Comparative analysis of nuclear transfer embryo-derived mouse embryonic stem cells. Part II: gene regulation

Abstract

In a mouse model nuclear transfer embryo-derived embryonic stem cell lines (ntESCs) of various genetic backgrounds and donor cell types were compared with reference ESCs and analyzed comprehensively at molecular level as a second part of a larger study. Expression profiles of ntESCs established by different NT-methods (piezoelectric microinjection or zona-free) were indistinguishable. However, expression profiling analyses identified differentially regulated genes between reference ESCs and ntESCs from different genetic backgrounds. A number of pluripotency and stemness marker genes significantly differed at the mRNA level between the cell lines. However, cluster and lineage analyses revealed that such differences had no effect on cell differentiation and cell fate. Regardless of the donor cell type, gene expression profiles of ntESCs were more similar to each other than to their counterpart fertilized embryo-derived ESCs of the same genotype. Overall, the results indicated that expression profile differences may be related to the genotype rather than to technical variations.

FIG. 1.

Relative gene expression levels of pluripotency mediating genes. Diagram represents the relative gene expression values of pluripotency genes. The gene expression levels of the HM1 as a standard was postulated to 1.0; thus, the relative expression values of further cell lines refer to the HM1. Gene expression differences were considered as significant at 2.5-fold change, labeled with an asterisk. +SD values are indicated on each column.

FIG. 2.

Number of regulated genes in distinct ESC line comparisons. Expression patterns of seven ESCs were analyzed in 13 sample comparisons. Numbers of significantly regulated genes between the different ESCs identified by a SAM one-class analyses. Significance was assigned using a FDR <10% in conjunction with a fold change threshold of >1.5.

FIG. 3.

Venn diagrams of ESC line comparisons. Venn diagrams indicate the overlap of differentially expressed genes across the sample comparisons, where HM1 ESC was used as reference.

FIG. 4.

Hierarchical clustering of ESC line comparison. The dendrogram reflects the process of clustering microarray samples according to the similarity of their gene expression profiles as measured by the Pearson correlation coefficient. Distances between array sample clusters are approximated. ESC line comparisons clustered by genetic background (octagons) and by the type of donor blastocysts (squares). NT, nuclear transfer; NC, noncloned; MEF, mouse embryonic fibroblast; CUM, cumulus; PEM, piezoelectric micromanipulation; PGA, parthenogenetic activation.