Expression Database

GUDMAP:14048

Theiler Stage

Images

Supplemental Data Files

CEL file:	13OctM2.CEL
CHP file:
RPT file:
TXT file:	13OctM2.TXT

Archive ID

234

Principal Investigator(s)

Blanche Capel, Duke University Medical Center, Durham, NC, USA, b.capel@cellbio.duke.edu

Submitted By

Samantha A Jameson, Duke University Medical Center, Durham, NC, USA, saj3@duke.edu

Specimen Details

Sample GEO ID:

GSM686272

Sample Description:

Gonad, 13.5 dpc male germ cells

Title:

13OctM2

Component(s) Sampled:

germ cell of the testis (EMAP:5539)

Organism:

Mus musculus

Strain:

Progeny of CD-1 crossed to C57BL/6J

Genotype:

Non-wild type Allele:
Gene		Pou5f1
MGI ID		MGI:3057158
MGI Symbol or lab name		Tg(Pou5f1-EGFP)2Mnn
Type		transgenic insertion
Notes:		Provided by JR Mann (Beckman Research Institute of the City of Hope, Duarte, CA). Szabo PE, Hubner K, Scholer H, Mann JR. (2002). Allele-specific expression of imprinted genes in mouse migratory primordial germ cells. Mech Dev 115 (1-2), 157-160. PMID: 12049782

Sex:

male

Development Age:

13.5 dpc

Theiler Stage:

Pool Size:

one or more litters

Pooled Sample:

Yes

Dissection Method:

Dissected out the gonad, trypsinized and FACS sorted. Capel protocol: FACS Protocol

Experimental Design:

Female mice were time-mated and the embryos were collected. The gonads were removed and the males and females were separately pooled. The gonads were incubated in Trypsin EDTA for 5-10 minutes. The trypsin was removed, PBS (often with DNase) was added, and the gonadal cells were dissociated by pipetting and pulling through a syringe. The samples were passed through a strainer and then FACS sorted. Capel protocol: FACS protocol.

Array Hybridization

Extracted Molecule:	Total RNA
A260:280 Ratio:	1.93
RNA Extraction Protocol:	Capel protocol: RNA extraction and sample preparation for Affymetrix Gene 1.0 ST arrays
Target Amplification Manufacturer/kit:	Nugen WT-Ovation Pico RNA Amplification System (3300) and WT-Ovation Exon Module (2000)
Target Amplification Protocol:	Capel protocol: RNA extraction and sample preparation for Affymetrix Gene 1.0 ST arrays
Rounds of Amplification:	1
Amount Labeled Target Hybridized To Array:	Approximately 2.5 ?g into the hybridization cocktail, approximately 2.1 ?g were hybridized to the array.
Label:	Biotin
Label Protocol:	Nugen Encore Biotin Module (4200)
Array Hyb/Wash Protocol:	The arrays were washed and stained using the Affymetrix Fluidics Station 450. Fluidics Protocol FS450-0007.
Scan Protocol:	Probe arrays were scanned using an Affymetrix GeneChip Scanner 3000 7G and Affymetrix GeneChip Command Console (AGCC) Software version 3.1.1.
GCOS Tgt Value:	500
Data Analysis Method:	Partek
Reference:	Universal: P0 mouse (from Steve Potter)

Series Details

Series GEO ID:	GSE27715
Number of Samples:	91 samples
Title:	Gene expression profiles of germ cells, supporting cells, interstitial cells (including steroidogenic precursors), and endothelial cells in the developing testis and ovary at 115, 125, and 135 dpc (GUDMAP Series ID: 43)
Summary:	The goal of this study is to determine the complete gene expression profile for each cell type of the developing gonad during the critical window in which it adopts the testis or ovarian fate.
Type:	Expression profiling of FACS sorted gonadal cells by microarray.
Overall Design:	Transgenic mice with cell type specific fluorescent markers were used to isolate germ cells, supporting cells, interstitial cells (including steroidogenic precursors), and endothelial cells in the developing testis and ovary. The gonads were dissociated in trypsin, and the fluorescent cells were isolated by FACS. The RNA was collected from the isolated cells and their gene expression profiles were determined by microarray analysis.

Platform Details

Platform GEO ID:	GPL6246
Title:	Affymetrix Mouse Gene 1.0 ST Array
Distribution:	commercial
Technology:	in situ oligonucleotide
Organism:	Mus musculus
Manufacturer:	Affymetrix
Manufacturer Protocol:	N/A
Catalogue Number:	N/A