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					    Up-Regulation of Genes in Murine Spinal Cord Transcriptome
                       Yan Z., Lathia K. B. and Clapshaw P. A.
   Solomon Park Research Institute, Frances and Wesley Johnson Laboratory, Seattle, WA
Abstract                                                        Results                                                                                                                                       Methods
BACKGROUND: Analysis of the transcriptome in motor                                                               CLONES OF NEURAL AND GLIAL ORIGIN                                                            MATERIALS: Male C57Bl6J mice (90-
neurons, the cells traditionally associated with amyotrophic                                                                                                                                                  110 days old) were sacrificed by
lateral sclerosis, is hampered both by the diversity of the                                                 Name                                                                       Function               cervical dislocation. Spinal cord
cell types in nervous tissue as well as the cloaking of the                                                                     Neural Clones                                                                 central cores were dissected out and
neural elements by the overwhelming glial matrix                AP2 associated kinase 1                                                                         clatherin mediated endocytosis                visual    cortices  were    manually
surrounding these cells.          Using a combination of        Proline argenine rich coiled coil 1                                                             exocitosis                                    dissected away from the remainder
suppression subtractive hybridization (SSH) and mirror          Pleckstrin homology domain containing, family B (evectins) member 1                             membrane                                      of the brain.
                                                                Dynein, cytoplasmic, intermediate chain 2                                                       motor protein
orientation selection (MOS), we have constructed a cDNA
                                                                Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation
library of differentially expressed genes that are up-          protein, epsilon polypeptide                                                                    signaling
                                                                                                                                                                                                              SUBTRACTIONS:             Suppression
regulated in spinal cord.                                       RIKEN cDNA 4930422J18 gene                                                                      signaling                                     subtractive     hybridization    was
                                                                Neurofilament, heavy polypedtide                                                                structural                                    performed using the BD Clontech
METHODS: The differentially expressed cDNA library from         Solute carrier family 12, member 2                                                              transport Na+K+Cl-                            PCR-Select cDNA Subtraction Kit
murine tissue was constructed using a combination of SSH        Granule cell antiserum positive 14                                                              unknown                                       with cDNA from spinal cord as tester
and MOS. Differential screening using dot blot arrays of        Expressed Sequence AI841971                                                                     unknown                                       and cDNA from visual cortex as
clones from these libraries were probed with cDNA derived       Intergenic between 4932416A11Rik and E230028L10Rik on chromosome 4                                                                            driver. Mirror orientation selection
                                                                mRNA expressed                                                                                  unknown/hypothetical protein
from forward and reverse subtracted spinal cord and visual                                                                                                                                                    was performed as described by
                                                                Phosphatase and actin regulator 4                                                               unknown/synaptosomes
cortical cDNA.    Positive clones from these screened                                                                                                                                                         (Rebrikov 2003). The preparations
                                                                G protein-coupled receptor 37-like 1                                                            vessel/synaptic & clathrin coated
libraries were confirmed with RT-PCR and Northern Blot          N-ethylmaleimide sensitive fusion protein                                                       vessicle fusion
                                                                                                                                                                                                              were finally amplified by PCR and the
Hybridization.                                                                                                                                                                                                products were ligated into TOPO 10
                                                                Stearoyl-Coenzyme A desaturase 1                                                                enzyme membrane
                                                                Secreted phosphoprotein 1 (osteopontin)                                                         extracellular matrix
                                                                                                                                                                                                              vector (InVitrogen) and subsequently
RESULTS: Two thousand randomly selected clones, from            Riken cDNA 2610024G14                                                                           unknown                                       used to transform TOP10 E. coli
10,000 colonies derived from forward subtracted libraries       DNA segment chromosome 8 ERATO Doi 325, expressed                                               unknown                                       (InVitrogen).
were surveyed. Sixty unique sequences were identified as                                                                        Glial Clones
up-regulated in spinal cord tissue when cDNA from spinal        N-myc downstream regulated gene 4                                                               axonal survival                               RT-PCR: Equal amounts of mRNA
cord was used as the probe. Using RT-PCR, we have               Potassium inwardly-rectifying channel, subfamilyJ, member 10                                    channel K                                     from spinal cord & visual cortex were
confirmed five clones that are marginally differentially        Creatinine kinase, brain                                                                        enzyme                                        used as templates. Primers specific
expressed (1~1.5 fold increased), one clone that is             Cyclic nucleotide phosphodiesterase 1                                                           enzyme                                        to each clone were designed for RT-
moderately differentially expressed (1.5~2 fold increased)      UDP glucuronosyltransferase                                                                     enzyme                                        PCR amplification.       The motor
and one clone that is strongly differentially expressed (>2     Crystaline, alpha B                                                                             hs protein                                    neuron specific Islet 1, neuron
fold increased). Twenty randomly selected clones from the       ATPase,Na+/K+ transporting, beta 2 polypeptide (AMOG)                                           membrane                                      specific NSE and non-specific
original 60 unique clones were confirmed as up-regulated in     Peripheral myelin protein                                                                       membrane                                      G3PDH were used as internal
                                                                Myelin-associated glycoprotein                                                                  membrane
spinal cord over visual cortex using Northern Blot                                                                                                                                                            controls. Aliquots were removed at
                                                                Tetraspan 2                                                                                     membrane
Hybridization.                                                                                                                                                                                                different cycles of the RT-PCR
                                                                Diazapam binding inhibitor                                                                      membrane
                                                                                                                                                                                                              reaction. Intensity of the product
                                                                Proteolipid protein (myelin)                                                                    structural/neuron survival
DISCUSSION: Several of the up-regulated genes are known         Myelin-associated oligodendrocytic basic protein                                                structural
                                                                                                                                                                                                              were analyzed by LabWorksTM Image
to be associated either directly or with conditions that        Glial fibrillary acidic protein                                                                 structural                                    Acquisition System.
could increase neuronal cell death, which plays a major role    Ferritin heavy chain 1                                                                          transport Fe
in many diseases. Most of the sequences we observed             Transferrin                                                                                     transport Fe                                  NORTHERN BLOTS: Total RNA
(78%), code for proteins of known and unknown function                                                                                                                                                        (10 µg) from murine spinal cord (SC),
while the remainder (12 %) are sequences that are of                                                                                                                                                          visual cortex (VC), liver (LV) and
hypothetical proteins with unknown functions. Whether                                                       RT-PCR CONFIRMATION OF POSITIVE CLONES                                                            kidney (KD) were electrophoresed on
these hypothetical protein sequences are of neural or glial                                                                                                                                                   1% formaldehyde gels using the
origin is difficult to determine at the present time.                                                                                                                                                         NorthernMax Kit from Ambion and
                                                                                SC      20E1                VC                                                                                                subsequently blotted onto a nylon

Introduction                                                      Subtraction KB20                C57Bl6J
                                                                                                                                                                                                              hybridization membrane from Perkin-
                                                                                                                                                                                                              Elmer.32[P]     labeled         probes
The spinal cord is the site of frequent injuries causing loss                                                                                                                                                 (1x106cpm/ml) were hybridized at
of function and paralysis as well as being an important site                                                                                                                                                  42˚C overnight followed by two 15
                                                                                                                                                                                                              minute washes with 2X SSC, 0.1%

of motor neuron diseases (MNDs), such as amyotrophic                           SC      NSE                  VC
lateral sclerosis (ALS), among others. Despite these                     FS           FU                                                                                                                      SDS at room temperature and 0.1X
considerations, little is known of the molecular differences                                                                                                                                                  SSC, 0.1% SDS at 42˚C. Intensity
between the spinal cord and other neural structures.                                                                                                                                                          was visualized with x-ray film.
Without this knowledge, many of the functions responsible                     SC      20CC12                VC
for the onset of these conditions or the lack of regeneration           RT-PCR cycles 18, 23, 28, 33, 38, 43                                         Image analysis of 20CC12/NSE RT-PCR. 20CC12
to injury or disease may be overlooked.
                                           SC      VC
                                                                        SC (spinal cord) & VC (visual cortex)                                           up-regulated 6.6 at cycle18 in spinal cord.
