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Status |
Public on Jan 13, 2007 |
Title |
ES cells tertiary clone no 2-41 |
Sample type |
genomic |
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Channel 1 |
Source name |
ES cells_tertiary clone_no 2-41
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Organism |
Mus musculus |
Characteristics |
Strain : (129/Sv x 129/Sv-CP) F1 Gender : male Passage: 29 Neomycin resistant, puromycin sensitive, hygromycin sensitive
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Biomaterial provider |
ES cells were engineered in the laboratory of Dr Guy Sauvageau, IRIC, Université de Montréal, Canada.
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Treatment protocol |
Infection of target cells. ES cells were infected with the anchor retrovirus A1 for 24h using 4ug ml-1 of polybren (Sigma). Fresh media was added the next day and puromycin (1.5 ug ml-1) selection started 48h after infection. 7-9 days later, colonies were isolated in 96 wells plates and expanded (primary clone). 10 millions ES cells were infected with the saturation retrovirus S1. 24h infections were carried out using 4ug ml-1 of polybren (Sigma). Fresh media were added the next day and hygromycin (150 ug ml-1) selection started 48h after infection. 7 days later, a polyclonal population of secondary clones was obtained. Retroviruses were produced by the GP+E-86 ecotropic packaging cell line. Viral titers were kept low to ensure low infection rate and to minimize chances of multiple integrations. Cre-induced recombination in ES cells. 10 millions ES cells were transfected with a cre plasmid. Cells were resuspended in 800 ul of ES cell media and electroporated with 25 ug of supercoiled pCX-cre or pOG231, using 225 V and 950 uF or 230 V and 500 uF parameters, respectively. After an incubation of 20 minutes at room temperature, cells were distributed on 3 dishes (100 mm) covered with irradiated MEFs. 48h after electroporation, G418 selection started at 300 ug ml-1. 7-9 days later, colonies were isolated in 96 wells plates and expanded in presence of G418. Fractions of these clones were put aside on gelatin coated plates and used to extract DNA.
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Growth protocol |
Prior to DNA extraction, ES cells were grown on gelatin coated dishes, in a media (DMEM high glucose with L-glutamine and pyruvate (Invitrogen), 15% fetal calf serum (Invitrogen),1.5 x 10-4 M alpha-monothioglycerol (Sigma) and 1x10-4 M non-essential amino acids (Invitrogen)) supplemented with 1000 U ml-1 of ESGRO (Chemicon) or leukemia inhibitory factor (LIF, conditioned media from transfected COS cells).
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Extracted molecule |
genomic DNA |
Extraction protocol |
Genomic DNA was isolated using DNAzol according to the manufacturer instructions (Invitrogen).
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Label |
Cy5
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Label protocol |
1μg of test sample genomic DNA was fluorescently labeled using the BioArray CGH Labeling System (Enzo Life Sciences). Initially, the DNA was denatured in the presence of the random primer at 99ºC for 10 minutes in a thermalcycler, and then quickly cooled to 4ºC. The tubes were transferred to ice and labeling occurred with the addition of dNTP-cyanine 5 mix and Klenow. Incubation took place overnight at 37ºC in a thermalcycler. The unincorporated nucleotides were removed using a QIAquick PCR purification column (Qiagen) and the labeled probe was eluted with 2 x 25 ul washes.
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Channel 2 |
Source name |
normal mouse
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Organism |
Mus musculus |
Characteristics |
Strain: C57BL/6J Gender: female
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Biomaterial provider |
Microarray and Genomics Facility of the Roswell Park Cancer Institute (Buffalo, NY)
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Treatment protocol |
no treatment
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Extracted molecule |
genomic DNA |
Extraction protocol |
DNA was extracted using the Quiagen FlexiGene DNA kit, according to the manufacturer instructions.
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Label |
Cy3
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Label protocol |
1μg of reference genomic DNA was fluorescently labeled using the BioArray CGH Labeling System (Enzo Life Sciences). Initially, the DNA was denatured in the presence of the random primer at 99ºC for 10 minutes in a thermalcycler, and then quickly cooled to 4ºC. The tubes were transferred to ice and labeling occurred with the addition of dNTP-cyanine 3 mix and Klenow. Incubation took place overnight at 37ºC in a thermalcycler. The unincorporated nucleotides were removed using a QIAquick PCR purification column (Qiagen) and the labeled probe was eluted with 2 x 25 ul washes.
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Hybridization protocol |
Prior to hybridization, the test and reference probes were combined with 100 ug mouse fluorimetric Cot-1 (Invitrogen) and ethanol precipitated. Following centrifugation, the probes were resuspended in 110ul SlideHyb Buffer #3 (Ambion) containing 5ul of 100μg/ul Yeast tRNA (Invitrogen), heated to 95ºC for 5 minutes and incubated for 30 minutes at 37ºC to block repetitive elements on the probe. Hybridization was performed for 16 hours at 55ºC using a GeneMachine hybridization station (Genomic Solutions, Inc.) as described (Cowell, J. K. and N. J. Nowak. 2003. High-resolution analysis of genetic events in cancer cells using bacterial artificial chromosome arrays and comparative genome hybridization. Adv Cancer Res 90:91-125). After hybridization, the slides were automatically washed in the GeneMachines station with reducing concentrations of SSC and SDS. The final wash was 30 seconds in 0.1X SSC, followed by a two second 100 % ethanol dip. The slides were spun dry and scanned immediately at 5µm resolution using a GenePix 4200A laser scanner (Axon Inc.)
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Scan protocol |
Slides were scanned on a GenePix 4200A laser Scanner (Axon Inc.) using GenePix Pro V5.0. Both lasers were set to 100% and the PMT was set to 500 for cy3 and 450 for cy5.
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Description |
no additional information
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Data processing |
Images were analyzed using Imagene 6.0 (BioDiscovery Inc.). The output of Image analysis was further processed by in-house developed programs. The log2 ratios of the test/control (values are not background subtracted) were normalized using a subgrid loess, with all flagged spots and clones on the sex chromosome are given a weight of zero. The mean of the replicate measures was taken and the final normalized log2 ratio was plotted against its genomic location.
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Submission date |
Jan 10, 2007 |
Last update date |
Jan 12, 2007 |
Contact name |
Guy Sauvageau |
E-mail(s) |
guy.sauvageau@gmail.com
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Organization name |
IRIC
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Department |
Université de Montréal
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Lab |
Laboratory of Molecular Genetics of Stem Cells
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Street address |
Marcelle-Coutu Building 2900 Boul. Edouard Montpetit Quai 20
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City |
Montreal |
State/province |
Quebec |
ZIP/Postal code |
H3T 1J4 |
Country |
Canada |
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Platform ID |
GPL4714 |
Series (1) |
GSE6706 |
A retroviral strategy that efficiently creates chromosomal deletions in mammalian cells |
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