Influence of cell permeable Tre-recombinase (CPTR) treatment on T-cell gene expression
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE32643 | GEO | 2012/02/22
SECONDARY ACCESSION(S): PRJNA147067
REPOSITORIES: GEO
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Project description:CEM-ss cells were treated with or without a recombinantly expressed recombinase and gene expression profiles were compared. Two-condition experiment, Untreated CEM-ss vs. CPTR treated CEM-ss cells
2012-02-22 | E-GEOD-32643 | biostudies-arrayexpress
Project description:Over the previous years, comprehensive studies on antiretroviral drugs resulted in the successful introduction of highly active antiretroviral therapy (HAART) into clinical practice for treatment of HIV/AIDS. However, there is still need for new therapeutic approaches, since HAART cannot eradicate HIV-1 from the infected organism and, unfortunately, can be associated with long-term toxicity and the development of drug resistance. In contrast, novel gene therapy strategies may have the potential to reverse the infection by eradicating HIV-1. For example, expression of long terminal repeat (LTR)-specific recombinase (Tre-recombinase) has been shown to result in chromosomal excision of proviral DNA and, in consequence, in the eradication of HIV-1 from infected cell cultures. However, the delivery of Tre-recombinase currently depends on the genetic manipulation of target cells, a process that is complicating such therapeutic approaches and, thus, might be undesirable in a clinical setting. In this report we demonstrate that E.coli expressed Tre-recombinases, tagged either with the protein transduction domain (PTD) from the HIV-1 Tat trans-activator or the translocation motif (TLM) of the Hepatitis B virus PreS2 protein, were able to translocate efficiently into cells and showed significant recombination activity on HIV-1 LTR sequences. Tre activity was observed using episomal and stable integrated reporter constructs in transfected HeLa cells. Furthermore, the TLM-tagged enzyme was able to excise the full-length proviral DNA from chromosomal integration sites of HIV-1-infected HeLa and CEM-SS cells. The presented data confirm Tre-recombinase activity on integrated HIV-1 and provide the basis for the non-genetic transient application of engineered recombinases, which may be a valuable component of future HIV eradication strategies.
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Project description:Stable integration of HIV proviral DNA into host cell chromosomes, a hallmark and essential feature of the retroviral life cycle, establishes the infection permanently. Current antiretroviral combination drug therapy cannot cure HIV infection. However, expressing an engineered HIV-1 long terminal repeat (LTR) site-specific recombinase (Tre), shown to excise integrated proviral DNA in vitro, may provide a novel and highly promising antiviral strategy. We report here the conditional expression of Tre-recombinase from an advanced lentiviral self-inactivation (SIN) vector in HIV-infected cells. We demonstrate faithful transgene expression, resulting in accurate provirus excision in the absence of cytopathic effects. Moreover, pronounced Tre-mediated antiviral effects are demonstrated in vivo, particularly in humanized Rag2?/??c?/? mice engrafted with either Tre-transduced primary CD4? T cells, or Tre-transduced CD34? hematopoietic stem and progenitor cells (HSC). Taken together, our data support the use of Tre-recombinase in novel therapy strategies aiming to provide a cure for HIV.
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Project description:The Bxb1 bacteriophage serine DNA recombinase is an efficient tool for engineering recombinant DNA into the genomes of cultured cells. Generally, a single engineered “landing pad” site is introduced into the cell genome, permitting the integration of transgenic circuits or libraries of transgene variants. While sufficient for many studies, the extent of genetic manipulation possible with a single recombinase site is limiting, and insufficient for more complex cell-based assays for protein function. Here, we harnessed two orthogonal Bxb1 recombinase sites to enable new avenues for mammalian synthetic biology. By designing plasmids with two recombinase sites, we demonstrate that we can avoid genomic integration of undesirable bacterial DNA elements. We also created “double landing pad” cells simultaneously harboring two orthogonal Bxb1 recombinase sites. These cells allow transgenic protein variant libraries to be readily paired with assay-specific protein partners or biosensors, opening up new functional readouts for large-scale functional assays.
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Project description:We describe a system for mosaic genetic analysis termed BEAM, which relies on exogenous DNA delivery in combination with sparse recombinase activation to generate two genetically distinct, non-overlapping populations of cells for comparative analysis. Following transfection or viral transduction, combinatorial recombinase activity generates two distinct populations of cells labeled with either green or red fluorescent protein. BEAM relies on recombinase-dependent signal amplification and delayed reporter expression to enable sharper delineation of control and experimental cells, and to improve reliability of relative to existing methods. We applied BEAM to a variety of known phenotypes to illustrate its advantages for identifying temporally or spatially aberrant phenotypes, for revealing changes in cell proliferation or death, and for controlling for procedural variability. In addition, we used BEAM to test the cortical protomap hypothesis at the individual radial unit level, revealing that area identity is cell-autonomously specified in adjacent radial units.
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2013-09-13 | GSE46599 | GEO
Project description:Analysis of interferon-stimulated genes (ISGs) in various primary cells and immortalized cell lines, following type 1 interferon (IFN) treatment. Some cell types become resistant to HIV-1 infection following type 1 interferon treatment (such as macrophages, THP-1, PMA-THP-1, U87-MG cells and to a lesser extent, primary CD4+ T cells) while others either become only partially resistant (e.g., HT1080, PMA-U937) or remain permissive (e.g., CEM, CEM-SS, Jurkat T cell lines and U937); for more information see (Goujon and Malim, Journal of Virology 2010) and (Goujon and Schaller et al., Retrovirology 2013). We hypothesized that the anti-HIV-1 ISGs are differentially induced and expressed in restrictive cells compared to permissive cells and performed a whole genome analysis following type 1 IFN treatment in cell types exhibiting different HIV-1 resistance phenotypes. 48 samples; design: 9 cell lines, primary CD4+ T cells and primary macrophages, untreated and IFN-treated; 2 replicate experiments per cell line; 3 replicate experiments per primary cell type
2013-09-13 | E-GEOD-46599 | biostudies-arrayexpress
Project description:Therapeutics delivery into cells has been hurdled due to the barrier of cytoplasmic membrane. Although cell penetrating peptide (CPP) can potentially serve as an intracellular drug delivery vehicle, the application of CPP-based delivery is limited because the unsatisfactory delivery efficiency of CPP conjugated potent cargos is challenging their applications in present. Thus, the development of strategies for enhancing the penetrating efficiency of CPP would therefore urgent need to be explored to increase the scope of potential applications. We report here the effects of glucose, sucrose and manntiol (abbreviated as GSM) combination facilitating the penetration efficiency of CPP peptide alone or CPP-GFP (green fluorescence protein) conjugation in cultured cell lines or primary cells. Moreover, osmoprotectants glycerol and glycine supplementation help cells cope with the stress from GSM combination. Thus, our present study suggests that GSM combination in the presence of osmoprotectant can work as a new strategy for CPP penetration enhancement.
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Project description:Cre-recombinase expression under control of an albumin promoter in the presence of floxed alleles is a highly effective and specific way to target gene mutations in hepatocytes. However, some concerns have been raised regarding off-target and/or toxic effects of cre itself, possibly confounding the interpretation of studies employing this approach. We have now used this tool to succesfully target gene deletions in hepatocytes during pneumonia, a condition which results in significant remodeling of the hepatic transcriptome. The goal of this study was to determine what effects, if any, cre expression alone has on hepatic gene expression during bacterial pneumonia. Mutant mice expressed CRE-recombinase under control of an albumin promoter. Results from mutants were compared to wild-type mice on the same genetic background (C57BL/6). Microarray analysis was performed on liver RNA collected from both genotypes of mice in the absence and presence of pneumococcal pneumonia.
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