Abstract
Tracking and isolating live cells based on their proliferative history in live animals remains a technical challenge in animal studies. We have designed a genetic marking system for tracking the proliferative frequency and history of lymphocytes during their development and homeostatic maintenance. This system is based on activation of a fluorescent marker after Cre-dependent recombination between sister chromatids at a specially designed tandem loxP site, named Tlox. We have demonstrated the utility of the Tlox system in tracking proliferative windows of B and T lymphocyte development. We have further applied the Tlox system in the analysis of the proliferative behavior and homeostatic maintenance of Vγ1.1 positive γδ T cells. Our data show that Vγ1.1 T cells generated in neonatal but not adult life are able to expand in the thymus. The expanded Vγ1.1 T cells are preferentially maintained in the liver but not in lymphoid organs. It has been shown that numbers of Vγ1.1 T cells were dramatically increased in the lymphoid organs of Id3 deficient mice. By combining BrdU and Tlox assays we show that this phenotype is primarily due to enhanced neonatal expansion and subsequent retention of Vγ1.1 T cells. Thus, the Tlox system provides a new genetic tool to track clonal expansion within a defined cell population or tissue type in live animals.
Highlights
Cell proliferation is a tightly regulated process in tissue development and maintenance of tissue functions
Identification and isolation of live cells based on their proliferative history remains a technical challenge in genetic analysis of animal models
We have successfully tested the reporter system in developing lymphocytes and revealed a unique phenomenon of population expansion involving the innate cd T lymphocytes generated in neonatal life
Summary
Cell proliferation is a tightly regulated process in tissue development and maintenance of tissue functions. The most commonly used lineage tracking methods are based on Cre mediated activation of reporters in progenitor cells [1,2,3]. The most commonly used methods for tracking cell proliferation are based on either incorporation of a nucleotide analog such as bromodeoxyurodine (BrdU) or tritiated thymidine during DNA replication [4] or natural dilution of a genetically activated protein marker such as GFP [5]. BrdU pulse labeling method has been successfully used to define the proliferative windows in thymic T cell development [6]. The BrdU method is simple and generally applicable to all tissues, but the detection of chemical labels is incompatible with retrieval of live cells for further studies. For cells that have undergone extensive rounds of proliferation, like lymphocytes during antigen responses or cancer stem cells coming out of a dormant phase, they will lose all GFP signals and become indistinguishable from unlabeled cells in the background
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