New fluorescently tagged cell lines available, including first ‘silent’ tagged line
June 19, 2018
The Allen Cell Collection now contains five new fluorescently tagged stem cell lines, including the first cells in the collection with a tag specific to heart muscle cells, or cardiomyocytes. To date, all the cell lines have been gene edited to carry a fluorescent marker that is produced in undifferentiated cells, before they go on to become specific cell types, like cardiomyocytes, nerve cells or liver cells.
Time-lapse movie of cardiomyocytes expressing a fluorescently tagged protein that lights up the cells’ sarcomeres, long muscle cell fibers.
Tagging a structure that is not present in the cell’s nascent, undifferentiated state means tagging something you can’t see. The gene that is tagged in the new line in the collection is known as troponin I1, slow skeletal type (TNNI1); it only becomes active as the cells develop into cardiomyocytes. The troponin I, slow skeletal muscle protein is part of muscle cell structures known as sarcomeres, long fibers that help the cells do the work of contracting when muscles are active.
To achieve this silent gene editing, the cell science team stitches the fluorescent tag specific to the troponin gene to another tag that will light up in the undifferentiated stem cells, allowing researchers to identify the cells where the gene editing has worked. They then use a genetic trick to cut out the initial selection tag which is active in the undifferentiated stem cell, leaving only the cardiomyocyte-specific gene and its fluorescent tag.
The researchers have several more heart cell-specific tags in the works that will be available in the collection in the coming months.
The cell lines released in May and June bear tags marking the proteins:
- Troponin I, slow skeletal muscle (TNNI1), the cardiomyocyte-specific protein.
- Connexin-43 (GJA1), which is part of cells’ gap junctions, the channels between cells that allow them to exchange small molecules.
- Nucleophosmin (NPM1), a protein that sits in the nucleolus, a structure inside the cell’s nucleus.
- Beta-catenin (CTNNB1), a protein that is also involved in cell-cell adhesion and differentiation.
- CAAX, the membrane-targeting domain of K-Ras, tagged with mTagRFP-T under the control of a CAG promoter inserted at the safe harbor locus (AAVS1). mTagRFP-T-CAAX localizes to the plasma membrane that surrounds the periphery of the cell.