Efficient subtractive cloning of genes activated by lipopolysaccharide and interferon γ in primary-cultured cortical cells of newborn mice.
Efficient subtractive cloning of genes activated by lipopolysaccharide and interferon γ in primary-cultured cortical cells of newborn mice.
复制标题
DOI：
10.1371/journal.pone.0079236
复制
发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Takiguchi M
中科院分区：
文献类型：
--
作者：
Miyauchi O;Iwase K;Itoh K;Kato M;Seki N;Braissant O;Bachmann C;Shozu M;Sekiya S;Osada H;Takiguchi M
在炎症过程中，先天免疫反应在神经保护和神经毒性中发挥着核心作用，炎症过程由病原体相关的分子模式展示剂如细菌脂多糖触发，并受炎症细胞因子如干扰素γ(干扰素γ)的调节。最近的发现描述了哺乳动物基因组和转录本出人意料的复杂性，促进了对参与特定生理和病理过程的新转录本的进一步鉴定，例如改变许多基因表达的神经先天免疫反应。我们开发了一个同时使用正义和反义cRNA驱动程序的高效消减克隆系统，并将其与内部的cDNA微阵列分析相结合。该系统能够从少量(0.5微克)的总RNA中有效地直接克隆差异表达的转录本。我们将该系统应用于分离原代培养的新生小鼠皮质细胞中由脂多糖和干扰素γ激活的基因，以探讨导致包括脑室周围白质软化症在内的各种脑损伤的母婴感染的神经保护和神经毒性机制。许多参与免疫和炎症反应的基因被鉴定出来，表明新生神经元/神经胶质细胞对脂多糖和干扰素γ具有高度的反应性。随后的核糖核酸印迹分析显示，已鉴定的基因被脂多糖和干扰素γ以协同或独特的方式激活，从而支持了这些细菌和细胞炎症介质可以通过直接但复杂的途径影响大脑的观点。我们还鉴定了几个明显非编码RNA的新克隆，它们可能具有各种调节功能。目前已确定的基因的特征将不仅对围产期感染引起的脑损伤的机制和干预措施有深入的了解，而且对许多其他与先天免疫相关的脑疾病也有帮助。
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.
登录
查看更多内容
影响因子：
5.3
作者：
Alfonso-Loeches, Silvia;Pascual-Lucas, Maya;Guerri, Consuelo
通讯作者：
Guerri, Consuelo
影响因子：
5.3
作者：
Bilbo, SD;Biedenkapp, JC;Maier, SF
通讯作者：
Maier, SF
影响因子：
37.8
作者：
Caso, Javier R.;Pradillo, Jesus M.;Lizasoain, Ignacio
通讯作者：
Lizasoain, Ignacio
影响因子：
16.2
作者：
Datwani, Akash;McConnell, Michael J.;Kanold, Patrick O.;Micheva, Kristina D.;Busse, Brad;Shamloo, Mehrdad;Smith, Stephen J.;Shatz, Carla J.
通讯作者：
Shatz, Carla J.
影响因子：
9.8
作者：
De Santa F;Barozzi I;Mietton F;Ghisletti S;Polletti S;Tusi BK;Muller H;Ragoussis J;Wei CL;Natoli G
通讯作者：
Natoli G