A number of studies have compared different regions in                        SC       G3PDH                 VC                                                                                               We have isolated 60 unique clones
                                                                                                                                                       1.40    SP 20-1CC12G3PDH
the mammalian spinal cord and brain using subtractive                                                                                                                                                         from spinal cord cDNA.          Twenty
hybridization (Kobayashi et al. 1991; Usui et al 1994;                                                                                                 1.20                                                   randomly chosen clones from these
                                                                                                                                                               VC 20-1CC12/G3PDH
Akopian & Wood 1995) serial analysis of gene expression                                                                                                1.00                                                   original positive sequences were
(de Chaldée et al. 2003) and microarray technology                             SC        CC12                VC
                                                                                                                                                       0.80                                                   verified by RT-PCR and Northern
                                                                                                                                   Mar1-05zy G3PDH

(Sandberg et al. 2000).                                                                                                                                                                                       Blots (see examples in figures). In all
                                                                                                                                                                                                              probability all 60 of these genes are
                                                                               SC      G3PDH                 VC                                        0.40
In the present study, we have used the spinal cord, without                                                                                                                                                   up-regulated in spinal cord when
 sensory ganglia to represent motor functions as tester and                                                                                            0.20                                                   compared to visual cortex. The types
 visual cortex which represents sensory functions as driver.                                                                                           0.00                                                   of molecules differentially expressed,
 We have separated mRNA from these two regions and have                       SC        VC                                                                                                                    as with other studies in the nervous
                                                                              SC       18E12                 VC                                                22 cycles 26 cycles 30 cycles        34cycle
 subtracted out the common sequences using suppression                                                                                                                                                        system, represent a broad range of
 subtractive hybridization (SSH) (Diatchenko et al. 1996).          RT-PCR cycles 14, 18, 22, 26, 30, 34,38                                             Image analysis of 20CC12/G3PDH RT-PCR                 products including cell cycle and
 This procedure has the advantage of identification of               SC (spinal cord) & VC (visual cortex)                                           20CC12 up-regulated 2.6 at 22 and 2.2 at cycle 26        differentiation proteins; enzymes;
 unknown genes which would not be detected in microarray                                                                                                                                                      heat    shock,     motor,    structural,
 analyses.                                                       PCR OF MOTOR NEURON MARKER ISLET 1                                                      NORTHERN BLOT HYBRIDIZATION                          membrane and transport proteins as
                                                                                                                                                        CONFIRMATION OF POSITIVE CLONES                       well as proteins that are both
Although the SSH method is a powerful procedure for                                                                                                                                                           hypothetical and previously observed
comparing cDNA sequences generated from differing                              SC                            VC          B Is                                                                                 with unknown functions. The use of
sources, it is thought to be susceptible to false positives                                                                                                                                                   differentially expressed genes will
from abundant sequences. Given the observation that the                                                                                                        SC VC KD                 SC VC LV KD

                                                                                                       Islet1                                                                                                 involve an analysis of the patterns of
cytological architecture of the mammalian nervous system                                                                                                      Clone 20CC12               Clone 20DD10         genes as well as a search for any
is highly variable and complex it is widely held that the                                                                                                                                                     unique products that may be used to
number and variety of RNA species is large opening the                                                20CC12                                                                                                  distinguish cells or cell populations.
possibility that any subtraction could be obscured by                                                                                                                                                         Several of the up-regulated genes are
background sequences. Mirror orientation selection ( MOS)         RT-PCR Islet1/CC12 at 18, 23, 28, 33, 38, 43                                                Clone 20M2B1               Clone 20M2A2         known to be associated either
is argued to decrease the presence of highly abundant              B (no template) Is (Islet 1 present in SC)                                                                                                 directly or with conditions that could
background sequences (Rebrikov et al. 2000), therefore to          SC (spinal cord) & VC visual cortex (VC)                                                                                                   increase neuronal cell death such as
identify sequences expressed in low abundance we have                                                                                                                                                         that observed in ALS.
applied this procedure in addition to SSH.                                                                                                                      SC VC KD        SC VC LV KD
                                                                                                                                                                    Beta Actin Controls