View Project marsh-affy-mouse-232749

Project Summary
Status:	Public
Publications:	1 Published
Project Detail		Data Detail
Platform:	Affymetrix	MIAME Areas	Compliance
Species:	Mouse	Array Design Detail	true
Organ/Tissue Type:	brain	Experiment Detail	true
Organ Region:	neocortex	Sample Detail	true
Cell Type:	neural cells	Hybridization Detail	false
Study Type:	subclassification	Measurement Detail	false
Disease/Condition:	normal
Replicates:	6
Expected Samples:

Available Actions

Investigator Contact Detail
Name	Eric Marsh
Institution:	Childrens Hospital of Phialdelphia
Street Address:	509 ARC, Childrens Hospital of Philadelphia
	34th Street and Civic Center Boulevard
City, State/Province:	Philadelphia , PA
Zip/Postal Code:	19104
Country:	United States
Work Phone:	215 590 5285
Fax:
E-mail:	marshe@email.chop.edu

Proposal Detail

Grant:

3R01NS045034-03S1

Status:

Public

Service Type:

Hybrization through Analysis

IACUC:

2005-4-547

IACUC date:

0005-01-13

Study Relevance:

Malformations of cortical development are the underlying eitiology of many cases of Mental Retardation and Epilepsy. Subtle, below the resolution of current MRI, cortical dysplasias are probably involved in many cases of MR, Epilepsy and Autism for which no diagnosis can currently be made. Therefore, understanding the process of cortical development will be vital in diagnosing and eventual treatment of many patients with these conditions. More specifically, the cortex forms from two major populations of neuroblasts which reach their final destination in the cortex by differerent mechanisms. One is radial migration from ventricular neuroblasts to the cortical plate. These cells are excititory projection neurons and glia. The second pathway is from the ventral ganglionic eminences and tangential migration of the interneuronal population of primarily inhibitory neurons. Much less is known about the control of the latter process, and many of these currently undiagnosed subtle malformations may stem from abnormalities of this tangential migration. This project focuses on the understanding the control of the tangentially migrating inhibitory interneurons.

Hypothesis:

We hypothesis that there will be distinct differences in mRNA transcription profiles between the ganglionic eminence and cortical plate within developing interneurons. The differences in transcript profiles will be genes that regulate the migration and differentiation of these developing neurons.

Specific Aim:

We aim to uncover the transcriptional differences between two distinct neuronal populations, GFP positive cells in the ganglionic emience and GFP positive cells within the cortical plate, in order to understand the genetic control of tangential migration. We will acomplish this aim by targeting two different labeled transcripts againts the affymetrix mouse genome arrays.

Experimental Procedure and Design:

Transgenic mice have been generated which express green flourscent protien (GFP) based on the activity of the Dlx 5-6 promotor. Dlx 5-6 are highly regulated genes, and are expressed only in developing interneurons with in the embryonic brain. The expression of this gene begins at around E8 within the medial and lateral ganglionic eminence (GE) and then remain present as cells born within these regions migrate out into the developing cortex. Therefore, we can take advantage of these mice by harvesting embryos at a midpoint in their development (E13.5-14.5) and dissect out GFP positive cells from the ganglionic eminence and from the cortical plate. In order to obtain enough cells to extract sufficent mRNA, we have choosen to perform microdisction of the entire GE and entire cortical plate then break up the tissue into single cell suspension and Florescent activated cell sort (FACs) the populations of GFP + and GFP - cells. We then will extract the total RNA from the GFP postive cell populations using a Trizol based method and purify the RNA with a Quiagen column purification method. The purified RNA will then be amplified using the Affymetrix 1 or 2 round T7 based amplification procedure in order to generate the microgram quantities of labelled cRNA. The labelled biotin cRNA will be sent to the microarray consortium for hybridization againt the Affy mouse genome chip. Three embryos will be pooled in order to obtain sufficent RNA and 6 GFP positive GE and cortical plate samples will be used. The six replicates will be obtained from at least 3 different pregnant mice.

Quality Control Description:

We have taken many steps to ensure quality microarray hybridizations. First, the areas are microdissected under direct visual guidence to clearly separate the cortex from the GE. The rostral migratory stream and olfactory regions are removed, as these represent a somewhat different population of cells. A sample of the cells are directly visualized before and after FACs sorting to ensure that the GFP+ cells are being used for the RNA extraction. During the amplification, a negative control of DEPC water is used to monitor for background activity of the reaction and any contamination. Pilot studies using the RNA hybridized againts 36 gene reverse northern blots were preformed to look at the quality of the amplified product and hybridization. This data can also be used to check the hybridization of these specific genes on the microarrays. We will use 3 pooled embryos to ensure sufficent quantity of the RNA and the harvested RNA is run out on a Agilent bioanalyzer before amplification to ensure its quality. To obtain maximally reliable bioinformatic analysis, we realize maximal biological replicates are necessary, therefore we have choose to use 6 (and possibly more) samples (of 3 embryos) from both the cortex and GE. For both the cortex and GE we will provide 2 technical replicates where the RNA population is split for hybrization. We will also provide 2 samples of the negitive control amplification product, if enough is generated, in order to test for non specific amplification products.

Quality Control Types:

biological_replicate

Replicate Description:

This is described in detail about, briefly we will be using 6 biological replicates from our two populations of cells. We will also use 2 technical replicates to ensure quality amplification and hybridization.

Replicate Types:

biological_replicate

Experimental Factors:

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

Factor Name	Description	Factor Category
developmental stage of inhibitory interneurons	We will compare the transcription pro...	developmental_stage

Project Samples

Samples associated with this project.

