CURRICULUM VITAE
JOHN ANTHONY O’BRIEN
WORK ADDRESS
MRC-Laboratory of Molecular Biology, Hills Road, Cambridge.
E-mail jaob@mrc-lmb.cam.ac.uc.uk Tel. 01223 402209
Web page www2.mrc-lmb.cam.ac.uk/personal/job/
EDUCATION
(1995) C.I.Biol. M.I.Biol (Charter member of the Institute of Biology)
(1987) M.I.Biol Honours Degree (Cambridge)
(1983) HNC Biological Science (specialist subject Microbiology)
EMPLOYMENT
MRC-LMB
1996 – Present Research Scientist
BABRAHAM INSTITUTE 1992 – 1995 Higher Scientific Officer BBSRC
1987 – 1992 Scientific Officer BBSRC
1983 – 1987 Assistant Scientific Officer BBSRC
COURSES AND ACHIEVEMENTS
An MRC-LMB achievement award for my work published in 2006 on “organisation of
spines on the dendrites of Purkinje cells”.
Interviewed by the editor of New Scientist relating to my work called “Setting a
New Benchmark”Nov.p60-61 (2006).
A sole inventor of a modified hand-held gene gun design to transfect intact
tissue. I have successfully gained sole patent rights in October 2002.
Two weeks in Heidelberg on an intensive “lives specimen light microscopy”
European molecular biology advance course working with the state-of-the-art confocal
microscopes.
Ten days at Kyoto University in Japan, giving lectures and workshop
demonstrations in relation to my work.
Member of the Institute of Biology in 1987.
Charter member of the Institute of Biology in 1995.
CURRENT RESEARCH
Spines on the dendrites of nerve cells are the sites at which most of the rapid
synaptic
communication
takes
place
in
the
brain,
yet
little
is
known
about
the
principles determining the numbers and organisation of spines along the dendritic
shaft.
By
investigating
Purkinje
cells
of
the
cerebellum
I
have
obtained
a
quantitative picture of the spine-dendrite architecture. This could play a vital role
in dendritic spine pathology.
The gene gun is a relatively new method of introducing DNA/dyes into culture cells or
into intact tissue. I have made modifications to the gene gun, it has improved the
accuracy and penetration allowing smaller and deeper tissues to be transfected, thus
making in-vivo biolistic transfection of tissue a possibility.
My research involves using the modified gene gun to shoot the cerebellum with a low
concentration of fluorescence dyes or DNA tagged constructs.After an incubation period
the
brain
is
cut
into
ultra-thin
slices
using
a
vibrotome
and
the
spines
are
investigated using the confocal microscope.
This
novel
approach
has
allowed
me
to
investigate
the
structural
and
synaptic
organisation of Purkinje cells in the mammalian cerebellum,in particular to evaluate
the functional significance of the spines on the dendritic shaft (see my web page). I
was able to investigate a defect cerebellum mouse called a “Weaver” which is depleted
of parallel axons.The investigation has lead to believe that the ordering of spines
are not controlled by external factors, but is an inherent property of the Purkinje
cell itself.
In collaboration with Dr Sarah Lummis at Cambridge University, using the modified gene
gun to investigate three DNA 5-HT3 receptor subunits (A-C) simultaneously placed onto
one gold particle.Gold DNA microcarriers are shot into either HEK293 cells or into
mouse brain cerebella.Observing the transfected images on the confocal microscope to
build up a quantify picture.Whilst much is known about the structure and function of
these proteins from work on the nACh receptor, there is still much to learn about the
assembly and translocation of these pentameric proteins to the membrane as problem in
these areas can cause serious neurological disorders. The data should clarify our
understanding
of
how
and
where
these
proteins
assemble,
why
they
do
not
always
translocate to the membrane, and differences between neuronal and non-neuronal tissue.
PUBLICATIONS
John O’Brien & Nigel Unwin. Organization of spines on the dendrites of Purkinje
cells. PNAS:103:1575-1580 (2006).
John A O’Brien & Sarah C.R.Lummis. Diolistic labelling of neuronal cultures and
in intact tissue using the hand-held gene gun. Nature protocols vol 1 (3) 1517-1521
(2006).
John A O’Brien & Sarah C.R.Lummis. Biolistic transfection of neuronal cultures
and modifications to hand held gene gun in Nature Protocols vol 1 (2) 977-981 (2006).
John A O’Brien & Sarah C.R.Lummis. Gene Gun Technology. Published a chapter in
the Encyclopaedia of Medical Genomics and Proteomics (EMGP) edited by Drs. Jürgen
Fuchs and Maurizio Podda of Klinikumder Johann Wolfgang Goethe Universität in
Frankfurt, German. Gene Gun technology. Page 1-7 August 2006.
Maywood ES, Reddy AB, Wong GKY, O’Neil J, O’Brien JA, McMahon DG, Harmar AJ &
Hastings MH. Synchronization and maintenance of timekeeping in superchiasmatic
circadian clock cells by neuropeptidergic signalling. Current Biology 16: 599-605.
