226 CELL LINE-DEPENDENT GENE EXPRESSION PROFILES IN MOUSE EMBRYONIC STEM CELLS

Abstract

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 × 129S1) blastocysts (Nagy et al. 1993 PNAS 90, 8424–8428) and HM-1 ESC established from an 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 a 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, USA) supplemented with Na pyruvate (0.11% w/w; GIBCO-Invitrogen), 0.1 mM 2-mercaptoethanol (Sigma-Aldrich, St Louis, MO, USA), fetal bovine serum (15% v/v; HyClone, Logan, UT, USA), 1000 U mL^-1 murine-LIF (Chemicon International, Temecula, CA, USA), and antibiotics (penicillin: 50 U mL^-1, streptomycin: 50 µg mL^-1; 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. Fifteen µg of total RNA each from the contrasting samples were used for reverse transcription and labeling with either Cy3 or Cy5 dyes (Amersham, Buckinghamshire, UK). The labeled samples were dissolved in hybridization buffer, added to the cDNA arrays (custom produced at GSF) containing over 21 000 sequences, and hybridized for 17 h at 42°C. The microarray results of 4 independent hybridizations were analyzed, and differentially regulated genes were identified. Finally, the results of 4 randomly selected genes were verified by real-time PCR analysis. The analysis revealed 55 transcripts that showed significant variation (P < 0.01) between the 2 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 over-represented in important biological processes such as 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 2 ES cell lines, and the identified transcripts can serve as molecular markers that explain biological differences between the 2 ES cell lines.

This work was supported by EU FP6 (MEXT-CT-2003-509582 and 518240), Wellcome Trust (Grant No.070246), and Hungarian National Science Fund (OTKA T046171).