Ufartes R.,Max Planck Institute for Experimental Medicine |
Schneider T.,University of Oxford |
Mortensen L.S.,Max Planck Institute for Experimental Medicine |
Romero C.J.,University Miguel Hernández |
And 10 more authors.
Human Molecular Genetics | Year: 2013
Kν10.1 (Eag1), member of the Kν10 family of voltage-gated potassium channels, is preferentially expressed in adult brain. The aim of the present study was to unravel the functional role of Kν10.1 in the brain by generating knockout mice, where the voltage sensor and pore region of Kv10.1 were removed to render non-functional proteins through deletion of exon 7 of the KCNH1 gene using the '3 Lox P strategy'. Kν10.1-deficient mice show no obvious alterations during embryogenesis and develop normally to adulthood; cortex, hippocampus and cerebellum appear anatomically normal. Other tests, including general health screen, sensorimotor functioning and gating, anxiety, social behaviour, learning and memory did not show any functional aberrations in Kν10.1 null mice. Kν10.1 null mice display mild hyperactivity and longer-lasting haloperidol-induced catalepsy, but there was no difference between genotypes in amphetamine sensitization and withdrawal, reactivity to apomorphine and haloperidol in the prepulse inhibition tests or to antidepressants in the haloperidol-induced catalepsy. Furthermore, electrical properties of Kν10.1 in cerebellar Purkinje cells did not show any difference between genotypes. Bearing in mind that Kν10.1 is overexpressed in over 70% of all human tumours and that its inhibition leads to a reduced tumour cell proliferation, the fact that deletion of Kν10.1 does not show a marked phenotype is a prerequisite for utilizing Kν10.1 blocking and/or reduction techniques, such as siRNA, to treat cancer. © The Author 2013. Published by Oxford University Press. All rights reserved.
Lim J.,Keele University |
Lim J.,University of Birmingham |
Clements M.A.,Keele University |
Clements M.A.,The Maynard Center |
And 2 more authors.
PLoS ONE | Year: 2012
Gene delivery technologies to introduce foreign genes into highly differentiated mammalian cells have improved significantly over the last few decades. Relatively new techniques such as magnetic nanoparticle-based gene transfection technology are showing great promise in terms of its high transfection efficiency and wide-ranging research applications. We have developed a novel gene delivery technique, which uses magnetic nanoparticles moving under the influence of an oscillating magnetic array. Herein we successfully introduced short interfering RNA (siRNA) against green fluorescent protein (GFP) or actin into stably-transfected GFP-HeLa cells or wild-type HeLa and rat aortic smooth muscle cells, respectively. This gene silencing technique occurred in a dose- and cell density- dependent manner, as reflected using fluorescence intensity and adhesion assays. Furthermore, using endocytosis inhibitors, we established that these magnetic nanoparticle-nucleic acid complexes, moving across the cell surface under the influence of an oscillating magnet array, enters into the cells via the caveolae-mediated endocytic pathway. © 2012 Lim et al.
Ogden S.J.,The Maynard Center |
Horton J.K.,The Maynard Center |
Stubbs S.L.,The Maynard Center |
Tatnell P.J.,The Maynard Center
Journal of Forensic Sciences | Year: 2015
The 1.2 mm Electric Coring Tool (e-Core™) was developed to increase the throughput of FTA™ sample collection cards used during forensic workflows and is similar to a 1.2 mm Harris manual micro-punch for sampling dried blood spots. Direct short tandem repeat (STR) DNA profiling was used to compare samples taken by the e-Core tool with those taken by the manual micro-punch. The performance of the e-Core device was evaluated using a commercially available PowerPlex™ 18D STR System. In addition, an analysis was performed that investigated the potential carryover of DNA via the e-Core punch from one FTA disc to another. This contamination study was carried out using Applied Biosystems AmpflSTR™ Identifiler™ Direct PCR Amplification kits. The e-Core instrument does not contaminate FTA discs when a cleaning punch is used following excision of discs containing samples and generates STR profiles that are comparable to those generated by the manual micro-punch. © 2014 American Academy of Forensic Sciences.
PubMed | The Maynard Center
Type: Journal Article | Journal: PloS one | Year: 2015
Definitive endoderm (DE) is one of the three germ layers which during in vivo vertebrate development gives rise to a variety of organs including liver, lungs, thyroid and pancreas; consequently efficient in vitro initiation of stem cell differentiation to DE cells is a prerequisite for successful cellular specification to subsequent DE-derived cell types [1, 2]. In this study we present a novel approach to rapidly and efficiently down regulate pluripotency genes during initiation of differentiation to DE cells by addition of dimethyl sulfoxide (DMSO) to Activin A-based culture medium and report its effects on the downstream differentiation to hepatocyte-like cells.Human embryonic stem cells (hESC) were differentiated to DE using standard methods in medium supplemented with 100ng/ml of Activin A and compared to cultures where DE specification was additionally enhanced with different concentrations of DMSO. DE cells were subsequently primed to generate hepatic-like cells to investigate whether the addition of DMSO during formation of DE improved subsequent expression of hepatic markers. A combination of flow cytometry, real-time quantitative reverse PCR and immunofluorescence was applied throughout the differentiation process to monitor expression of pluripotency (POUF5/OCT4 & NANOG), definitive endoderm (SOX17, CXCR4 & GATA4) and hepatic (AFP & ALB) genes to generate differentiation stage-specific signatures.Addition of DMSO to the Activin A-based medium during DE specification resulted in rapid down regulation of the pluripotency genes OCT4 and NANOG, accompanied by an increase expression of the DE genes SOX17, CXCR4 and GATA4. Importantly, the expression level of ALB in DMSO-treated cells was also higher than in cells which were differentiated to the DE stage via standard Activin A treatment.