In situ hybridization

In situ hybridization (ISH) is a type of hybridization that uses a labeled complementary DNA, RNA or modified nucleic acids strand (i.e., probe) to localize a specific DNA or RNA sequence in a portion or section of tissue (in situ) or if the tissue is small enough (e.g., plant seeds, Drosophila embryos), in the entire tissue (whole mount ISH), in cells, and in circulating tumor cells (CTCs). This is distinct from immunohistochemistry, which usually localizes proteins in tissue sections. In situ hybridization is used to reveal the location of specific nucleic acid sequences on chromosomes or in tissues, a crucial step for understanding the organization, regulation, and function of genes. The key techniques currently in use include in situ hybridization to mRNA with oligonucleotide and RNA probes (both radio-labeled and hapten-labeled), analysis with light and electron microscopes, whole mount in situ hybridization, double detection of RNAs and RNA plus protein, and fluorescent in si

Insight into the Regulation of Telomerase Access to Telomeres by Shelterin Proteins

Elena Aritonovska

Recruitment to telomeres is a pivotal step in the function and regulation of human telomerase. Impaired telomerase function can lead to premature organismal aging, development of cancer and multisystem disorders such as dyskeratosis congenita. Telomerase access to telomeres may be regulated by a telomere-binding complex termed shelterin, which is composed of TRF1, TRF2, RAP1, TIN2, TPP1 and POT1 proteins. However the molecular basis for human telomerase recruitment to telomeres is not known. Here, we have directly investigated the process of telomerase recruitment via chromatin immunoprecipitation (ChIP) and fluorescence in situ hybridization (FISH). We find that depletion of two components of the shelterin complex – TPP1 and the protein that tethers TPP1 to the complex, TIN2 – results in a loss of telomerase recruitment. On the other hand, we find that the majority of the observed telomerase association with telomeres does not require POT1, the shelterin protein that links TPP1 to the single-stranded region of the telomere. Furthermore, we find that the double-stranded telomere binding protein, TRF2 is dispensable for telomerase association with telomeres. Deletion of the oligonucleotide/oligosaccharide-binding fold (OB-fold) of TPP1 further disrupts telomerase recruitment. In addition, while loss of TPP1 results in the appearance of DNA damage factors at telomeres, the DNA damage response per se does not account for the telomerase recruitment defect observed in the absence of TPP1. Our findings indicate that TIN2-anchored TPP1 plays a major role in the recruitment of telomerase to telomeres in human cells and that the recruitment does not depend on POT1 or interaction of the shelterin complex with the single-stranded region of the telomere. We propose that the loss of TRF2 can be compensated by the second double-stranded telomere binding protein, TRF1 that anchors TIN2/TPP1-recruited telomerase onto the telomeres. Dyskeratosis congenita (DC) is a multisystem disorder characterized with bone marrow failure, cancer predisposition and defective telomere maintenance. The TINF2 gene that encodes for the TIN2 shelterin protein is one of seven mutated genes identified in DC patients. Here, we have examined the effects of TIN2 DC mutants on telomere protection and shelterin protein stability. We find that exogenous expression of TIN2 DC mutants can rescue the TIN2-depleted phenotype, restoring TPP1 protein levels and telomere protection. Our findings indicate that TIN2 DC mutants preserve the intact telomere structure and protection. We propose that defective telomere elongation may be the underlying cause of impaired telomere maintenance in TIN2 DC patients.

EPFL2011

Study of Cell Adhesion and Tracking Methods for in Vivo Cell Localisation in a Tissue-Engineering Scaffold

Thaïs Cléa Laura Séverine Clouet, Tanja Cloé Hausherr

Bone fracture healing problems, because of the loss of tissue regeneration, can affect many old or multi-pathologic patients around the world. Among the therapeutic strategies, bone substitutes, such as poly-(L-lactitve acid) with 5% wt. % ß-tricalcium phosphate (PLA/5%ß-TCP) scaffolds, are promising as a clinical solution. One strategy to tune their osteoinductive properties is the addition of bone progenitor cells. Therefore, cell- free and cell-seeded PLA/5% ß-TCP scaffolds were implanted into femoral condyles of rats to promote bone regeneration. The in vivo study showed that the bone growth inside a cell-seeded scaffold with human bone progenitor cells was delayed compared to cell-free scaffold. What happened to the cells? Did they trigger an immune response, migrate or die? Therefore, the goals of this project are to evaluate the cell adhesion in vitro to the scaffold and to propose a reliable cell labeling method to track them in future in vivo studies. In the first aim, the adhesion of the cells was qualitatively assessed by the cell morphology and the presence of fibronectin using fluorescence imaging. To quantify their adherence, seeded scaffolds were washed up to 3 times with a 0.9 % NaCl solution. The luminescence of the scaffolds and the washing solutions was measured using a cell viability assay. These experiments demonstrated that after 72h of culture, the measured adherent bone progenitor cells on the scaffolds decreased after 3 washes down to 50 % while it stayed stable after 14 days of culture. In the second aim, we focused on methods to label the cells either once implanted or prior to implantation. The first method was to translate the in situ hybridization protocol with the human specific Alu sequence from paraffin embedded samples to resin embedded ones. The obtained results were so far inconclusive. Concerning the labeling of the cells before implantation, the transmembrane label PKH26 was investigated. Its staining efficacy was high just after labeling, while it decreased drastically over time due to cell division.

2015

Study of Cell Adhesion and Tracking Methods for in Vivo Cell Localisation in a Tissue-Engineering Scaffold

Thaïs Cléa Laura Séverine Clouet, Tanja Cloé Hausherr

2015

Insight into the Regulation of Telomerase Access to Telomeres by Shelterin Proteins

Elena Aritonovska

EPFL2011

Tracing toluene-assimilating sulfate-reducing bacteria using 13C-incorporation in fatty acids and whole-cell hybridization

Insight into the Regulation of Telomerase Access to Telomeres by Shelterin Proteins

Study of Cell Adhesion and Tracking Methods for in Vivo Cell Localisation in a Tissue-Engineering Scaffold

Study of Cell Adhesion and Tracking Methods for in Vivo Cell Localisation in a Tissue-Engineering Scaffold

Tracing toluene-assimilating sulfate-reducing bacteria using 13C-incorporation in fatty acids and whole-cell hybridization

Insight into the Regulation of Telomerase Access to Telomeres by Shelterin Proteins