A new generation of pPRIG-based retroviral vectors

<p>Abstract</p> <p>Background</p> <p>Retroviral vectors are valuable tools for gene transfer. Particularly convenient are IRES-containing retroviral vectors expressing both the protein of interest and a marker protein from a single bicistronic mRNA. This coupled expression increases the relevance of tracking and/or selection of transduced cells based on the detection of a marker protein. pAP2 is a retroviral vector containing eGFP downstream of a modified IRES element of EMCV origin, and a CMV enhancer-promoter instead of the U3 region of the 5'LTR, which increases its efficiency in transient transfection. However, pAP2 contains a limited multicloning site (MCS) and shows weak eGFP expression, which previously led us to engineer an improved version, termed pPRIG, harboring: i) the wild-type ECMV IRES sequence, thereby restoring its full activity; ii) an optimized MCS flanked by T7 and SP6 sequences; and iii) a HA tag encoding sequence 5' of the MCS (pPRIG HAa/b/c).</p> <p>Results</p> <p>The convenience of pPRIG makes it a good basic vector to generate additional derivatives for an extended range of use. Here we present several novel pPRIG-based vectors (collectively referred to as PRIGs) in which : i) the HA tag sequence was inserted in the three reading frames 3' of the MCS (3'HA PRIGs); ii) a functional domain (ER, VP16 or KRAB) was inserted either 5' or 3' of the MCS (« modular » PRIGs); iii) eGFP was replaced by either eCFP, eYFP, mCherry or puro-R (« single color/resistance » PRIGs); and iv) mCherry, eYFP or eGFP was inserted 5' of the MCS of the IRES-eGFP, IRES-eCFP or IRES-Puro-R containing PRIGs, respectively (« dual color/selection » PRIGs). Additionally, some of these PRIGs were also constructed in a pMigR MSCV background which has been widely used in pluripotent cells.</p> <p>Conclusion</p> <p>These novel vectors allow for straightforward detection of any expressed protein (3'HA PRIGs), for functional studies of chimeric proteins (« modular » PRIGs), for multiple transductions and fluorescence analyses of transduced cells (« single color/resistance » PRIGs), or for quantitative detection of studied proteins in independently identified/selected transduced cells (« dual color/selection » PRIGs). They maintain the original advantages of pPRIG and provide suitable tools for either transient or stable expression and functional studies in a large range of experimental settings.</p

Extracellular acidification stimulates GPR68 mediated IL-8 production in human pancreatic β cells.

International audienceAcute or chronic metabolic complications such as diabetic ketoacidosis are often associated with extracellular acidification and pancreatic β-cell dysfunction. However, the mechanisms by which human β-cells sense and respond to acidic pH remain elusive. In this study, using the recently developed human β-cell line EndoC-βH2, we demonstrate that β-cells respond to extracellular acidification through GPR68, which is the predominant proton sensing receptor of human β-cells. Using gain- and loss-of-function studies, we provide evidence that the β-cell enriched transcription factor RFX6 is a major regulator of GPR68. Further, we show that acidic pH stimulates the production and secretion of the chemokine IL-8 by β-cells through NF-кB activation. Blocking of GPR68 or NF-кB activity severely attenuated acidification induced IL-8 production. Thus, we provide mechanistic insights into GPR68 mediated β-cell response to acidic microenvironment, which could be a new target to protect β-cell against acidosis induced inflammation

BCL6-mediated Attenuation of DNA Damage Sensing Triggers Growth Arrest and Senescence through a p53-dependent Pathway in a Cell Context-dependent Manner*S⃞

The BCL6 oncogenic transcriptional repressor is required for development of germinal center centroblasts, which undergo simultaneous genetic recombination and massive clonal expansion. Although BCL6 is required for survival of centroblasts, its expression in earlier B-cells is toxic. Understanding these opposing effects could provide critical insight into normal B-cell biology and lymphomagenesis. We examined the transcriptional and biological effects of BCL6 in various primary cells. BCL6 repression of ATR was previously shown to play a critical role in the centroblast phenotype. Likewise, we found that BCL6 could impose an ATR-dependent phenotype of attenuated DNA damage sensing and repair in primary fibroblasts and B-cells. BCL6 induced true genomic instability because DNA repair was delayed and was qualitatively impaired, which could be critical for BCL6-induced lymphomagenesis. Although BCL6 can directly repress TP53 in centroblasts, BCL6 induced TP53 expression in primary fibroblasts and B-cells, and these cells underwent p53-dependent growth arrest and senescence in the presence of physiological levels of BCL6. This differential ability to trigger a functional p53 response explains at least in part the different biological response to BCL6 expression in centroblasts versus other cells. The data suggest that targeted re-activation of TP53 could be of therapeutic value in centroblast-derived lymphomas

RFX6 Regulates Insulin Secretion by Modulating Ca(2+) Homeostasis in Human β Cells.