Action Button Key

View Sample

Name	Description	Bio-Source	Extracts
1-FACS6cort2+	gfp pos. sorted cells from ...	Biosource 1	1
2-FACS6cort2-	gfp neg. sorted cells from ...	BioSource2	1
3-FACS6GE2+	gfp pos. sorted cells from ...	BioSource3	1
4-FACS8cort1+	gfp pos. sorted cells from ...	BioSource4	1
5-FACS8cort1-	gfp neg. sorted cells from ...	BioSource5	1
6-FACS8GE1+	gfp pos. sorted cells from ...	BioSource6	1
7-FACS9cort1+	gfp pos. sorted cells from ...	BioSource7	1
8-FACS9cort1-	gfp neg. sorted cells from ...	BioSource8	1
9-FACS9GE1+	gfp pos. sorted cells from ...	BioSource9	1
10-FACS10cort1+	gfp pos. sorted cells from ...	BioSource10	1
11-FACS10GE1+	gfp pos. sorted cells from ...	BioSource11	1
12-FACS10cort2+	gfp pos. sorted cells from ...	BioSource12	1
13-FACS10cort2-	gfp neg. sorted cells from ...	BioSource13	1
14-FACS10GE2+	gfp pos. sorted cells from ...	BioSource14	1
15-FACS10cort3+	gfp pos. sorted cells from ...	BioSource15	1
16-FACS10cort3-	gfp neg. sorted cells from ...	BioSource16	1
17-FACS10GE3+	gfp pos. sorted cells from ...	BioSource17	1
18-FACS10GE2+R2	gfp pos. sorted cells from ...	BioSource18	1
19-FACS10GE2+R3	gfp pos. sorted cells from ...	BioSource19	1
20-FACS10cort3+R2	gfp pos. sorted cells from ...	BioSource20	1
21-FACS10cort3+R3	gfp pos. sorted cells from ...	BioSource21	1

Project Hybridizations

Action Button Key

View Hybridization

Name	Array	Labeled Extract
Hybridization42		Affy labeling kit
Hybridization43		Sample2_e1_le1
Hybridization44		Sample3_e1_le1
Hybridization45		Sample4_e1_le1
Hybridization46		Sample5_e1_le1
Hybridization47		Sample6_e1_le1
Hybridization48		Sample7_e1_le1
Hybridization49		Sample8_e1_le1
Hybridization50		Sample9_e1_le1
Hybridization51		Sample10_e1_le1
Hybridization52		Sample11_e1_le1
Hybridization53		Sample12_e1_le1
Hybridization54		Sample13_e1_le1
Hybridization55		Sample14_e1_le1
Hybridization56		Sample15_e1_le1
Hybridization57		Sample16_e1_le1
Hybridization58		Sample17_e1_le1
Hybridization59		Sample18_e1_le1
Hybridization60		Sample19_e1_le1
Hybridization61		Sample20_e1_le1
Hybridization62		Sample21_e1_le1
Mouse Genome 430 2.0 Array_1_hyb	Mouse Genome 430 2.0 Array_1	Affy labeling kit
Mouse Genome 430 2.0 Array_2_hyb	Mouse Genome 430 2.0 Array_2	Sample2_e1_le1
Mouse Genome 430 2.0 Array_3_hyb	Mouse Genome 430 2.0 Array_3	Sample3_e1_le1
Mouse Genome 430 2.0 Array_4_hyb	Mouse Genome 430 2.0 Array_4	Sample4_e1_le1
Mouse Genome 430 2.0 Array_5_hyb	Mouse Genome 430 2.0 Array_5	Sample5_e1_le1
Mouse Genome 430 2.0 Array_6_hyb	Mouse Genome 430 2.0 Array_6	Sample6_e1_le1
Mouse Genome 430 2.0 Array_7_hyb	Mouse Genome 430 2.0 Array_7	Sample7_e1_le1
Mouse Genome 430 2.0 Array_8_hyb	Mouse Genome 430 2.0 Array_8	Sample8_e1_le1
Mouse Genome 430 2.0 Array_9_hyb	Mouse Genome 430 2.0 Array_9	Sample9_e1_le1
Mouse Genome 430 2.0 Array_10_hyb	Mouse Genome 430 2.0 Array_10	Sample10_e1_le1
Mouse Genome 430 2.0 Array_11_hyb	Mouse Genome 430 2.0 Array_11	Sample11_e1_le1
Mouse Genome 430 2.0 Array_12_hyb	Mouse Genome 430 2.0 Array_12	Sample12_e1_le1
Mouse Genome 430 2.0 Array_13_hyb	Mouse Genome 430 2.0 Array_13	Sample13_e1_le1
Mouse Genome 430 2.0 Array_14_hyb	Mouse Genome 430 2.0 Array_14	Sample14_e1_le1
Mouse Genome 430 2.0 Array_15_hyb	Mouse Genome 430 2.0 Array_15	Sample15_e1_le1
Mouse Genome 430 2.0 Array_16_hyb	Mouse Genome 430 2.0 Array_16	Sample16_e1_le1
Mouse Genome 430 2.0 Array_17_hyb	Mouse Genome 430 2.0 Array_17	Sample17_e1_le1
Mouse Genome 430 2.0 Array_18_hyb	Mouse Genome 430 2.0 Array_18	Sample18_e1_le1
Mouse Genome 430 2.0 Array_19_hyb	Mouse Genome 430 2.0 Array_19	Sample19_e1_le1
Mouse Genome 430 2.0 Array_20_hyb	Mouse Genome 430 2.0 Array_20	Sample20_e1_le1
Mouse Genome 430 2.0 Array_21_hyb	Mouse Genome 430 2.0 Array_21	Sample21_e1_le1

Version 3.2.13

Site built by