Generation of stably expressing IRF5 spliced isoform in Jurkat cells

Lentiviral transduction enables the generation of gain-of-function of a targeted gene in mammalian cells. Single-cell cloning through limiting dilution can establish a population of cells with homogenous transgene expression for exploring protein function. Here, we describe step by step optimized protocols for generating clonal stably expressing using crude lentiviral supernatant in Jurkat cells. Although the protocol is for general use, we will detail how to create stable cell lines based on Jurkat cells expressing IRF5 spliced isoform. These protocols will be broadly useful for researchers seeking to apply overexpression by viral transduction and generation of stable clone to study gene function in mammalian cells.

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Estimation of the Frequency of Self-reactive T cells in Health and Inflammatory Diseases by Limiting Dilution Analysis and Single Cell Cloning

Journal of Autoimmunity ◽

10.1006/jaut.1996.0087 ◽

1996 ◽

Vol 9 (5) ◽

pp. 667-675 ◽

Cited By ~ 17

Author(s):

A Lohse

Keyword(s):

T Cells ◽

Single Cell ◽

Inflammatory Diseases ◽

Cell Cloning ◽

Limiting Dilution Analysis ◽

Single Cell Cloning ◽

Limiting Dilution

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Single-Cell Cloning of Hybridoma Cells by Limiting Dilution

Cold Spring Harbor Protocols ◽

10.1101/pdb.prot103192 ◽

2019 ◽

Vol 2019 (11) ◽

pp. pdb.prot103192 ◽

Cited By ~ 2

Author(s):

Edward A. Greenfield

Keyword(s):

Single Cell ◽

Hybridoma Cells ◽

Cell Cloning ◽

Single Cell Cloning ◽

Limiting Dilution

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An epigenetic GPI anchor defect impairs TLR4 signaling in the B cell transdifferentiation model for primary human monocytes BLaER1

Scientific Reports ◽

10.1038/s41598-021-94386-z ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Julia Wegner ◽

Thomas Zillinger ◽

Thais Schlee-Guimaraes ◽

Eva Bartok ◽

Martin Schlee

Keyword(s):

Single Cell ◽

Human Monocyte ◽

Surface Expression ◽

Human Monocytes ◽

Innate Immune Responses ◽

Loss Of Function ◽

Cell Cloning ◽

Gpi Anchor ◽

Single Cell Cloning ◽

Antigen Presenting

AbstractAntigen-presenting myeloid cells like monocytes detect invading pathogens via pattern recognition receptors (PRRs) and initiate adaptive and innate immune responses. As analysis of PRR signaling in primary human monocytes is hampered by their restricted expandability, human monocyte models like THP-1 cells are commonly used for loss-of-function studies, such as with CRISPR-Cas9 editing. A recently developed transdifferentiation cell culture system, BLaER1, enables lineage conversion from malignant B cells to monocytes and was found superior to THP-1 in mimicking PRR signaling, thus being the first model allowing TLR4 and inflammasome pathway analysis. Here, we identified an important caveat when investigating TLR4-driven signaling in BLaER1 cells. We show that this model contains glycosylphosphatidylinositol (GPI) anchor-deficient cells, which lack CD14 surface expression when differentiated to monocytes, resulting in diminished LPS/TLR4 but not TLR7/TLR8 responsiveness. This GPI anchor defect is caused by epigenetic silencing of PIGH, leading to a random distribution of intact and PIGH-deficient clones after single-cell cloning. Overexpressing PIGH restored GPI-anchored protein (including CD14) expression and LPS responsiveness. When studying CD14- or other GPI-anchored protein-dependent pathways, researchers should consider this anomaly and ensure equal GPI-anchored protein expression when comparing cells that have undergone single-cell cloning, e. g. after CRISPR-Cas9 editing.

