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Tracking proliferative history in lymphocyte development with cre-mediated sister chromatid recombination.

Zhang B, Dai M, Li QJ, Zhuang Y - PLoS Genet. (2013)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Duke University Medical Center, Durham, North Carolina, United States of America.

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.

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Related in: MedlinePlus

Assessing proliferation frequency and history of Vγ1.1 T cells on Id3 deficient background.(A) Analysis of Vγ1.1 T cells in one week old LckCre;Tolx;Id3f/f thymus. Vγ1.1 T cells were first gated from analysis of total thymocytes (left) and then displayed for BrdU incorporation (middle) and tdTomato expression (right). Below each FACS plot is statistic analysis against Id3 wild type samples described in Fig. 3. Four wild type and seven Id3 mutant samples were used in the unpaired student t-test. (B) Analysis of Vγ1.1 T cells in five week old LckCre;Tolx;Id3f/f thymus. Sample display is as in (A). Student t tests were based on three wild type and three Id3 mutant samples for the left and center plot and five wild type and six Id3 mutant samples for the right plot.
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pgen-1003887-g004: Assessing proliferation frequency and history of Vγ1.1 T cells on Id3 deficient background.(A) Analysis of Vγ1.1 T cells in one week old LckCre;Tolx;Id3f/f thymus. Vγ1.1 T cells were first gated from analysis of total thymocytes (left) and then displayed for BrdU incorporation (middle) and tdTomato expression (right). Below each FACS plot is statistic analysis against Id3 wild type samples described in Fig. 3. Four wild type and seven Id3 mutant samples were used in the unpaired student t-test. (B) Analysis of Vγ1.1 T cells in five week old LckCre;Tolx;Id3f/f thymus. Sample display is as in (A). Student t tests were based on three wild type and three Id3 mutant samples for the left and center plot and five wild type and six Id3 mutant samples for the right plot.

Mentions: Recently, several studies have shown that deletion of the Id3 gene led to a significant expansion of Vγ1.1 T cells in adult animals [28], [29]. The Id3 gene encodes a nuclear protein, which regulates lymphocyte development through direct inhibition of E-protein transcription factors [30]. How Id3 knockout promotes development and/or expansion of Vγ1.1 T cells is still not clear. We thought to further investigate this issue by combining the traditional BrdU method with the newly established Tlox system. LckCre was used to induce T lineage specific deletion of Id3 and activation of the Tlox marker. We first analyzed cell cycle status with BrdU pulse labeling. A significant increase in BrdU positive cells was observed in Id3 deficient one-week old neonates in comparison with the wild type controls (Fig. 4A, middle column). However, analysis of young adult mice showed a moderate decrease in BrdU incorporation in Id3 deficient mice (Fig. 4B, middle column). We then used the Tlox system to track the proliferative history of Vγ1.1 cells. Analysis of Vγ1.1 T cells from one-week old neonates revealed a similar frequency of tdTomato expression between Id3 deficient and wild type controls (Fig. 4A, right column), suggesting that Vγ1.1 T cells on both backgrounds have gone through similar numbers of cell cycles at the neonatal stage. This result is in contrast to the BrdU data, which detects a higher percentage of proliferating cells in Id3 deficient Vγ1.1 T cells than in wild type controls within the 4 hour window of pulse labeling. Thus, the proliferation rate revealed by BrdU pulse labeling may not reflect the proliferative history of the cell population. The differential outcomes from these two assays become even more dramatic in the analysis of 5-week old mice. In contrast to the neonates, the frequency of tdTomato positive Vγ1.1 T cells in 5-week old mice was significantly increased in Id3 deficient mice even though BrdU labeling frequency has decreased (Fig. 4B, middle and right panels). This result supports the idea that Id3 deficiency promotes development and expansion of Vγ1.1 T cells during neonatal life and their subsequent maintenance in postnatal life.


Tracking proliferative history in lymphocyte development with cre-mediated sister chromatid recombination.

Zhang B, Dai M, Li QJ, Zhuang Y - PLoS Genet. (2013)

Assessing proliferation frequency and history of Vγ1.1 T cells on Id3 deficient background.(A) Analysis of Vγ1.1 T cells in one week old LckCre;Tolx;Id3f/f thymus. Vγ1.1 T cells were first gated from analysis of total thymocytes (left) and then displayed for BrdU incorporation (middle) and tdTomato expression (right). Below each FACS plot is statistic analysis against Id3 wild type samples described in Fig. 3. Four wild type and seven Id3 mutant samples were used in the unpaired student t-test. (B) Analysis of Vγ1.1 T cells in five week old LckCre;Tolx;Id3f/f thymus. Sample display is as in (A). Student t tests were based on three wild type and three Id3 mutant samples for the left and center plot and five wild type and six Id3 mutant samples for the right plot.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3814321&req=5

pgen-1003887-g004: Assessing proliferation frequency and history of Vγ1.1 T cells on Id3 deficient background.(A) Analysis of Vγ1.1 T cells in one week old LckCre;Tolx;Id3f/f thymus. Vγ1.1 T cells were first gated from analysis of total thymocytes (left) and then displayed for BrdU incorporation (middle) and tdTomato expression (right). Below each FACS plot is statistic analysis against Id3 wild type samples described in Fig. 3. Four wild type and seven Id3 mutant samples were used in the unpaired student t-test. (B) Analysis of Vγ1.1 T cells in five week old LckCre;Tolx;Id3f/f thymus. Sample display is as in (A). Student t tests were based on three wild type and three Id3 mutant samples for the left and center plot and five wild type and six Id3 mutant samples for the right plot.
Mentions: Recently, several studies have shown that deletion of the Id3 gene led to a significant expansion of Vγ1.1 T cells in adult animals [28], [29]. The Id3 gene encodes a nuclear protein, which regulates lymphocyte development through direct inhibition of E-protein transcription factors [30]. How Id3 knockout promotes development and/or expansion of Vγ1.1 T cells is still not clear. We thought to further investigate this issue by combining the traditional BrdU method with the newly established Tlox system. LckCre was used to induce T lineage specific deletion of Id3 and activation of the Tlox marker. We first analyzed cell cycle status with BrdU pulse labeling. A significant increase in BrdU positive cells was observed in Id3 deficient one-week old neonates in comparison with the wild type controls (Fig. 4A, middle column). However, analysis of young adult mice showed a moderate decrease in BrdU incorporation in Id3 deficient mice (Fig. 4B, middle column). We then used the Tlox system to track the proliferative history of Vγ1.1 cells. Analysis of Vγ1.1 T cells from one-week old neonates revealed a similar frequency of tdTomato expression between Id3 deficient and wild type controls (Fig. 4A, right column), suggesting that Vγ1.1 T cells on both backgrounds have gone through similar numbers of cell cycles at the neonatal stage. This result is in contrast to the BrdU data, which detects a higher percentage of proliferating cells in Id3 deficient Vγ1.1 T cells than in wild type controls within the 4 hour window of pulse labeling. Thus, the proliferation rate revealed by BrdU pulse labeling may not reflect the proliferative history of the cell population. The differential outcomes from these two assays become even more dramatic in the analysis of 5-week old mice. In contrast to the neonates, the frequency of tdTomato positive Vγ1.1 T cells in 5-week old mice was significantly increased in Id3 deficient mice even though BrdU labeling frequency has decreased (Fig. 4B, middle and right panels). This result supports the idea that Id3 deficiency promotes development and expansion of Vγ1.1 T cells during neonatal life and their subsequent maintenance in postnatal life.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Duke University Medical Center, Durham, North Carolina, United States of America.

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.

Show MeSH
Related in: MedlinePlus