Construction of spinal cord cDNA library and application for subtractive cloning of spinal cord-specific cDNAs.
Neurodegenerative diseases are characterized by neuronal degeneration of specific neurons, e.g., degeneration of motoneurons in amyotrophic lateral sclerosis. As an approach to understand molecular mechanisms of neuronal degeneration of human spinal cord motoneurons in various motor neuron diseases, we have constructed a human spinal cord cDNA library and developed a strategy for isolating spinal cord-specific genes by subtractive cloning. We constructed human spinal cord and brain cDNA libraries from postmortem human spinal cord and brain. To isolate human spinal cord-specific cDNAs, a spinal cord-enriched [32P]cDNA probe was generated by the phenol emulsion reassociation technique. Forty-eight cDNA clones out of 10,000 colonies gave strong signals with the subtracted probe, and individual spinal cord cDNA clones were isolated. Northern blotting analysis confirmed that two spinal cord cDNA clones are, in fact, more abundant in spinal cord compared to brain.
Kobayashi
H
H
Department of Neurology, Niigata University, Japan.
Takahashi
H
H
Oyanagi
K
K
Ikuta
F
F
Miyatake
T
T
Tsuji
S
S
D015152:Blotting, Northern; D001923:Brain Chemistry; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000235:genetics*; D015342:DNA Probes; D005786:Gene Expression Regulation; D015723:Gene Library; D006801:Humans; D009046:Motor Neurons; D009468:Neuromuscular Diseases / Q000235:genetics*; D009928:Organ Specificity; D011061:Poly A / Q000235:genetics* / Q000302:isolation & purification; D012333:RNA, Messenger / Q000235:genetics* / Q000302:isolation & purification; D013116:Spinal Cord / Q000737:chemistry*
D015342:DNA Probes; D012333:RNA, Messenger; D011061:Poly A; D004247:DNA