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Efficient subtractive cloning of genes activated by lipopolysaccharide and interferon γ in primary-cultured cortical cells of newborn mice.

Miyauchi O, Iwase K, Itoh K, Kato M, Seki N, Braissant O, Bachmann C, Shozu M, Sekiya S, Osada H, Takiguchi M - PLoS ONE (2013)

Bottom Line: This system enabled effective direct cloning of differentially expressed transcripts, from a small amount (0.5 µg) of total RNA.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Genetics, Chiba University Graduate School of Medicine, Chiba, Japan ; Department of Reproductive Medicine, Chiba University Graduate School of Medicine, Chiba, Japan.

ABSTRACT
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.

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Time course of changes in mRNA levels of several genes in response to LPS and IFNγ.Total RNAs were prepared from primary-cultured neuronal/glial cells stimulated by IFNγ (I), LPS (L), or both (I/L) for indicated periods. RNAs (0.5 µg per lane) were electrophoresed and subjected to Northern analysis for the indicated mRNAs and for GAPDH mRNA as a control. Below the chemiluminogram, the densitometrically quantified band intensities are shown. “−” indicates that the band intensity was below the detectable level.
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pone-0079236-g004: Time course of changes in mRNA levels of several genes in response to LPS and IFNγ.Total RNAs were prepared from primary-cultured neuronal/glial cells stimulated by IFNγ (I), LPS (L), or both (I/L) for indicated periods. RNAs (0.5 µg per lane) were electrophoresed and subjected to Northern analysis for the indicated mRNAs and for GAPDH mRNA as a control. Below the chemiluminogram, the densitometrically quantified band intensities are shown. “−” indicates that the band intensity was below the detectable level.

Mentions: For several selected genes, the time-dependent changes in the mRNA levels in response to LPS and/or IFNγ were examined by Northern analysis (Fig. 4). In the present experimental system, cDNA clones of the subtracted libraries were ready to serve as templates for antisense strand-specific cRNA probes, as described in the Materials and Methods. Coadministration of LPS and IFNγ was required for strong induction of the mRNAs of the chemokines CCL5 (No. 1), CXCL9 (No. 4), and CCL2 (No. 8). The mRNA levels of serum amyloid A3 (SAA3; No. 2) and lipocalin 2 (No. 6) were mainly responsive to LPS, while those for the GTPases GBP-2 (No. 10) and IFI47 (No. 16) were mainly responsive to IFNγ. Therefore, these genes displayed differential patterns with respect to their responsiveness to LPS and IFNγ. The gene activation profiles in Fig. 4 are concordant with the notion that both LPS and IFNγ can affect the brain cells through direct but complicated means. It remains to be investigated what cell type is responsible for each gene activation in the present mixed neuronal/glial culture system.


Efficient subtractive cloning of genes activated by lipopolysaccharide and interferon γ in primary-cultured cortical cells of newborn mice.

Miyauchi O, Iwase K, Itoh K, Kato M, Seki N, Braissant O, Bachmann C, Shozu M, Sekiya S, Osada H, Takiguchi M - PLoS ONE (2013)

Time course of changes in mRNA levels of several genes in response to LPS and IFNγ.Total RNAs were prepared from primary-cultured neuronal/glial cells stimulated by IFNγ (I), LPS (L), or both (I/L) for indicated periods. RNAs (0.5 µg per lane) were electrophoresed and subjected to Northern analysis for the indicated mRNAs and for GAPDH mRNA as a control. Below the chemiluminogram, the densitometrically quantified band intensities are shown. “−” indicates that the band intensity was below the detectable level.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3823591&req=5

pone-0079236-g004: Time course of changes in mRNA levels of several genes in response to LPS and IFNγ.Total RNAs were prepared from primary-cultured neuronal/glial cells stimulated by IFNγ (I), LPS (L), or both (I/L) for indicated periods. RNAs (0.5 µg per lane) were electrophoresed and subjected to Northern analysis for the indicated mRNAs and for GAPDH mRNA as a control. Below the chemiluminogram, the densitometrically quantified band intensities are shown. “−” indicates that the band intensity was below the detectable level.
Mentions: For several selected genes, the time-dependent changes in the mRNA levels in response to LPS and/or IFNγ were examined by Northern analysis (Fig. 4). In the present experimental system, cDNA clones of the subtracted libraries were ready to serve as templates for antisense strand-specific cRNA probes, as described in the Materials and Methods. Coadministration of LPS and IFNγ was required for strong induction of the mRNAs of the chemokines CCL5 (No. 1), CXCL9 (No. 4), and CCL2 (No. 8). The mRNA levels of serum amyloid A3 (SAA3; No. 2) and lipocalin 2 (No. 6) were mainly responsive to LPS, while those for the GTPases GBP-2 (No. 10) and IFI47 (No. 16) were mainly responsive to IFNγ. Therefore, these genes displayed differential patterns with respect to their responsiveness to LPS and IFNγ. The gene activation profiles in Fig. 4 are concordant with the notion that both LPS and IFNγ can affect the brain cells through direct but complicated means. It remains to be investigated what cell type is responsible for each gene activation in the present mixed neuronal/glial culture system.

Bottom Line: This system enabled effective direct cloning of differentially expressed transcripts, from a small amount (0.5 µg) of total RNA.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Genetics, Chiba University Graduate School of Medicine, Chiba, Japan ; Department of Reproductive Medicine, Chiba University Graduate School of Medicine, Chiba, Japan.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus