Reproducible doxycycline-inducible transgene expression at specific loci generated by Cre-recombinase mediated cassette exchange

Comparative analysis of mutants using transfection is complicated by clones exhibiting variable levels of gene expression due to copy number differences and genomic position effects. Recombinase-mediated cassette exchange (RMCE) can overcome these problems by introducing the target gene into pre-determined chromosomal loci, but recombination between the available recombinase targeting sites can reduce the efficiency of targeted integration. We developed a new LoxP site (designated L3), which when used with the original LoxP site (designated L2), allows highly efficient and directional replacement of chromosomal DNA with incoming DNA. A total of six independent LoxP integration sites introduced either by homologous recombination or retroviral delivery were analyzed; 70–80% of the clones analyzed in hamster and human cells were correct recombinants. We combined the RMCE strategy with a new, tightly regulated tetracycline induction system to produce a robust, highly reliable system for inducible transgene expression. We observed stable inducible expression for over 1 month, with uniform expression in the cell population and between clones derived from the same integration site. This system described should find significant applications for studies requiring high level and regulated transgene expression and for determining the effects of various stresses or oncogenic conditions in vivo and in vitro

The impact of cell architecture on activation and output of the p53 stress response pathway

Most studies of cell signaling in vitro use cells grown as monolayers on rigid and non-physiological substrata. However, epithelial cells in vivo are attached on malleable extracellular matrix (ECM) and are organized into three dimensional (3D) structures with the aid of cell-cell and cell-ECM interactions. The rigidity of the ECM and the 3D cellular architecture have a profound influence on cell growth, survival and differentiation mediated through changes in cell signaling and gene transcription. P53 is a transcription factor capable of responding to a variety of stresses and signals. Furthermore, the level of P53 negative regulator Hdm2 is influenced by mitogenic signalings from the environment. Since the activity of P53 is influenced by the levels of its upstream regulators, changes in cell architecture may influence Hdm2 level and P53 activity. To study the modulation of P53 activity by different transgenes, an improved Cre-loxP recombination system was characterized. The new LoxP site (designated L3), which when used with the original LoxP site (designated L2), allows highly efficient and directional replacement of chromosomal DNA at a defined locus with incoming DNA. When used in combination with a stringent inducible system, the level of transgene expression can be controlled at physiologically relevant levels in an in vitro setting. The study of the influence of cell architecture and the impact of neighboring stromal cells on P53 activity and levels of its upstream regulators were described in subsequent chapters. We found that cells grown in 3D were more resistant to apoptosis induced by a combination of doxorubicin and Nutlin. The sensitivity to apoptosis correlated with the efficiency of Hdmx downregulation. Lowering Hdmx level by shRNA in 3D cells sensitizes these cells to apoptosis induced by doxorubicin and Nutlin. Hence, the cell architecture is one factor to consider in determining the efficacy of chemotherapeutic agents as it can impact on Hdmx level to reduce P53-dependent apoptosi

Cre-recombinase

Reproducible doxycycline-inducible transgen

A Versatile Platform to Analyze Low-Affinity and Transient Protein-Protein Interactions in Living Cells in Real Time

Summary: Protein-protein interactions (PPIs) play central roles in orchestrating biological processes. While some PPIs are stable, many important ones are transient and hard to detect with conventional approaches. We developed ReBiL, a recombinase enhanced bimolecular luciferase complementation platform, to enable detection of weak PPIs in living cells. ReBiL readily identified challenging transient interactions between an E3 ubiquitin ligase and an E2 ubiquitin-conjugating enzyme. ReBiL’s ability to rapidly interrogate PPIs in diverse conditions revealed that some stapled α-helical peptides, a class of PPI antagonists, induce target-independent cytosolic leakage and cytotoxicity that is antagonized by serum. These results explain the requirement for serum-free conditions to detect stapled peptide activity, and define a required parameter to evaluate for peptide antagonist approaches. ReBiL’s ability to expedite PPI analysis, assess target specificity and cell permeability, and reveal off-target effects of PPI modifiers should facilitate the development of effective, cell-permeable PPI therapeutics and the elaboration of diverse biological mechanisms. : Li et al. developed a recombinase-enhanced bimolecular luciferase complementation platform, termed ReBiL, to evaluate low-affinity protein-protein interactions (PPIs) that are not detectable by other methods and to analyze PPI antagonists in living cells. ReBiL showed that small-molecule p53-Mdm2 antagonists disrupt their intended targets effectively in cells, whereas stapled peptides did not. Stapled peptides unexpectedly induced cell membrane disruption resulting in p53-independent death associated with cytoplasmic leakage. ReBiL is also valuable for high-throughput screening and for deciphering signaling mechanisms mediated by protein interactions

E-vapor aerosols do not compromise bone integrity relative to cigarette smoke after 6-month inhalation in an ApoE–/– mouse model

Cigarette smoke (CS) exposure is one of the leading risk factors for human health. Nicotine-containing inhalable products, such as e-cigarettes, can effectively support tobacco harm reduction approaches. However, there are limited comparative data on the effects of the aerosols generated from electronic vapor products (e-vapor) and CS on bone. Here, we report the effects of e-vapor aerosols and CS on bone morphology, structure, and strength in a 6-month inhalation study. Eight-week-old Apo

WIP1 phosphatase is a negative regulator of NF-κB signalling

10.1038/ncb1873Nature Cell Biology115659-666NCBI