Establishment and application of cell proliferation tracing technology ProTracer

On June 2, Nature Protocols published online the results of Genetic recording of in vivo cell proliferation by ProTracer, a collaboration between Zhou Bin’s research group at the Center for Excellence in Molecular Cell Science of the Chinese Academy of Sciences and He Lingjuan’s research group at Westlake University. This study explains the construction and application of ProTracer, a cell proliferation tracing technology, and discusses how to use ProTracer technology to trace cell proliferation in adult mammals in the process of organ homeostasis and repair regeneration.

Cell proliferation is the basis of cell source in the process of development, homeostasis maintenance, repair and regeneration of various tissues and organs. Cell proliferation in vivo varies greatly depending on the cell type and time period. Previously, the more commonly used methods for detecting cell proliferation in the field were mainly divided into cell proliferation marker staining, nucleotide analogue incorporation and isotope incorporation. The above methods have certain limitations for detecting cell proliferation in vivo: cell proliferation marker staining methods can only detect a certain instantaneous cell proliferation state, nucleotide analogues can be incorporated for a long time but have certain cytotoxicity, and the detection method of isotope incorporation is more complex and inconvenient. In addition, none of the above assays can detect cell proliferation specific to cell type. When the proliferation rate of the target cell is relatively slow, the proliferation signal of the target cell is easily interfered with or overwhelmed by other cell proliferation signals with a faster proliferation rate by the above detection method. In order to solve the above problems, Zhou Bin’s research group recently established a genetic tracing technology that can trace cell proliferation in vivo – Proliferation Tracer (ProTracer). This technology has achieved breakthroughs in long-term and uninterrupted tracing of cell proliferation, cell type-specific cell proliferation detection, and in vivo detection of cell proliferation.

This work examines the technical details of cell proliferation tracing using ProTracer technology, including mouse strain construction, identification, mating strategies, and final assays for cell proliferation tracing. In order to achieve seamless capture cell proliferation in vivo, a CreER mouse strain that can be induced to become Cre was constructed, Ki67-Cre-rox-ER-rox (Ki67-CrexER), where rox is the recognition site of Dre homologous recombinase. When Ki67-CrexER mice are combined with specific DreER mice, DreER is able to induce nucleation in Tamoxifen and identify the rox site in Ki67-CrexER, and a Dre-rox homologous recombination reaction occurs to cut out the ER sequence located between the two rox sites, thereby transforming the inducibly expressed Ki67-CrexER into persistently expressed Ki67-Cre in DreER-expressing cells. Achieve uninterrupted capture of spatiotemporal controllable and cell-specific cell proliferation. In addition, in combination with luciferase reporters, ProTracer technology can also enable lifelong non-invasive detection of the proliferation of specific cell types in living animals without the need to sacrifice the animals.

Taking adult mouse livers as examples, this work explores the tracer of cell proliferation in adult mouse tissues, including mouse mating strategies, tamoxifen induction strategies, mouse injury models and tissue sample analysis. The researchers used ProTracer technology to explore the proliferation of liver cells in adult tissue homeostasis and injury regeneration, and quantified the number of neonatal hepatocytes in liver homeostasis and injury, and found the regional enrichment phenomenon of hepatocyte proliferation, revealing the main source of neonatal hepatocytes in adult liver. The publication of this work facilitates the use of ProTracer technology in vivo proliferation tracing for specific cell types in the field.

The research work is supported by the Chinese Academy of Sciences, the National Natural Science Foundation of China, the Ministry of Science and Technology, the Shanghai Municipal Science and Technology Commission, and the Animal Platform and Cell Platform of the Center of Excellence for Molecular Cells. (Source: Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences)

Related paper information:

Red: GS+ hepatocytes; Purple: E-Cad+ hepatocytes; Green: GFP+ hepatocytes

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