionality of the DNA and to score aberrant staining patterns indicative of C-SCE. The Cen-CO-FISH method applied to human centromeres revealed that human centromeres indeed undergo recombination in cycling cells resulting in C-SCE, and centromere instability is enhanced in cancer cell lines and primary cells undergoing senescence (Giunta and Funabiki, 2017). Here, I present the detailed protocol of the preparation, experimental procedure and data acquisition for the Cen-CO-FISH method in human cells. It also includes a conceptual overview of the technique, with examples of representative images and scoring guidelines. The Cen-CO-FISH represents a valuable tool to facilitate exploration of centromere repeats.

Global Mapping of Human RNA-RNA Interactions

The majority of the human genome is transcribed intonon-coding (nc)RNAs that lack known biologicalfunctions or else are only partially characterized. Numerous characterized ncRNAs function via base pairing with target RNA sequences to direct their biological activities, which include critical roles in RNA processing, modification, turnover, and translation. To define roles for ncRNAs, we have developed a method enabling the global-scale mapping of RNA-RNA duplexes crosslinked invivo, "LIGation of interacting RNA followed by high-throughput sequencing" (LIGR-seq). Applying this method in human cells reveals a remarkable landscape of RNA-RNA interactions involving all major classes of ncRNA and mRNA. LIGR-seq data reveal unexpected interactions between small nucleolar (sno)RNAs and mRNAs, including those involving the orphan C/D box snoRNA, SNORD83B, that control steady-state levels of its target mRNAs. LIGR-seq thus represents a powerful approach for illuminating the functions of the myriad of uncharacterized RNAs that act via base-pairing interactions.

Regulation of global gene expression and cell proliferation by APP.

Down syndrome (DS), caused by trisomy of chromosome 21, is one of the most common genetic disorders. Patients with DS display growth retardation and inevitably develop characteristic Alzheimer’s disease (AD) neuropathology, including neurofibrillary tangles and neuritic plaques. The expression of amyloid precursor protein (APP) is increased in both DS and AD patients. To reveal the function of APP and elucidate the pathogenic role of increased APP expression in DS and AD, we performed gene expression profiling using microarray method in human cells overexpressing APP. A set of genes are significantly altered, which are involved in cell cycle, cell proliferation and p53 signaling. We found that overexpression of APP inhibits cell proliferation. Furthermore, we confirmed that the downregulation of two validated genes, PSMA5 and PSMB7, inhibits cell proliferation, suggesting that the downregulation of PSMA5 and PSMB7 is involved in APP-induced cell proliferation impairment. Taken together, this study suggests that APP regulates global gene expression and increased APP expression inhibits cell proliferation. Our study provides a novel insight that APP overexpression may contribute to the growth impairment in DS patients and promote AD pathogenesis by inhibiting cell proliferation including neural stem cell proliferation and neurogenesis.

Targeted chromosomal deletions in human cells using zinc finger nucleases

We present a novel approach for generating targeted deletions of genomic segments in human and other eukaryotic cells using engineered zinc finger nucleases (ZFNs). We found that ZFNs designed to target two different sites in a human chromosome could introduce two concurrent DNA double-strand breaks (DSBs) in the chromosome and give rise to targeted deletions of the genomic segment between the two sites. Using this method in human cells, we were able to delete predetermined genomic DNA segments in the range of several-hundred base pairs (bp) to 15 mega-bp at frequencies of 10(-3) to 10(-1). These high frequencies allowed us to isolate clonal populations of cells, in which the target chromosomal segments were deleted, by limiting dilution. Sequence analysis revealed that many of the deletion junctions contained small insertions or deletions and microhomologies, indicative of DNA repair via nonhomologous end-joining. Unlike other genome engineering tools such as recombinases and meganucleases, ZFNs do not require preinsertion of target sites into the genome and allow precise manipulation of endogenous genomic scripts in animal and plant cells. Thus, ZFN-induced genomic deletions should be broadly useful as a novel method in biomedical research, biotechnology, and gene therapy.