Supplementary Material for: Maternal Diet Enriched with α-Linolenic or Saturated Fatty Acids Differentially Regulates Gene Expression in the Liver of Mouse Offspring

<p><b><i>Background/Aims:</i></b> Lipid metabolic disarray in the liver of young and adult mice offspring is induced by saturated fatty acids (SFA) but prevented by α-linolenic acid (ALA, 18:3 ω-3) in the maternal diet during pregnancy and lactation. The aim of the present study was to analyze the impact of maternal dietary ALA compared to an SFA diet on the liver gene expression in the newborn offspring. <b><i>Methods:</i></b> C57Bl6/J dams were fed with diets normal in calories but rich in ALA or SFA before mating and during pregnancy. Pups were sacrificed at birth and liver parameters were assessed. Gene expression was characterized by microarray analysis and validated by real-time quantitative PCR. <b><i>Results:</i></b> ALA, compared to SFA, in maternal diets during pregnancy increased polyunsaturated fatty acids, while it differentially modified fatty acid desaturase activities in offspring liver. Overall, 474 and 662 genes from the liver of newborn pups were differentially regulated by ALA and SFA compared to control diet (p < 0.05; fold change 2), respectively. Notably, Per3 was upregulated by ALA, whereas it was downregulated by SFA, compared to control diet. <b><i>Conclusions:</i></b> ALA- and SFA-enriched diets differentially affect the gene expression pattern in the offspring's liver. ALA, in particular, upregulates genes associated with low adiposity.</p

Efficient Subtractive Cloning of Genes Activated by Lipopolysaccharide and Interferon γ in Primary-Cultured Cortical Cells of Newborn Mice.

Innate immune responses play a central role in neuroprotection and neurotoxicity during inflammatory processes that are triggered by pathogen-associated molecular pattern-exhibiting agents such as bacterial lipopolysaccharide (LPS) and that are modulated by inflammatory cytokines such as interferon γ (IFNγ). Recent findings describing the unexpected complexity of mammalian genomes and transcriptomes have stimulated further identification of novel transcripts involved in specific physiological and pathological processes, such as the neural innate immune response that alters the expression of many genes. We developed a system for efficient subtractive cloning that employs both sense and antisense cRNA drivers, and coupled it with in-house cDNA microarray analysis. This system enabled effective direct cloning of differentially expressed transcripts, from a small amount (0.5 µg) of total RNA. We applied this system to isolation of genes activated by LPS and IFNγ in primary-cultured cortical cells that were derived from newborn mice, to investigate the mechanisms involved in neuroprotection and neurotoxicity in maternal/perinatal infections that cause various brain injuries including periventricular leukomalacia. A number of genes involved in the immune and inflammatory response were identified, showing that neonatal neuronal/glial cells are highly responsive to LPS and IFNγ. Subsequent RNA blot analysis revealed that the identified genes were activated by LPS and IFNγ in a cooperative or distinctive manner, thereby supporting the notion that these bacterial and cellular inflammatory mediators can affect the brain through direct but complicated pathways. We also identified several novel clones of apparently non-coding RNAs that potentially harbor various regulatory functions. Characterization of the presently identified genes will give insights into mechanisms and interventions not only for perinatal infection-induced brain damage, but also for many other innate immunity-related brain disorders