ABSTRACT
BACKGROUND: The transcriptome of the cerebral cortex is remarkably homogeneous, with variations being stronger between individuals than between areas. It is thought that due to the presence of many distinct cell types, differences within one cell population will be averaged with the noise from others. Studies of sorted cells expressing the same transgene have shown that cell populations can be distinguished according to their transcriptional profile. METHODOLOGY: We have prepared a low-redundancy set of 16,209 full-length cDNA clones which represents the transcriptome of the mouse visual cortex in its coding and non-coding aspects. Using an independent tag-based approach, CAGE, we confirmed the cortical expression of 72% of the clones. Clones were amplified by PCR and spotted on glass slides, and we interrogated the microarrays with RNA from flow-sorted fluorescent cells from the cerebral cortex of parvalbumin-egfp transgenic mice. CONCLUSIONS: We provide an annotated cDNA clone collection which is particularly suitable for transcriptomic analysis in the mouse brain. Spotting it on microarrays, we compared the transcriptome of EGFP positive and negative cells in a parvalbumin-egfp transgenic background and showed that more than 30% of clones are differentially expressed. Our clone collection will be a useful resource for the study of the transcriptome of single cell types in the cerebral cortex.
Subject(s)
Brain/physiology , Gene Expression Profiling , Transcription, Genetic , Visual Cortex/physiology , Animals , Cerebellum/physiology , DNA, Complementary/genetics , Expressed Sequence Tags , Gene Library , Mice , Models, Statistical , Nerve Tissue Proteins/geneticsABSTRACT
Expression of human telomerase reverse transcriptase (hTERT) in normal human fibroblast cell strain, TIG-3, extends their replicative life span. We found that expression levels of interleukin (IL)-1alpha, IL-1beta, IL-6, IL-8, and granulocyte-macrophage colony-stimulating factor (GM-CSF) mRNA were up-regulated in hTERT-induced fibroblasts irrespective of population doubling level (PDL). Expression levels of these cytokines were low in growing young TIG-3 cells and in control vector-transfected TIG-3 cells but were up-regulated in growth-arrested young cells maintained at high cell density. In senescent TIG-3 cells, expression of IL-1beta, IL-6, and GM-CSF was moderately increased. These results indicate that the introduction of hTERT into normal fibroblasts up-regulates the expression of some inflammatory cytokines, and caution should be paid when introducing the hTERT gene to establish cell lines with normal phenotype.
Subject(s)
Cytokines/biosynthesis , Telomerase/metabolism , Up-Regulation , Cell Line , Cellular Senescence , Clone Cells , DNA-Binding Proteins , Fibroblasts/metabolism , Gene Expression Regulation , Granulocyte-Macrophage Colony-Stimulating Factor/biosynthesis , Granulocyte-Macrophage Colony-Stimulating Factor/genetics , Humans , Interleukin-1/biosynthesis , Interleukin-1/genetics , Interleukin-6/biosynthesis , Interleukin-6/genetics , Interleukin-8/biosynthesis , Interleukin-8/genetics , RNA, Messenger/biosynthesis , Telomerase/genetics , TransfectionABSTRACT
Changes in expression levels of various cytokines, growth factors, and related genes were examined by reverse transcriptase polymerase chain reaction in a normal human fibroblast cell strain, TIG-3, along with in vitro aging. The expression levels of KGF and IGF-II were decreased with proliferative aging but not by growth arrest of young cells. In telomere-elongated cells prepared by transfection with human telomerase reverse transcriptase cDNA, high expression levels of these two genes were maintained, suggesting a causal relation between telomere shortening and reduced expression of KGF and IGF-II. The expression level of HGF was high in both growing and growth-arrested young cells but low in both senescent and telomere-elongated cells. The expression levels of follistatin and HB-EGF were high in both young growing and telomere-elongated cells but low in both senescent and growth-arrested young cells, indicating a growth-dependent expression. Expression levels of FGF-1, FGF-2, VEGF, BMP-3, and amphiregulin did not change with proliferative aging, growth arrest of young cells, or telomere elongation and life-span extension.
