Dr John O'Brien CBiol FSB University positionDivisional Laboratory Coordinator DepartmentsInstitutesother Home pagehttp://www2.mrc-lmb.cam.ac.uk/persona... Research ThemesInterestsWorking on the application of improving biolistic delivery of getting DNA or fluorescent dyes into live cells. Biolistic transfection is proving an increasingly popular method of incorporating DNA or dyes into cells that are difficult to transfect using traditional methods. The technique routinely uses ‘microparticles’, which are 1-2 microns in diameter, fired into tissues using pressurised helium gas. Fluorescent dyes delivered into living cells by a biolistic delivery enables a detailed map of the anatomical connections between individual cells and groups of cells providing a “wiring diagram” of connections. More recently fluorescent dye coated nanosensors introduced into cells by the gene gun has the potential to implement novel measurement techniques where currently none are available. I’ve made modifications to the hand-held gene gun which has enhanced its accuracy by restricting its target area, and increased the depth penetration achieved by gold dye-coated particles.  Biolistically transfected pyramidal nerve cell in the CA1 region of the hippocampus part of the brain. Organotypic slices were 300?m in thickness, cut by the vibroslicer. Resolutions of smaller cellular structures was enhanced by using gold nanoparticles as a biolistic delivery. Synaptic contacts were visible on the spines (arrow). Blue is DAPI stain. Green is EYFP. Scale bar (—) 10?m. Insert: A lower magnification of transfected EYFP cells in the CA1 hippocampal regions of the brain. Scale bar (—) = 200?m Click image to view full-size Research Focus
Equipmentacute slices confocal microscope culture cells gene gun Microscopy organotypic slices vibroslicer CollaboratorsNo collaborators listed Associated News ItemsPublications2013Arsenault J, Cuijpers SA, Ferrari E, Niranjan D, Rust A, Leese C, O'Brien JA, Binz T, Davletov B (2013), “Botulinum protease-cleaved SNARE fragments induce cytotoxicity in neuroblastoma cells.” J Neurochem Details Arsenault J, Ferrari E, Niranjan D, Cuijpers SA, Gu C, Vallis Y, O'Brien J, Davletov B (2013), “Stapling of the botulinum type A protease to growth factors and neuropeptides allows selective targeting of neuroendocrine cells.” J Neurochem 126(2):223-33 Details Arsenault J, O'Brien JA (2013), “Optimized heterologous transfection of viable adult organotypic brain slices using an enhanced gene gun.” BMC Res Notes 6:544 Details Ferrari E, Gu C, Niranjan D, Restani L, Rasetti-Escargueil C, Obara I, Geranton SM, Arsenault J, Goetze TA, Harper CB, Nguyen TH, Maywood E, O'Brien J, Schiavo G, Wheeler DW, Meunier FA, Hastings M, Edwardson JM, Sesardic D, Caleo M, Hunt SP, Davletov B (2013), “Synthetic self-assembling clostridial chimera for modulation of sensory functions.” Bioconjug Chem 24(10):1750-9 Details O'Brien JA, Lummis SC (2013), “Biolistic transfection of neurons in organotypic brain slices.” Methods Mol Biol 940:157-66 Details 2012Ferrari E, Soloviev M, Niranjan D, Arsenault J, Gu C, Vallis Y, O'Brien J, Davletov B (2012), “Assembly of protein building blocks using a short synthetic peptide.” Bioconjug Chem 23(3):479-84 Details 2011Maywood ES, Chesham JE, O'Brien JA, Hastings MH (2011), “A diversity of paracrine signals sustains molecular circadian cycling in suprachiasmatic nucleus circuits.” Proc Natl Acad Sci U S A 108(34):14306-11 Details Münch C, O'Brien J, Bertolotti A (2011), “Prion-like propagation of mutant superoxide dismutase-1 misfolding in neuronal cells.” Proc Natl Acad Sci U S A 108(9):3548-53 Details O'Brien JA, Lummis SC (2011), “Nano-biolistics: a method of biolistic transfection of cells and tissues using a gene gun with novel nanometer-sized projectiles.” BMC Biotechnol 11:66 Details Wheeler DW, Thompson AJ, Corletto F, Reckless J, Loke JC, Lapaque N, Grant AJ, Mastroeni P, Grainger DJ, Padgett CL, O'Brien JA, Miller NG, Trowsdale J, Lummis SC, Menon DK, Beech JS (2011), “Anaesthetic impairment of immune function is mediated via GABA(A) receptors.” PLoS One 6(2):e17152 Details 2007O'Brien JA, Lummis SC (2007), “Diolistics: incorporating fluorescent dyes into biological samples using a gene gun.” Trends Biotechnol 25(11):530-4 Details 2006Maywood ES, Reddy AB, Wong GK, O'Neill JS, O'Brien JA, McMahon DG, Harmar AJ, Okamura H, Hastings MH (2006), “Synchronization and maintenance of timekeeping in suprachiasmatic circadian clock cells by neuropeptidergic signaling.” Curr Biol 16(6):599-605 Details O'Brien J, Unwin N (2006), “Organization of spines on the dendrites of Purkinje cells.” Proc Natl Acad Sci U S A 103(5):1575-80 Details O'Brien JA, Lummis SC (2006), “Diolistic labeling of neuronal cultures and intact tissue using a hand-held gene gun.” Nat Protoc 1(3):1517-21 Details O'Brien JA, Lummis SC (2006), “Biolistic transfection of neuronal cultures using a hand-held gene gun.” Nat Protoc 1(2):977-81 Details 2004O'Brien J, Lummis SC (2004), “Biolistic and diolistic transfection: using the gene gun to deliver DNA and lipophilic dyes into mammalian cells.” Methods 33(2):121-5 Details 2003Maywood ES, O'Brien JA, Hastings MH (2003), “Expression of mCLOCK and other circadian clock-relevant proteins in the mouse suprachiasmatic nuclei.” J Neuroendocrinol 15(4):329-34 Details 2002O'Brien J, Lummis SC (2002), “An improved method of preparing microcarriers for biolistic transfection.” Brain Res Brain Res Protoc 10(1):12-5 Details 2001O'Brien JA, Holt M, Whiteside G, Lummis SC, Hastings MH (2001), “Modifications to the hand-held Gene Gun: improvements for in vitro biolistic transfection of organotypic neuronal tissue.” J Neurosci Methods 112(1):57-64 Details 2000Field MD, Maywood ES, O'Brien JA, Weaver DR, Reppert SM, Hastings MH (2000), “Analysis of clock proteins in mouse SCN demonstrates phylogenetic divergence of the circadian clockwork and resetting mechanisms.” Neuron 25(2):437-47 Details Nuesslein-Hildesheim B, O'Brien JA, Ebling FJ, Maywood ES, Hastings MH (2000), “The circadian cycle of mPER clock gene products in the suprachiasmatic nucleus of the siberian hamster encodes both daily and seasonal time.” Eur J Neurosci 12(8):2856-64 Details 1999Palmer JA, De Felipe C, O'Brien JA, Hunt SP (1999), “Disruption of the substance P receptor (neurokinin-1) gene does not prevent upregulation of preprotachykinin-A mRNA in the spinal cord of mice following peripheral inflammation.” Eur J Neurosci 11(10):3531-8 Details 1998Ahluwalia A, De Felipe C, O'Brien J, Hunt SP, Perretti M (1998), “Impaired IL-1beta-induced neutrophil accumulation in tachykinin NK1 receptor knockout mice.” Br J Pharmacol 124(6):1013-5 Details De Felipe C, Herrero JF, O'Brien JA, Palmer JA, Doyle CA, Smith AJ, Laird JM, Belmonte C, Cervero F, Hunt SP (1998), “Altered nociception, analgesia and aggression in mice lacking the receptor for substance P.” Nature 392(6674):394-7 Details 1997Livesey FJ, O'Brien JA, Li M, Smith AG, Murphy LJ, Hunt SP (1997), “A Schwann cell mitogen accompanying regeneration of motor neurons.” Nature 390(6660):614-8 Details 1995Shmakov AN, Morey AL, Ferguson DJ, Fleming KA, O'Brien JA, Savidge TC (1995), “Conventional patterns of human intestinal proliferation in a severe-combined immunodeficient xenograft model.” Differentiation 59(5):321-30 Details Valverde MA, O'Brien JA, Sepúlveda FV, Ratcliff RA, Evans MJ, Colledge WH (1995), “Impaired cell volume regulation in intestinal crypt epithelia of cystic fibrosis mice.” Proc Natl Acad Sci U S A 92(20):9038-41 Details 1993Muir EM, Lawson DE, Sepúlveda FV, O'Brien JA, Harding M (1993), “Calbindin D28K expression in transfected mouse NIH3T3 cells.” Cell Calcium 14(4):349-58 Details O'Brien JA, Walters RJ, Valverde MA, Sepúlveda FV (1993), “Regulatory volume increase after hypertonicity- or vasoactive-intestinal-peptide-induced cell-volume decrease in small-intestinal crypts is dependent on Na(+)-K(+)-2Cl- cotransport.” Pflugers Arch 423(1-2):67-73 Details Valverde MA, O'Brien JA, Sepúlveda FV, Ratcliff R, Evans MJ, Colledge WH (1993), “Inactivation of the murine cftr gene abolishes cAMP-mediated but not Ca(2+)-mediated secretagogue-induced volume decrease in small-intestinal crypts.” Pflugers Arch 425(5-6):434-8 Details 1992Walters RJ, O'Brien JA, Valverde MA, Sepúlveda FV (1992), “Membrane conductance and cell volume changes evoked by vasoactive intestinal polypeptide and carbachol in small intestinal crypts.” Pflugers Arch 421(6):598-605 Details 1991O'Brien JA, James PS, Torre M (1991), “Primary cultures of guinea-pig gall-bladder epithelial cells suitable for patch-clamp studies.” Exp Physiol 76(5):807-10 Details O'Brien JA, Walters RJ, Sepúlveda FV (1991), “Regulatory volume decrease in small intestinal crypts is inhibited by K+ and Cl- channel blockers.” Biochim Biophys Acta 1070(2):501-4 Details 1988López-Burillo S, O'Brien JA, Ilundain A, Wreggett KA, Sepúlveda FV (1988), “Activation of basolateral membrane K+ permeability by bradykinin in MDCK cells.” Biochim Biophys Acta 939(2):335-42 Details 1987Ilundain A, O'Brien JA, Burton KA, Sepúlveda FV (1987), “Inositol trisphosphate and calcium mobilisation in permeabilised enterocytes.” Biochim Biophys Acta 896(1):113-6 Details |