Development and function of pancreatic β cells involve the regulated activity of specific transcription factors. RFX6 is a transcription factor essential for mouse β cell differentiation that is mutated in monogenic forms of neonatal diabetes. However, the expression and functional roles of RFX6 in human β cells, especially in pathophysiological conditions, are poorly explored. We demonstrate the presence of RFX6 in adult human pancreatic endocrine cells. Using the recently developed human β cell line EndoC-βH2, we show that RFX6 regulates insulin gene transcription, insulin content, and secretion. Knockdown of RFX6 causes downregulation of Ca(2+)-channel genes resulting in the reduction in L-type Ca(2+)-channel activity that leads to suppression of depolarization-evoked insulin exocytosis. We also describe a previously unreported homozygous missense RFX6 mutation (p.V506G) that is associated with neonatal diabetes, which lacks the capacity to activate the insulin promoter and to increase Ca(2+)-channel expression. Our data therefore provide insights for understanding certain forms of neonatal diabetes

Virus-like infection induces human &beta; cell dedifferentiation.

Type 1 diabetes (T1D) is a chronic disease characterized by an autoimmune-mediated destruction of insulin-producing pancreatic &beta; cells. Environmental factors such as viruses play an important role in the onset of T1D and interact with predisposing genes. Recent data suggest that viral infection of human islets leads to a decrease in insulin production rather than &beta; cell death, suggesting loss of &beta; cell identity. We undertook this study to examine whether viral infection could induce human &beta; cell dedifferentiation. Using the functional human &beta; cell line EndoC-&beta;H1, we demonstrate that polyinosinic-polycytidylic acid (PolyI:C), a synthetic double-stranded RNA that mimics a byproduct of viral replication, induces a decrease in &beta; cell-specific gene expression. In parallel with this loss, the expression of progenitor-like genes such as SOX9 was activated following PolyI:C treatment or enteroviral infection. SOX9 was induced by the NF-&kappa;B pathway and also in a paracrine non-cell-autonomous fashion through the secretion of IFN-&alpha;. Lastly, we identified SOX9 targets in human &beta; cells as potentially new markers of dedifferentiation in T1D. These findings reveal that inflammatory signaling has clear implications in human &beta; cell dedifferentiation

The pupal specifier broad directs progressive morphogenesis in a direct-developing insect

A key regulatory gene in metamorphosing (holometabolous) insect life histories is the transcription factor broad (br), which specifies pupal development. To determine the role of br in a direct-developing (hemimetabolous) insect that lacks a pupal stage, we cloned br from the milkweed bug, Oncopeltus fasciatus (Of’br). We find that, unlike metamorphosing insects, in which br expression is restricted to the larval–pupal transition, Of’br mRNA is expressed during embryonic development and is maintained at each nymphal molt but then disappears at the molt to the adult. Induction of a supernumerary nymphal stage with a juvenile hormone (JH) mimic prevented the disappearance of br mRNA. In contrast, induction of a precocious adult molt by application of precocene II to third-stage nymphs caused a loss of br mRNA at the precocious adult molt. Thus, JH is necessary to maintain br expression during the nymphal stages. Injection of Of’br dsRNA into either early third- or fourth-stage nymphs caused a repetition of stage-specific pigmentation patterns and prevented the normal anisometric growth of the wing pads without affecting isometric growth or molting. Therefore, br is necessary for the mutable (heteromorphic) changes that occur during hemimetabolous development. Our results suggest that metamorphosis in insects arose as expression of br, which conveys competence for change, became restricted to one postembryonic instar. After this shift in br expression, the progressive changes that occur within the nymphal series in basal insects became compressed to the one short period of morphogenesis seen in the larva-to-pupa transition of holometabolous insects