Dr John O'Brien CBiol FSB

John O'Brien

University position

Divisional Laboratory Coordinator

Departments

Department of Medicine

Institutes

other

Email

jao47@medschl.cam.ac.uk

Home page

http://www2.mrc-lmb.cam.ac.uk/persona...

Research Themes

Cellular and Molecular Neuroscience

Developmental Neuroscience

Interests

Working 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 300μm 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 (—) 10μm. Insert: A lower magnification of transfected EYFP cells in the CA1 hippocampal regions of the brain. Scale bar (—) = 200μm
Biolistically transfected pyramidal nerve cell in the CA1 region of the hippocampus part of the brain. Organotypic slices were 300μm 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 (—) 10μm. Insert: A lower magnification of transfected EYFP cells in the CA1 hippocampal regions of the brain. Scale bar (—) = 200μm
Click image to view full-size

Research Focus

Keywords

biolistics

culture cells

nano-biolistics

confocal

vibroslicer

Clinical conditions

Prion diseases

Equipment

acute slices

confocal microscope

culture cells

gene gun

Microscopy

organotypic slices

vibroslicer

Collaborators

No collaborators listed

Associated News Items


    Publications

    2013

    Arsenault 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

    2012

    Ferrari 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

    2011

    Maywood 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

    2007

    O'Brien JA, Lummis SC (2007), “Diolistics: incorporating fluorescent dyes into biological samples using a gene gun.” Trends Biotechnol 25(11):530-4 Details

    2006

    Maywood 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

    2004

    O'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

    2003

    Maywood 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

    2002

    O'Brien J, Lummis SC (2002), “An improved method of preparing microcarriers for biolistic transfection.” Brain Res Brain Res Protoc 10(1):12-5 Details

    2001

    O'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

    2000

    Field 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

    1999

    Palmer 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

    1998

    Ahluwalia 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

    1997

    Livesey 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

    1995

    Shmakov 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

    1993

    Muir 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

    1992

    Walters 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

    1991

    O'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

    1988

    López-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

    1987

    Ilundain 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