(2006),
O’Brien JA, Harmar AJ, McMahon DG, Maywood ES & Hastings MH Fluorescence imaging
of the suprachiasmatic circadian clockwork: dependence on peptidergic inter-neuronal
signalling. XCONGRESS OF THE EPBR conference J.W.Goethe-university Frankfurt/main
(2005).
John A O’Brien & Sarah C.R.Lummis. Biolistic and diolistic transfection: using
the gene gun to deliver DNA and lipophilic dyes into mammalian cells. Methods 33:121125 (2004).
J.A.O’Brien,A.B.Reddy, M.Garabette,V.M.King, S.Chahad-Ehlers, E.S.Maywood &
Hastings MH. Expression of clock gene products in the SCN nucleus in relation to
circadian behaviour.Novartis foundation symposium Vol 253:203-222 (2003).
John A O’Brien, E.S Maywood & M.H.Hastings. Expression of mCLOCK and other
circadian clock-relevant proteins in the mouse superchiasmatic nuclei. Journal of
Neuroendocrinology 15: 329-334 (2003).
John A O’Brien & Sarah C.R.Lummis. An improved method of preparing microcarriers
for biolistic transfection. Brain Research Protocols. 10: 12-15 (2002).
John A O’Brien,Sarah C Lummis,Garth T.Whiteside,Ray W.Colburn and Michael H
Hastings.Modifications to the handheld gene gun. Improvements for in-vitro Biolistic
transfection of neuronal tissue. J Neuroscience Methods 112:57-64 (2001).
Barbara Nusslein-Hildesheim, John A O’Brien, Francis J.P. Ebling, Elizabeth S.
Maywood, Michael H. Hastings.The circadian cycle of mPER clock products in the
suprachiasmatic nucleus of the Siberian hamster encodes both daily and seasonal time.
Eur Journal of Neuroscience
12 :2856-2864 (2000).
MD Field, ES Maywood, John A O’Brien,DR Weaver,SM Reppert and MH
Hastings.Analysis of clock proteins in mourse SCN demonstrates phylogenetic divergence
of the circadian clockwork and resetting mechanisms. Neuron. 25:437-47 (2000).
James A. Palmer, Carmen De Felipe, John A O’Brien and Stephen P. Hunt.
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. Euro. J of Neuroscience.Vol. 11:3531-3538 (1999).
Amrita Ahluwalia, Carmen De Felipe, John A O’Brien, Stephen Hunt & Mauro
Perretti. Impaired IL-1beta-induced neutrophil accumulation in tachykinin NK-1
receptor knockout mice. British Journal of Pharmacology. 124: 1013-1015 (1998).
De Felipe, C., O’Brien, J.A., Palmer, J.A., Doyle, C.A., Smith, A.J.H., and
Hunt, S.P. Characterization of the NK-1 receptor Knockout mouse: alterations in
nociceptive behaviour. Soc. Neurosci Abstract 27:2:2354 (1997).
Doyle, C.A., De Felipe, C., O’Brien, J.A., Palmer, J.A. and Hunt, S.P. The role
of Substance P in nociception, analgesia and aggression in molecular genetic
mechanisms in nociceptor development and function (Ed Wood, J.) Springer/Londes
Bioscience,Texas. (1998).
Carmen De Felipe, Juan F. Herrero, John A O’Brien, James A. Palmer, Christopher
A. Doyle et al. Altered nociception, analgesia and aggression in mice lacking the
receptor for Substance P. Nature.Vol. 392. (1998).
F.J. Livesey, John A O’Brien, M.Li, A.G. Smith, L.J. Murphy and S.P. Hunt. A
Schwaan cell mitogen accompanying regeneration of moter neurons. Nature.Vol. 390.
(1997).
Shmakov, A.N., Morey, A.L., Ferguson, D.J.P., Fleming, V.A., O’Brien, J.A.,
Savidge, T.C. Conventional patterns of human intestinal proliferation in a severecombined immunodeficient Xenograft model. Differentiation. 5:321-330 (1995).
Valverde, M.A., O’Brien, J.A., Sepúlveda, F.V., Ratcliff, R., Evans, M.J.,
Colledge, W.H. Impaired cell volume regulation in intestinal crypt epithelia of cystic
fibrosis mice.Proc.Natl.Acad.Sci.USA 92:9038-9041 (1995).
Valverde, M.A., O’Brien, J.A., Sepúlveda, F.V., Ratcliff, R., Evans, M.J.,
Colledge, W.H. Inactivation of the murine cftr gene abolishes cAMP-mediated but not
Ca – mediated secretagogue-induced volume decrease in small-intestinal crypts.
2+
Pflügers Arch
425:434-438 (1993).
O’Brien, J.A., Muir, E.M., Lawson, D.E.M., Sepúlveda, F.V. & Harding, M.
