https://www.cell.com/cell/pdf/S0092-8674(13)01602-4.pdf

Dynamic Imaging of Genomic Loci in Living Human Cells by an Optimized CRISPR/Cas System

The recent advent of methods for high-throughput single-cell molecular profiling has catalyzed a growing sense in the scientific community that the time is ripe to complete the 150-year-old effort to identify all cell types in the human body. The Human Cell Atlas Project is an international collaborative effort that aims to define all human cell types in terms of distinctive molecular profiles (such as gene expression profiles) and to connect this information with classical cellular descriptions (such as location and morphology). An open comprehensive reference map of the molecular state of cells in healthy human tissues would propel the systematic study of physiological states, developmental trajectories, regulatory circuitry and interactions of cells, and also provide a framework for understanding cellular dysregulation in human disease. Here we describe the idea, its potential utility, early proofs-of-concept, and some design considerations for the Human Cell Atlas, including a commitment to open data, code, and community.

The Human Cell Atlas

Chromosomes can be specifically stained in metaphase spreads and interphase nuclei by in situ hybridization with entire chromosome-specific DNA libraries. Unlabeled human genomic DNA is used to inhibit the hybridization of sequences in the library that bind to multiple chromosomes. The target chromosome can be made at least 20 times brighter per unit length than the others. Trisomy 21 and translocations involving chromosome 4 can be detected in metaphase spreads and interphase nuclei by using this technique.

https://www.pnas.org/content/pnas/85/23/9138.full.pdf

Fluorescence in situ hybridization with human chromosome-specific libraries: detection of trisomy 21 and translocations of chromosome 4.

Mass cytometry enables high-dimensional, single-cell analysis of cell type and state. In mass cytometry, rare earth metals are used as reporters on antibodies. Analysis of metal abundances using the mass cytometer allows determination of marker expression in individual cells. Mass cytometry has previously been applied only to cell suspensions. To gain spatial information, we have coupled immunohistochemical and immunocytochemical methods with high-resolution laser ablation to CyTOF mass cytometry. This approach enables the simultaneous imaging of 32 proteins and protein modifications at subcellular resolution; with the availability of additional isotopes, measurement of over 100 markers will be possible. We applied imaging mass cytometry to human breast cancer samples, allowing delineation of cell subpopulations and cell-cell interactions and highlighting tumor heterogeneity. Imaging mass cytometry complements existing imaging approaches. It will enable basic studies of tissue heterogeneity and function and support the transition of medicine toward individualized molecularly targeted diagnosis and therapies.

Highly multiplexed imaging of tumor tissues with subcellular resolution by mass cytometry

The toll gene of drosophila, required for dorsal-ventral embryonic polarity, appears to encode a transmembrane protein

A method is described for localizing DNA sequences hybridized in situ to Drosophila polytene chromosomes. This procedure utilizes a biotin-labeled analog of TTP that can be incorporated enzymatically into DNA probes by nick-translation. After hybridization in situ, the biotin molecules in the probe serve as antigens which bind affinity-purified rabbit antibiotin antibodies. The site of hybridization is then detected either fluorimetrically, by using fluorescein-labeled goat anti-rabbit IgG, or cytochemically, by using an anti-rabbit IgG antibody conjugated to horseradish peroxidase. When combined with Giemsa staining, the immunoperoxidase detection method provides a permanent record that is suitable for detailed cytogenetic analysis. This immunological approach offers four advantages over conventional autoradiographic procedures for detecting in situ hybrids: (i) the time required to determine the site of hybridization is decreased markedly, (ii) biotin-labeled probes are chemically stable and give reproducible results for many months; (iii) biotin-labeled probes appear to produce less background noise than do radiolabeled probes; and (iv) the resolving power is equal to and often greater than that achieved autoradiographically.

https://www.pnas.org/content/pnas/79/14/4381.full.pdf

Pennina R Langer-Safer

Papers

Immunological method for mapping genes on Drosophila polytene chromosomes

Modified nucleotides and methods for their preparation and use