What are the Techniques Used in Lineage Tracing Cells? In lineage tracing cells, the cells are marked in order to identify by the expression of a reporter gene. It involves the marking of cells either with dyes or nucleoside analogs. Also, the genetic marking of cells involves the marking with fluorescent or colorimetric protein reporters. When the cell divides, the expression of the reporter gene is passed to the progeny. Therefore, with the marker gene, the migration of cells can be easily identified.
Lineage tracing is a technique to investigate biological processes such as embryonic development, stem cell function in regeneration and disease, homeostatic tissue turnover, etc. Typical reporter genes include β-galactosidase or fluorescent protein.
Key Areas Covered
2. What is the Principle of Lineage Tracing cells
– Definition, Features, Importance
2. What are the Techniques Used in Lineage Tracing Cells
– Description of Techniques
Key Terms
Lineage Tracing Cells, Techniques
What is the Principle of Lineage Tracing cells
Lineage tracing cells is a technique that describes all progeny of a single cell or a group of cells. It is important in developmental biology and stem cell biology. In lineage tracing cell experiments, cells are marked at a one-time point, and that marked cell reveals another time point of its development. Therefore, a cell lineage describes the developmental history of a particular cell in the embryo until it becomes a tissue or organ. Hence, in lineage tracing cells, the cellular ancestry of an organism can be determined by means of cell division and relocation of cells. It originates from the stem cells and finishes from mature cells. It also gives the developmental fate of the early cleavage egg to its clonal progeny.
Figure 1: Liver Cell Lineage
Furthermore, developmental biologists used numerous ways to track cells from their origin to their differentiation into specialized cell types. There are three requirements for a successful lineage tracing experiment. They include careful assessment of cells for the marking as the starting population at the initial time point, careful selection of a marker to mark the original cell that passes through the progeny without leaking to other neighboring cells, and the markers being stable and nontoxic to cells. However, the violation of these requirements can result in alterations in cell behavior, labeling of unrelated cells, and misinterpretation of tracing cell results.
What are the Techniques Used in Lineage Tracing Cells
Several techniques are involved in lineage tracing cells. They include the use of nonselective markers, including vital dye, carbocyanine dyes and dextrans, nucleotide pulse-chase, carbon dating, sequencing-based technology, several genetic markers, and direct observation.
Nonselective Markers
These markers allow the marking of a specific subset of cells by marking cell membranes, cytoplasm, and the nucleus.
- In vital dye, a colored substance label the cells, and the labeled cells can be distinguished from other cells very easily. Here, vital dye only stains the cell but does not kill the cell. However, this dye is water soluble and can diffuse to neighboring cells.
- In contrast, carbocyanine dyes and dextrans are lipid soluble, and these substances need to be injected into the target cells. But, they dilute in every round of cell division.
- In nucleotide pulse-chase, at a specific time (pulse), a thymidine (3H-Thymidine) analog is given. Then, this 3H-thymidine is incorporated into the cells during cell division. Significantly, the fast-dividing cells incorporate 3H-Thymidine at higher rates, while slow-dividing cells incorporate 3H-Thymidine at slow rates.
- In carbon dating, stable carbon isotopes are used to label cells. Moreover, carbon isotopes are nontoxic to cells.
- In sequencing-based technology, naturally-occurring sequence variants such as mutations pass to the progeny, and these mutations are important in building cell lineages.
Cell Fate Mapping
Genetic Marking
The rapid development of genetic tools allows the determination of cell lineages as well. There, genetic markers are introduced into the cells, such as fluorescent proteins and enzymes such as beta-galactosidase and alkaline phosphatase.
- In transplantation, transplanted cells can be identified by genetic markers by marking their nucleus. Moreover, hematopoietic stem cells can be marked in different embryos with similar development.
- In the Cre-lox-based strategy, Cre-recombinase is expressed as a reporter gene by site-specific genetic recombination.
Direct Observation
Specific cells can undergo direct observation in methods like live imaging in two-photon microscopy along with genetic markers. This allows the direct observation of cell fate.
Conclusion
In brief, cell lineage is a progeny of cells with a common origin. Moreover, the development of these cells can be experimented with in lineage tracing cells. The three main techniques of lineage trace cells include nonselective markers, genetic markers, and direct observation. Nonselective markers include vital dyes, carbocyanine dyes and dextrans, nucleotide pulse-chase, carbon dating, and sequencing-based technology. Genetic markers involve the marking of cells with fluorescent proteins and enzymes that develop color. In addition to that, direct observation is done by the two-photon microscope along with genetic markers. Observation of cell lineages in lineage tracing cells is important in determining the developmental and relocation fate of cell progeny.
References:
- Hsu YC. Theory and Practice of Lineage Tracing. Stem Cells. 2015 Nov;33(11):3197-204. doi: 10.1002/stem.2123. Epub 2015 Aug 18. PMID: 26284340; PMCID: PMC4618107.
Image Courtesy:
- “Liver cell lineage” By Zorn, A.M – Own work (CC-BY 3.0) via Commons Wikimedia
- “Fate mapping fig2” By Pookana2004 – Own Work (CC-BY 3.0) via Commons Wikimedia
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