Calbindin D28K, expression in transfected mouse NIH 3T3 cells. Cell calcium 14:349-358
(1993).
O’Brien, J.A.,Walters,R.J.,Valverde,M.A.& Sepúlveda,F.V.Regulatory volume
increase after hypertonicity- or VIP-induced cell volume decrease in small intestinal
crypts is dependent on Na /K /2Cl cotransport. Eur. J. Physiol 423:67-73 (1993).
+
+
–
O’Brien, J.A.,Walters, R.J.,Valverde, M.A. & Sepúlveda, F.V. Membrane
conductance and cell volume changes evoked by vasoactive intestinal polypeptide and
carbachol in small intestinal crypts. Eur. J. Physiol. 421:598-605 (1992).
O’Brien, J.A.,Walters, R.J. & Sepúlveda, F.V. Regulatory volume decrease in
small intestinal crypts is inhibited by K and Cl channel blockers. Biochim. Biophys.
+
–
Acta 1070:501-504 (1991).
O’Brien, J.A., James, P.S. & Torre, M. Primary cultures of guinea-pig gallbladder epithelial cells suitable for patch-clamp studies. Exp. Physiol 76:807-810
(1991).
Lopez-Burillo, S., O’Brien, J.A., Ilundain, A., Wreggett, K.A. & Sepúlveda, F.V.
Activation of basolateral membrane K permeability by bradykinin in MDCK cells.
+
Biochim. Biophys. Acta 939:335-342.7. (1988).
Lopez-Burillo, S., O’Brien, J.A. & Sepúlveda, F.V. Effects of Ni of Ca 2+
2+
dependent activation of K permeability in MDCK cells. J. Physiol. 396:150P. (1988).
+
Ilundain, A., O’Brien, J.A., Burton, K.A. & Sepúlveda, F.V. Inositol
trisphosphate and calcium mobilisation in permeabilised enterocytes. Biochim. Biophys.
Acta. 896: 113-116 (1987).
Lopez-Burillo, S., O’Brien, J.A.,Ilundain,A.& Sepúlveda,F.V.Ca -dependent
2+
stimulation of K permeability in MDCK cells by bradykinin. Z. Gastroenterol. 25:635-636
+
(1987).
Lopez-Burillo, S., O’Brien, J.A., Ilundain, A. & Sepúlveda, F.V. Ca -dependent
2+
stimulation of basolateral membrane K permeability in MDCK cells by bradykinin. Z.
+
Gastroenterol. 25:635-636. 1. (1987.)
Burton, K.A., Ilundain,A., O’Brien, J.A. & Sepúlveda, F.V. Calcium mobilisation
by inositol 1, 4, 5-trisphosphate in chicken enterocytes. J. Physiol. 378:77P (1986).
ABSTRACTS
J.A.O’Brien, E.S.Maywood, M.D.Field & M.H.Hastings (2000). Molecular
associations at the heart of the mammalian clockwork. Neuroscience meeting Brighton
U.K.
J.A.O’Brien, G.Whiteside, M.H.Hastings (2000). Modifications to the hand-held
gene gun: Improvements for in-vitro biolistic transfection of neuronal tissue.
Neuroscience meeting New Orleans U.S.A.
O’Brien, J., Hastings, M. & Lummis, S.C. (2000). Gene gun mediated expression of
green fluorescent protein-linked 5HT3A receptors in brain slices. Neurosci. Abs. 26,
811.2.37
J.A.O’Brien, S.C.R.Lummis & M.H.Hastings (2001). Gene gun mediated expression of
yellow fluorescent protein-linked 5-HT3A receptors in HEK293 cells and brain slices.
Proceedings of the British Pharmacology Society 132: p106
J.A.O’Brien, M.H.Hastings (2001). Constructing a circadian clockwork. Manchester
U.K
O’Brien, J., Lummis, S.C. & Hastings, M. (2001). Further characterisation of the
performance of the modified Bio-Rad hand-held gene gun. Neurosci. Abs. 27, 816.4
J.A.O’Brien, M.Holt, G.Whiteside, S.C.R.Lummis, M.H.Hastings (2002).
Modifications made to the hand-held gene gun: improvements for biolistic transfection
on organotypic neuronal tissue. Cambridge neuroscience meeting.
J.A.O’Brien, S.C.R.Lummis (2004). Using the Gene Gun to transfect ligand-gated
channels. Cambridge Neuroscience meeting.
Hastings MH, O’Brien JA, Harmar AJ, McMahon DG+ & Maywood ES (2005).
Fluorescence imaging of the suprachiasmatic circadian clockwork: dependence on
peptidergic inter-neuronal signalling.
J.A.O’Brien, S.C.R.Lummis (2006) ABC of the Gene Gun. MRC-LMB internal symposium
9-13th October.
J.A.O’Brien & N.Unwin (2006) Organisation of spines on the dendrites of Purkinje
cells. MRC Showcase “Breakthrough in Neuroscience and Mental Health”. 7-8th December.