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1.
PLoS One ; 10(7): e0132856, 2015.
Article in English | MEDLINE | ID: mdl-26177549

ABSTRACT

Much progress has been made in understanding the important cis-mediated controls on mouse TCRα gene function, including identification of the Eα enhancer and TCRα locus control region (LCR). Nevertheless, previous data have suggested that other cis-regulatory elements may reside in the locus outside of the Eα/LCR. Based on prior findings, we hypothesized the existence of gene regulatory elements in a 3.9-kb region 5' of the Cα exons. Using DNase hypersensitivity assays and TCRα BAC reporter transgenes in mice, we detected gene regulatory activity within this 3.9-kb region. This region is active in both thymic and peripheral T cells, and selectively affects upstream, but not downstream, gene expression. Together, these data indicate the existence of a novel cis-acting regulatory complex that contributes to TCRα transgene expression in vivo. The active chromatin sites we discovered within this region would remain in the locus after TCRα gene rearrangement, and thus may contribute to endogenous TCRα gene activity, particularly in peripheral T cells, where the Eα element has been found to be inactive.


Subject(s)
Gene Expression Regulation , Genetic Loci , Receptors, Antigen, T-Cell, alpha-beta/genetics , T-Lymphocytes/cytology , Thymus Gland/cytology , Animals , Chromatin/genetics , Deoxyribonuclease I/metabolism , Genes, Reporter , Humans , Mice, Inbred C57BL , Mice, Transgenic , Sequence Deletion , T-Lymphocytes/metabolism
2.
J Vis Exp ; (92): e52119, 2014 Oct 14.
Article in English | MEDLINE | ID: mdl-25349888

ABSTRACT

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic progenitors of both mouse and human origin. It is now used to address a growing variety of complex genetic, cellular and molecular questions related to hematopoiesis, and is at the cutting edge of efforts to translate these basic findings to therapeutic applications. The procedures are straightforward and routinely yield robust results. However, achieving successful hematopoietic differentiation in vitro requires special attention to the details of reagent and cell culture maintenance. Furthermore, the protocol features technique sensitive steps that, while not difficult, take care and practice to master. Here we focus on the procedures for differentiation of T lymphocytes from mouse embryonic stem cells (mESC). We provide a detailed protocol with discussions of the critical steps and parameters that enable reproducibly robust cellular differentiation in vitro. It is in the interest of the field to consider wider adoption of this technology, as it has the potential to reduce animal use, lower the cost and shorten the timelines of both basic and translational experimentation.


Subject(s)
Cytological Techniques/methods , Embryonic Stem Cells/cytology , Hematopoietic Stem Cells/cytology , T-Lymphocytes/cytology , Animals , Cell Differentiation/physiology , Cell Line , Coculture Techniques/methods , Mice
3.
J Immunol Methods ; 407: 135-45, 2014 May.
Article in English | MEDLINE | ID: mdl-24681242

ABSTRACT

Numerous locus control region (LCR) activities have been discovered in gene loci important to immune cell development and function. LCRs are a distinct class of cis-acting gene regulatory elements that appear to contain all the DNA sequence information required to establish an independently and predictably regulated gene expression program at any genomic site in native chromatin of a whole animal. As such, LCR-regulated transgenic reporter systems provide invaluable opportunities to investigate the mechanisms of gene regulatory DNA action during development. Furthermore the qualities of LCR-driven gene expression, including spatiotemporal specificity and "integration site-independence" would be highly desirable to incorporate into vectors used in therapeutic genetic engineering. Thus, advancement in the methods used to investigate LCRs is of considerable basic and translational significance. We study the LCR present in the mouse T cell receptor (TCR)-α gene locus. Until recently, transgenic mice provided the only experimental model capable of supporting the entire spectrum of LCR activities. We have recently reported complete manifestation of TCRα LCR function in T cells derived in vitro from mouse embryonic stem cells (ESC), thus validating a complete cell culture model for the full range of LCR activities seen in transgenic mice. Here we discuss the critical parameters involved in studying LCR-regulated gene expression during in vitro hematopoietic differentiation from ESCs. This advance provides an approach to speed progress in the LCR field, and facilitate the clinical application of its findings, particularly to the genetic engineering of T cells.


