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Library ID

Library Name:Bain Rancourt retinoic acid induced ES cell neural differentiation subtraction library
Organism:Mus musculus
Strain:129
UniGene Library ID:2757
Tissue Description:
Library Keywords:normal, cell line, subtracted, retinoic acid, EST, embryonic stem cell, embryonic tissue, unknown embryonic stage, PCR directed strategy, cDNA template, plasmid vector, 129

Clones and Sequences

#Clones Generated to Date:
#Sequences Generated to Date:604

Library Preparation Details

Description:Library constructed by Dr. Gerard Bain (present address: Hoechst-ARIAD Genomic Center, ARIAD Pharmaceuticals Inc., 26 Landsdowne Street, Cambridge, Massachusetts, 02139-4234, U.S.A.). To isolate cDNAs corresponding to mRNAs which are upregulated during the neural differentiation of ES cells in vitro, the subtractive hybridization technique of Wang and Brown [1] was employed. Poly(A)+ RNA was prepared from both undifferentiated ES cells and from embryoid bodies which had been cultured for 4 days in the absence of RA followed by an additional 3 days in the presence of 0.5 (M RA (4-/3+ cells). These poly(A)+ RNAs were converted to double-stranded cDNA using the Superscript Choice System (Gibco). Aliquots of both cDNAs were digested with the restriction enzymes AluI and AluI plus RsaI. An adaptor oligo [1] containing an EcoRI site was ligated to the ends of the restricted cDNAs to provide primer binding sites and large amounts of each cDNA population were then produced by the polymerase chain reaction (PCR) as described [1]. Amplified cDNA from undifferentiated ES cells was biotinylated using Photoprobe biotin (Vector Laboratories) according to the manufacturer's protocol. 2.5 ug of amplified cDNA from 4-/3+ cells was mixed with 50 ug of biotinylated ES cell cDNA, denatured by boiling, and hybridized for 20 h. Double stranded cDNAs containing biotin were removed by streptavidin/phenol treatment as described [1]. The remaining subtracted cDNA was mixed with an additional 25 mg of biotinylated ES cell cDNA, denatured by boiling, and hybridized for 2 h. The streptavidin/phenol treatment was repeated and the remaining cDNA was amplified by PCR [Wang and Brown, 1991]. Two additional rounds of subtraction were repeated exactly as described above. The cDNA obtained from this subtraction procedure was digested with EcoRI and ligated to pBS II SK+ (Stratagene) followed by transformation into E. coli DH5 cells. Individual colonies were picked and the corresponding plasmids were isolated either by an alkaline lysis miniprep procedure [2], or using the Qiaprep spin miniprep kit (Qiagen). Sequence analysis was performed using the Big Dye Cycle Sequencing kit and an ABI373 sequencer (University Core DNA Services, University of Calgary). 1. Wang,Z; Brown,DD (1991) A gene expression screen. Proc. Natl. Acad. Sc i. USA 88, 11505-11509. 2. Sambrook,J; Fritsch,EF; Maniatis,T. (1989) Molecular Cloning:A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor,New York.
R. Site1:EcoR1
R. Site2:EcoR1
Lab Host:DH5 alpha
Vector:pBluescript II SK+ (Stratagene)
Vector Type:plasmid
Tissue Supplier:
Library Producer: