Cargando…
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...
Autores principales: | , , , , , , , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2013
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3823591/ https://www.ncbi.nlm.nih.gov/pubmed/24244457 http://dx.doi.org/10.1371/journal.pone.0079236 |
_version_ | 1782290591752650752 |
---|---|
author | Miyauchi, Osamu Iwase, Katsuro Itoh, Kanako Kato, Masaki Seki, Naohiko Braissant, Olivier Bachmann, Claude Shozu, Makio Sekiya, Souei Osada, Hisao Takiguchi, Masaki |
author_facet | Miyauchi, Osamu Iwase, Katsuro Itoh, Kanako Kato, Masaki Seki, Naohiko Braissant, Olivier Bachmann, Claude Shozu, Makio Sekiya, Souei Osada, Hisao Takiguchi, Masaki |
author_sort | Miyauchi, Osamu |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-3823591 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2013 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-38235912013-11-15 Efficient Subtractive Cloning of Genes Activated by Lipopolysaccharide and Interferon γ in Primary-Cultured Cortical Cells of Newborn Mice Miyauchi, Osamu Iwase, Katsuro Itoh, Kanako Kato, Masaki Seki, Naohiko Braissant, Olivier Bachmann, Claude Shozu, Makio Sekiya, Souei Osada, Hisao Takiguchi, Masaki PLoS One Research Article 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. Public Library of Science 2013-11-11 /pmc/articles/PMC3823591/ /pubmed/24244457 http://dx.doi.org/10.1371/journal.pone.0079236 Text en © 2013 Miyauchi et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. |
spellingShingle | Research Article Miyauchi, Osamu Iwase, Katsuro Itoh, Kanako Kato, Masaki Seki, Naohiko Braissant, Olivier Bachmann, Claude Shozu, Makio Sekiya, Souei Osada, Hisao Takiguchi, Masaki Efficient Subtractive Cloning of Genes Activated by Lipopolysaccharide and Interferon γ in Primary-Cultured Cortical Cells of Newborn Mice |
title | Efficient Subtractive Cloning of Genes Activated by Lipopolysaccharide and Interferon γ in Primary-Cultured Cortical Cells of Newborn Mice |
title_full | Efficient Subtractive Cloning of Genes Activated by Lipopolysaccharide and Interferon γ in Primary-Cultured Cortical Cells of Newborn Mice |
title_fullStr | Efficient Subtractive Cloning of Genes Activated by Lipopolysaccharide and Interferon γ in Primary-Cultured Cortical Cells of Newborn Mice |
title_full_unstemmed | Efficient Subtractive Cloning of Genes Activated by Lipopolysaccharide and Interferon γ in Primary-Cultured Cortical Cells of Newborn Mice |
title_short | Efficient Subtractive Cloning of Genes Activated by Lipopolysaccharide and Interferon γ in Primary-Cultured Cortical Cells of Newborn Mice |
title_sort | efficient subtractive cloning of genes activated by lipopolysaccharide and interferon γ in primary-cultured cortical cells of newborn mice |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3823591/ https://www.ncbi.nlm.nih.gov/pubmed/24244457 http://dx.doi.org/10.1371/journal.pone.0079236 |
work_keys_str_mv | AT miyauchiosamu efficientsubtractivecloningofgenesactivatedbylipopolysaccharideandinterferonginprimaryculturedcorticalcellsofnewbornmice AT iwasekatsuro efficientsubtractivecloningofgenesactivatedbylipopolysaccharideandinterferonginprimaryculturedcorticalcellsofnewbornmice AT itohkanako efficientsubtractivecloningofgenesactivatedbylipopolysaccharideandinterferonginprimaryculturedcorticalcellsofnewbornmice AT katomasaki efficientsubtractivecloningofgenesactivatedbylipopolysaccharideandinterferonginprimaryculturedcorticalcellsofnewbornmice AT sekinaohiko efficientsubtractivecloningofgenesactivatedbylipopolysaccharideandinterferonginprimaryculturedcorticalcellsofnewbornmice AT braissantolivier efficientsubtractivecloningofgenesactivatedbylipopolysaccharideandinterferonginprimaryculturedcorticalcellsofnewbornmice AT bachmannclaude efficientsubtractivecloningofgenesactivatedbylipopolysaccharideandinterferonginprimaryculturedcorticalcellsofnewbornmice AT shozumakio efficientsubtractivecloningofgenesactivatedbylipopolysaccharideandinterferonginprimaryculturedcorticalcellsofnewbornmice AT sekiyasouei efficientsubtractivecloningofgenesactivatedbylipopolysaccharideandinterferonginprimaryculturedcorticalcellsofnewbornmice AT osadahisao efficientsubtractivecloningofgenesactivatedbylipopolysaccharideandinterferonginprimaryculturedcorticalcellsofnewbornmice AT takiguchimasaki efficientsubtractivecloningofgenesactivatedbylipopolysaccharideandinterferonginprimaryculturedcorticalcellsofnewbornmice |