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Volumn 289, Issue 5477, 2000, Pages 313-316

Induction and maintenance of the neuronal cholinergic phenotype in the central nervous system by BMP-9

Author keywords

[No Author keywords available]

Indexed keywords

ACETYLCHOLINE; BONE MORPHOGENETIC PROTEIN; BONE MORPHOGENETIC PROTEIN 9; MESSENGER RNA; UNCLASSIFIED DRUG;

EID: 0039598632     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.289.5477.313     Document Type: Article
Times cited : (208)

References (46)
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    • Holley, S.A.1
  • 8
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    • D. Bachiller et al., Nature 403, 658 (2000).
    • (2000) Nature , vol.403 , pp. 658
    • Bachiller, D.1
  • 23
    • 0343783817 scopus 로고    scopus 로고
    • note
    • BMP-9 mRNA was measured in the septum, cortex, mesencephalon, and spinal cord dissected from E14 mice and snap-frozen in liquid nitrogen. Total RNA was prepared with a Quiagen RNeasy Mini Kit. Each sample contained RNA prepared from a pool of at least 10 animals to minimize animal-to-animal variations. cDNA was prepared with a Superscript Preamplification System (Gibco-BRL, Gaithersburg, MD). Real-time semiquantitative polymerase chain reaction (PCR) was performed with the SYBR Green PCR Reagents and an ABI PRISM 7700 Sequence Detection System (PE Applied Biosystems, Foster City, CA). BMP-9 mRNA levels were normalized to levels of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The PCR primers used were as follows: BMP-9: forward primer, 5′-TC-CCCACCGACTTGTTCTTC-3′; reverse primer, 5′-GAG-AGTCAGCTGGGAGCTTGA-3′; GAPDH: forward primer, 5′-TGTGTCCGTCGTGGATCTGA-3′: and reverse primer, 5′-CCTGCTTCACCACCTTCTTGA-3′. Sequencing of PCR products confirmed their identity.
  • 24
    • 0343348128 scopus 로고    scopus 로고
    • note
    • 2 inhalation. Brains were dissected and placed flat in ice-cold physiological salt solution. The forebrain was obtained by means of a coronal cut performed under microscope, 2.5 mm from the frontal pole at the level of the thalamus, including the telencephalic vesicles. ACh content of the forebrain was measured by high-performance liquid chromatography (30). All experiments with animals were approved by the Boston University Institutional Animal Care and Use Committee.
  • 25
    • 0343348127 scopus 로고    scopus 로고
    • note
    • Immunofluorescence studies were performed with goat affinity-purified polyclonal antibodies for ChAT (1:100), TH (1:200), GAD (1:1000), and VAChT (1:500) and a mouse monoclonal antibody against βIII-tubulin (1:500) (all from Chemicon), done in parallel with negative and positive controls. Briefly, cells plated on Biocoat chamber slides (Falcon) or cover-glass coated with poly-D-lysine and laminin were fixed in methanol for 5 min at -10°C and incubated at room temperature with 0.1% Triton-X 100 solution. The cells were washed with phosphate-buffered saline, blocked for 30 min with 1% bovine serum albumin (BSA), and incubated overnight with primary antibodies in 1% BSA and for 40 min with the appropriate fluorescent Alexa-conjugated secondary antibodies (Molecular Probes). Slides were mounted with Prolong Antifade (Molecular Probes) and visualized on an epifluorescence-equipped microscope.
  • 31
    • 0343348124 scopus 로고    scopus 로고
    • note
    • Total RNA was extracted from the cells with the acid guanidinium thiocyanate-phenol-chloroform method. Northern blotting was performed as described (30). Signal intensities were quantified directly from the blots with a Phospholmager 400E and Image-Quant software (Molecular Dynamics). To control for total mRNA content and lack of degradation, we stripped the blots and subsequently hybridized them with a mouse GAPDH cDNA probe.
  • 33
    • 0342912907 scopus 로고    scopus 로고
    • note
    • Transfections and reporter plasmid experiments were carried out on mouse cholinergic septal SN56T17 cells. The cells were maintained in DMEM containing 10% fetal bovine serum and gentamicin (50 μg/ml). Reverse transcription (RT)-PCR for the M exon of ChAT was performed with the Access RT-PCR system from Promega. The forward primer (5′-GGGGTGCCTGGTTTGCTTGCAGTCA-3′) was designed specifically for detection of transcripts originating at the M promoter, and the reverse primer (5′-GGGGGCACTGGCAACTTAGGTAAG-3′) was derived from the coding region of the ChAT gene. After PCR, amplification products were subjected to Southern blotting with a ChAT-specific probe and visualized by autoradiography. To make a reporter construct, we inserted a 4.8-kb Xho I-Hind III fragment of the ChAT promoter (a gift from J. Naciff) upstream of the luciferase coding region in the plasmid pGL3-Basic from Promega. SN56T17 cells were transfected with LipofectAMINE (Gibco-BRL), and luciferase activity was measured with the Luciferase Assay System from Promega.
  • 46
    • 0342912903 scopus 로고    scopus 로고
    • note
    • We thank D. Larson for help with immunofluorescence studies, J. Naciff for the ChAT plasmid, and L. Heines for sequencing of PCR products. These studies were supported by NIH grant P01 AG09525 (J.K.B.) and NSF grant IBN-9907572 (J.K.B. and B.B.).


* 이 정보는 Elsevier사의 SCOPUS DB에서 KISTI가 분석하여 추출한 것입니다.