Subject(s)
Cell Differentiation , Embryonic Stem Cells/cytology , Gene Expression Regulation , Locus Control Region/genetics , Receptors, Antigen, T-Cell, alpha-beta/genetics , Animals , Cell Culture Techniques/methods , Cells, Cultured , Genetic Engineering , Genetic Therapy , Humans , Immune System/physiology , Mice , Mice, Transgenic
4.
J Immunol ; 191(1): 472-9, 2013 Jul 01.
Article in English | MEDLINE | ID: mdl-23720809

ABSTRACT

Locus control regions (LCRs) are cis-acting gene regulatory elements with the unique, integration site-independent ability to transfer the characteristics of their locus-of-origin's gene expression pattern to a linked transgene in mice. LCR activities have been discovered in numerous T cell lineage-expressed gene loci. These elements can be adapted to the design of stem cell gene therapy vectors that direct robust therapeutic gene expression to the T cell progeny of engineered stem cells. Currently, transgenic mice provide the only experimental approach that wholly supports all the critical aspects of LCR activity. In this study, we report the manifestation of all key features of mouse TCR-α gene LCR function in T cells derived in vitro from mouse embryonic stem cells. High-level, copy number-related TCR-α LCR-linked reporter gene expression levels are cell type restricted in this system, and upregulated during the expected stage transition of T cell development. We also report that de novo introduction of TCR-α LCR-linked transgenes into existing T cell lines yields incomplete LCR activity. These data indicate that establishing full TCR-α LCR activity requires critical molecular events occurring prior to final T lineage determination. This study also validates a novel, tractable, and more rapid approach for the study of LCR activity in T cells, and its translation to therapeutic genetic engineering.


Subject(s)
Cell Differentiation/genetics , Cell Lineage/genetics , Embryonic Stem Cells/immunology , Locus Control Region/immunology , Receptors, Antigen, T-Cell, alpha-beta/genetics , T-Lymphocyte Subsets/immunology , Animals , Cell Differentiation/immunology , Cell Line , Cell Lineage/immunology , Cells, Cultured , Coculture Techniques , Embryonic Stem Cells/cytology , Embryonic Stem Cells/metabolism , Genes, Reporter/immunology , Mice , Mice, Transgenic , Random Allocation , T-Lymphocyte Subsets/cytology , T-Lymphocyte Subsets/metabolism
5.
J Immunol ; 186(4): 2201-9, 2011 Feb 15.
Article in English | MEDLINE | ID: mdl-21239722

ABSTRACT

Autophagy is a highly regulated and evolutionarily conserved process of cellular self-digestion. Recent evidence suggests that this process plays an important role in regulating T cell homeostasis. In this study, we used Rag1(-/-) (recombination activating gene 1(-/-)) blastocyst complementation and in vitro embryonic stem cell differentiation to address the role of Beclin 1, one of the key autophagic proteins, in lymphocyte development. Beclin 1-deficient Rag1(-/-) chimeras displayed a dramatic reduction in thymic cellularity compared with control mice. Using embryonic stem cell differentiation in vitro, we found that the inability to maintain normal thymic cellularity is likely caused by impaired maintenance of thymocyte progenitors. Interestingly, despite drastically reduced thymocyte numbers, the peripheral T cell compartment of Beclin 1-deficient Rag1(-/-) chimeras is largely normal. Peripheral T cells displayed normal in vitro proliferation despite significantly reduced numbers of autophagosomes. In addition, these chimeras had greatly reduced numbers of early B cells in the bone marrow compared with controls. However, the peripheral B cell compartment was not dramatically impacted by Beclin 1 deficiency. Collectively, our results suggest that Beclin 1 is required for maintenance of undifferentiated/early lymphocyte progenitor populations. In contrast, Beclin 1 is largely dispensable for the initial generation and function of the peripheral T and B cell compartments. This indicates that normal lymphocyte development involves Beclin 1-dependent, early-stage and distinct, Beclin 1-independent, late-stage processes.


Subject(s)
Apoptosis Regulatory Proteins/physiology , Autophagy/immunology , Cell Differentiation/immunology , Lymphocyte Subsets/immunology , Animals , Apoptosis Regulatory Proteins/deficiency , Apoptosis Regulatory Proteins/genetics , B-Lymphocyte Subsets/cytology , B-Lymphocyte Subsets/immunology , B-Lymphocyte Subsets/pathology , Beclin-1 , Cell Differentiation/genetics , Coculture Techniques , Embryonic Stem Cells/immunology , Embryonic Stem Cells/pathology , Embryonic Stem Cells/transplantation , Female , Humans , Lymphocyte Subsets/metabolism , Lymphocyte Subsets/pathology , Male , Mice , Mice, 129 Strain , Mice, Inbred C57BL , Mice, Knockout , Radiation Chimera/genetics , Radiation Chimera/immunology , T-Lymphocyte Subsets/cytology , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/pathology , Time Factors
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