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Position Effects Are Influenced by the Orientation of a Transgene with Respect to Flanking Chromatin

Molecular and Cellular Biology ◽

10.1128/mcb.21.1.298-309.2001 ◽

2001 ◽

Vol 21 (1) ◽

pp. 298-309 ◽

Cited By ~ 55

Author(s):

Yong-Qing Feng ◽

Matthew C. Lorincz ◽

Steve Fiering ◽

John M. Greally ◽

Eric E. Bouhassira

Keyword(s):

Mammalian Cells ◽

Transgene Expression ◽

Methylation Status ◽

Regulatory Elements ◽

Cell Populations ◽

Methylation Analysis ◽

Position Effects ◽

Methylation State ◽

Cassette Exchange

ABSTRACT We have inserted two expression cassettes at tagged reference chromosomal sites by using recombinase-mediated cassette exchange in mammalian cells. The three sites of integration displayed either stable or silencing position effects that were dominant over the different enhancers present in the cassettes. These position effects were strongly dependent on the orientation of the construct within the locus, with one orientation being permissive for expression and the other being nonpermissive. Orientation-specific silencing, which was observed at two of the three site tested, was associated with hypermethylation but not with changes in chromatin structure, as judged by DNase I hypersensitivity assays. Using CRE recombinase, we were able to switch in vivo the orientation of the transgenes from the permissive to the nonpermissive orientation and vice versa. Switching from the permissive to the nonpermissive orientation led to silencing, but switching from the nonpermissive to the permissive orientation did not lead to reactivation of the transgene. Instead, transgene expression occurred dynamically by transcriptional oscillations, with 10 to 20% of the cells expressing at any given time. This result suggested that the cassette had been imprinted (epigenetically tagged) while it was in the nonpermissive orientation. Methylation analysis revealed that the methylation state of the inverted cassettes resembled that of silenced cassettes except that the enhancer had selectively lost some of its methylation. Sorting of the expressing and nonexpressing cell populations provided evidence that the transcriptional oscillations of the epigenetically tagged cassette are associated with changes in the methylation status of regulatory elements in the transgene. This suggests that transgene methylation is more dynamic than was previously assumed.

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Adeno-associated viral vectors engineered for macrolide-adjustable transgene expression In mammalian cells and mice

BMC Biotechnology ◽

10.1186/1472-6750-7-75 ◽

2007 ◽

Vol 7 (1) ◽

pp. 75 ◽

Cited By ~ 6

Author(s):

David A Fluri ◽

Marie Baba ◽

Martin Fussenegger

Keyword(s):

Mammalian Cells ◽

Transgene Expression ◽

Viral Vectors ◽

Expression In Mammalian Cells

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Usp12 stabilizes the T-cell receptor complex at the cell surface during signaling

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1521763113 ◽

2016 ◽

Vol 113 (6) ◽

pp. E705-E714 ◽

Cited By ~ 23

Author(s):

Akhee S. Jahan ◽

Maxime Lestra ◽

Lee Kim Swee ◽

Ying Fan ◽

Mart M. Lamers ◽

...

Keyword(s):

T Cell ◽

T Cell Receptor ◽

Posttranslational Modifications ◽

Protein Function ◽

Adaptor Proteins ◽

Cell Receptor ◽

Surface Expression ◽

Jurkat Cells ◽

Tcr Signaling ◽

Primary Mouse

Posttranslational modifications are central to the spatial and temporal regulation of protein function. Among others, phosphorylation and ubiquitylation are known to regulate proximal T-cell receptor (TCR) signaling. Here we used a systematic and unbiased approach to uncover deubiquitylating enzymes (DUBs) that participate during TCR signaling in primary mouse T lymphocytes. Using a C-terminally modified vinyl methyl ester variant of ubiquitin (HA-Ub-VME), we captured DUBs that are differentially recruited to the cytosol on TCR activation. We identified ubiquitin-specific peptidase (Usp) 12 and Usp46, which had not been previously described in this pathway. Stimulation with anti-CD3 resulted in phosphorylation and time-dependent translocation of Usp12 from the nucleus to the cytosol. Usp12−/− Jurkat cells displayed defective NFκB, NFAT, and MAPK activities owing to attenuated surface expression of TCR, which were rescued on reconstitution of wild type Usp12. Proximity-based labeling with BirA-Usp12 revealed several TCR adaptor proteins acting as interactors in stimulated cells, of which LAT and Trat1 displayed reduced expression in Usp12−/− cells. We demonstrate that Usp12 deubiquitylates and prevents lysosomal degradation of LAT and Trat1 to maintain the proximal TCR complex for the duration of signaling. Our approach benefits from the use of activity-based probes in primary cells without any previous genome modification, and underscores the importance of ubiquitin-mediated regulation to refine signaling cascades.

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