SPINAL CORD TRANSCRIPTOME ANALYZED BY SUPPRESSION SUBTRACTIVE HYBRIDIZATION AND MIRROR ORIENTATION S

1
SPINAL CORD TRANSCRIPTOME ANALYZED BY
SUPPRESSION SUBTRACTIVE HYBRIDIZATION AND MIRROR
ORIENTATION SELECTION Lathia K. B., Yan Z. and
Clapshaw P. A. Solomon Park Research Institute,
Frances and Wesley Johnson Laboratory, Seattle, WA
Methods
Discussion
Introduction
Analysis of the transcriptome in motor neurons,
the cells traditionally associated with motor
neuron disease (MND)also known as amyotrophic
lateral sclerosis (ALS), is hampered both by the
diversity of the cell types in nervous tissue as
well as the masking of the neural elements by the
overwhelming glial matrix surrounding these
cells.  Additionally, it is not known if the
occurrence of such conditions as MND can be
attributed to the motor neurons alone or are a
product of external influences on these cells
such as would be found in the surrounding glial
cells.  Using a combination of suppression
subtractive hybridization (SSH) and mirror
orientation subtraction (MOS), we have
constructed a library of expressed genes that are
spinal cord specific.  Dot blot arrays of clones
from libraries of spinal cord and visual cortex
origin were probed with forward and reverse
subtracted cDNA from these structures.  Positive
clones from these libraries were confirmed with
Northern analyses.  Sixty unique clones from 139
randomly selected colonies from a total
background of 2000 colonies derived from the
forward subtracted libraries were up-regulated in
spinal cord tissue.  Sequencing analysis of the
cDNA derived from these clones exhibited a
mixture of known and novel genes differentially
expressed in the spinal cord.  It can be
concluded that the spinal cord, as a structure,
exhibits many up-regulated genes when compared to
an area of the brain that does not contain motor
elements.   
In the current study, we have demonstrated
abundant myelin associated with the spinal cord
and the visual cortex which would lead one to
believe that all myelin derived cDNA sequences
(including the sequences for PLP, CNP, MBP, etc.)
should be highly represented in both tissues and
should subtract against each other leaving
sequences specific to the remaining neurons to be
differentially expressed.  The strong
up-regulation of myelin protein derived sequences
(at least 45 percent of all isolated sequences)
in any one part of the adult mouse brain when
compared to any other part of the brain is
surprising.  Additionally, why all myelin
proteins are not represented and why they are not
represented in the order of abundance that these
proteins are found in the nervous system (1) is
not immediately clear, however, we have high
confidence in our findings that subtraction
process is not simply recording background
sequences as we have demonstrated previously (2).
That myelin may be different in different areas
of the nervous system is somewhat surprising,
however, it has been shown that there is a large
and significant difference in the density,
turnover, molecular biological and biochemical
properties of oligodendrocytes in male versus
female rodents (4) It is clear from these studies
that the role of myelin in difference regions of
the brain may be of importance and should be
given appropriate consideration.  The large
preponderance of these sequences may explain why
there is so little difference in expressed genes
regionally in the brain (4).
Perform SSH
Suppression Subtractive Hybridization
Initial subtractions using Suppression
Subtractive Hybridization (SSH) were carried out
using the BD Clontech PCR-Select cDNA Subtraction
Kit from BD Clontech. Subtraction efficiencies
of 10,000 or greater were routinely achieved as
is demonstrated below . Excess prevalent cDNA
sequences identified in the initial subtractions
were used to generate reverse complement 20-25mer
5 biotinylated oligomers and subsequently
exposed to Streptavidin coated beads to remove
the complementary sequences before performing
further subtractions. These sequences were
tested for their presence in the samples pre- and
post- exposure to the Streptavidin beads as shown
in the gels below.
References 1) Trotter, J.L., Wegescheide, C.L.
and Garvey, W.F. (1984) Regional studies of
myelin     proteins in human brain and spinal
cord.  Neurochemical Research 9(1)133-146. 2)
Lathia, K.B., Yan,Z. and Clapshaw, P.A. (2006)
Spinal cord transcriptome analyzed by suppression
subtractive hybridization and mirror orientation
selection. Cellular and Molecular Neurobiology
submitted. 3) Cerghet, M., Skoff, R.P.,
Sessert, D., Zhang, Z., Mullins, C., (2006)
Ghandour, M.S. Proliferation and death of
oligodendrocytes and myelin proteins are
differentially regulated in male and female
rodents.  Neurosci.  126(5)1439-47. 4)
Sandberg, R., Yasuda, R., Pankratz, D.G. Carter,
T.A., Del Rio, J.A. Wodicka, L., Mayford, M.,
Lockhart, D.J. and Barlow, C.  (2000) Regional
and strain-specific gene expression mapping in
the adult mouse brain.  Proc. Natl. Acad. Sci.
USA 9711038-11043.
Results
INDIRECT CAPTURE OF REPRESENTATIVE MYELIN
SEQUENCES ON MAGNETIC BEADS
NORTHERN BLOT CONFIRMATION OF MYELIN SEQUENCES
UP-REGULATED CLONES BY SSH MOS
Pre-Beads
Post-Beads
SC VC
0.5KB
15 20 25 30
15 20 25 30
CNP
CNP
CNP
Pre-Beads
Post-Beads
15 20 25 30
15 20 25 30
PLP
PLP
PLP
SUBTRACTION EFFICIENCY
Efficiency of beads was estimated by PCR at
different cycles on the tester Spinal Cord
G3PDH CONTROL ON THE BEADS
18 22 28 32
18 22 28 32
Post-Beads
Pre-Beads
PLP
Subtraction efficiency gt 1x106
18 22 28 32
Efficiency of the beads was proved with G3PDH
showing same intensity pre and post beads
18 22 28 32
Efficiency was estimated by PCR using G3PDH
Forward Reverse Subtracted (FS) (RS) Forward
Reverse UnSubtracted (FU) (RU)