View Project Burden-2R01NS036193-06A1

Project Summary
Status:	Public
Publications:	2 Published
Project Detail		Data Detail
Platform:	Affymetrix	MIAME Areas	Compliance
Species:	Mouse	Array Design Detail	true
Organ/Tissue Type:	Diaphragm-skeletal muscle	Experiment Detail	false
Organ Region:	left hemi-diaphragm	Sample Detail	false
Cell Type:	mixed skeletal muscle cells	Hybridization Detail	true
Study Type:	subclassification	Measurement Detail	true
Disease/Condition:	motor axon growth and differentiation
Replicates:	3
Expected Samples:

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Investigator Contact Detail
Name	Steven J Burden
Street Address:	Molecular Neurobiology Skirball Institute NYU Medical School
	540 First Avenue
City, State/Province:	New York , NY
Zip/Postal Code:	10016
Country:	United States
Work Phone:	212-263-7341
Fax:	212-263-2842
E-mail:	burden@saturn.med.nyu.edu

Proposal Detail

Grant:

2R01NS036193-06A1

Status:

Public

Service Type:

Start to Finish Profiling

IACUC:

A3435-01

IACUC date:

2002-09-11

Study Relevance:

These experiments are designed to discover genes that are expressed selectively by synaptic nuclei in skeletal muscle with the particular goal of identifying genes that regulate motor axon growth and differentiation.

Hypothesis:

Previously, we showed that motor axons fail to stop and differentiate in mice lacking MuSK, a receptor tyrosine kinase that is activated by motor neuron-derived Agrin. We hypothesize that MuSK activation normally leads to the production of a retrograde stop/differentiation signal that is encoded by a gene that is expressed preferentially in synaptic nuclei. In the absence of MuSK signaling, the retrograde signaling is not produced by synaptic nuclei, and consequently motor axons wander aimlessly over the muscle.

Specific Aim:

We plan to isolate RNA from the dissected synaptic region of skeletal muscle and from the non-synaptic region of skeletal muscle and to identify the genes that are expressed at higher levels in the synaptic than non-synaptic region.

Experimental Procedure and Design:

We obtain 6 to 8 micrograms of total RNA from the dissected synaptic or non-synaptic region from a single P21 mouse diaphragm muscle. This is a standard procedure in the lab, and we have used these methods to analzye gene expression and to generate high-quality cDNA libraries. Because the synaptic zone is narrower in the left hemi-diaphragm, we will isolate RNA from this half of the diaphragm. In order to isolate sufficient RNA (5 micrograms from each sample), we will pool the synaptic and non-synaptic regions from two hemi-diaphragms. In order to reduce experimental variability, we wish to analzye expression in six samples: three samples of synaptic RNA and three samples of non-synaptic RNA. We will ship the isolated RNA samples to the Consortium in order to generate labeled cDNA, to screen Affymetrix mouse oligo arrays and to assist in the analysis. Several genes, including the subunits of the acetylcholine receptor, MuSK, acetylcholinesterase, and utrophin are known to be expressed preferentially in synaptic nuclei; thus, these genes serve as internal controls for the reliability and effectiveness of the screen. Most other genes, several of which we have analyzed in previous studies, including actin, GAPDH, runx1, nogoC, creatine kinase, etc. are expressed uniformly in skeletal muscle; thus, expression of these genes should be equally represented in synaptic and non-synaptic regions.

Experimental Factors:

Conditions that are tested in the experiment. At least one is required. Experimental factors are the independent variables in the experiment.


Experimental Factors is empty.

Project Samples

Samples associated with this project.

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Name	Description	Extracts
synaptic region	mouse left hemi-diaphragm	1
non-synaptic region	mouse left hemi-diaphragm	1

Project Hybridizations

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Name	Array	Labeled Extract	Hybridization Protocol
Hybridization2	Mouse Genome 430 2.0 Array_1	synaptic region_e1_le1	Affymetrix
Hybridization3	Mouse Genome 430 2.0 Array_3	non-synaptic region_e1_le1	Affymetrix
Mouse Genome 430 2.0 Array_2_hyb	Mouse Genome 430 2.0 Array_2	synaptic region_e1_le2	Affymetrix
Mouse Genome 430 2.0 Array_4_hyb	Mouse Genome 430 2.0 Array_4	non-synaptic region_e1_le2	Affymetrix

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