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Mouse ES cells versus XEN cells

ABSTRACT: Comparison of mouse ES cells and three different XEN cell cultures. Three XEN cell cultures: Two different strains (IM8A1 is PO, and XEN1-3 is ICR). And two different culture conditions (IM8A1-I versus IM8A1-II). Three biological replicates of XEN cell RNA were submitted to the Centre for Applied Genomics at the Hospital for Sick Children (Toronto, Canada) for preparation of cRNA and hybridization to the mouse U74Av2 Affymetrix gene array. QIAGEN RNeasy midi kit (QIAGEN Inc., Santa Clarita, CA) was used to extract total RNA from all samples according to manufacturer's instructions. The samples were (1) XEN1-3 cells at passage 18 (ICR strain, male) cultured on gelatin with 70% EMFI-CM, (2) IM8A1 cells at passage 27 (PO strain) cultured on gelatin with 70% EMFI-CM, (3) IM8A1 cells at passage 27 cultured on tissue culture plastic in RPMI 1640 (Gibco) supplemented with 20% FBS (CanSera, Rexdale, Canada), 1 mM sodium pyruvate, 2 mM L-glutamine, 50 mg/ml each of penicillin/streptomycin (all from Gibco), 100 uM b-mercaptoethanol (Sigma) for 4 days. RNA was also obtained from R1 ES cells grown in the absence of EMFIs in the above medium with LIF. Keywords = ES cells Keywords = XEN cells Keywords: ordered

ORGANISM(S): Mus musculus

PROVIDER: GSE2204 | GEO | 2005/01/29

SECONDARY ACCESSION(S): PRJNA91333

REPOSITORIES: GEO

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Project description:Molecular phenotyping studies carried out so far on different embryonic stem cells (ESC) have focused mainly on the identification of molecular markers responsible for pluripotency. Unlike these, the goals of our study were to compare and functionally characterize the gene expression profiles of R1 ESC established from F1 (129X1/SvJ x 129S1) blastocyst (Nagy et al. 1993 PNAS 90: 8424-8428) and HM-1 ESC established from inbred strain (Selfridge et al. 1992 Somat Cell Mol Genet 18: 325-336), as our earlier study showed performance variations between these cells. ES cells were cultured on mitomycin C treated mouse embryonic fibroblast feeder cell layer. Cells were grown in standard ES cell medium changed daily [high glucose DMEM (Gibco-Invitrogen, Carlsbad, CA) supplemented with Na-pyruvate (0.11% w/w; Gibco-Invitrogen), 0.1mM 2-mercaptoethanol (Sigma-Aldrich, St. Louis, MO), fetal bovine serum (15% v/v; HyClone; Logan,UT), 1000 U/ml murine-LIF (CHEMICON International, Temecula, CA) and antibiotics (penicillin: 50 U/ml, streptomycin: 50 μg/ml (Sigma-Aldrich)]. Total RNA was isolated from aliquots of R1 ES cells at passage 13 and HM-1 ES cells at passage 23 using RNeasy Midi kit (Qiagen, Düsseldorf, Germany) procedures. 15 µg of total RNA each from the contrasting samples were used for reverse transcription and labeling either with Cy3 or Cy5 dyes (Amersham, Buckinghamshire, UK). The labeled samples were dissolved in hybridization buffer and added to the cDNA arrays (custom produced at GSF) containing over 21,000 sequences and hybridized for 17 hours at 42°C. The microarray results of four independent hybridizations were analyzed and differentially regulated genes were identified. Finally, the results of four randomly selected genes were verified by real time PCR analysis. The analysis revealed 55 transcripts that showed significant variation (p < 0.01) between the two ES cell lines. Of these, 8 transcripts were up regulated and the rest down regulated in the HM-1 ES cells. Most of these genes were overrepresented in important biological processes like growth and development (21%), cell organization and biogenesis (11%), regulatory roles (21%), and organogenesis (14%). Moreover, the verification analysis using real-time PCR has confirmed the results of microarray. Thus based on the detailed analysis, and confirmation of the results with independent analysis, it is possible to conclude that the expression profile reflected the true molecular variations between the two ES cell lines, and the identified transcripts can serve as molecular markers that explain biological differences between the two ES cell lines. Keywords: ES cell lines, murine, nuclear embryo derived, Nocodazol,

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Mouse ES cells versus XEN cells

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