Limits...
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.

Show MeSH

Related in: MedlinePlus

Schematic illustration of cDNA amplification (A) and subsequent subtraction (B).See the text for explanation.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3823591&req=5

pone-0079236-g001: Schematic illustration of cDNA amplification (A) and subsequent subtraction (B).See the text for explanation.

Mentions: Total RNA was prepared from the stimulated (LPS and IFNγ) and non-stimulated primary-cultured neuronal/glial cells using the acid-guanidine-phenol-chloroform method [38], and was processed to amplify the total cDNA using PCR as schematically illustrated in Fig. 1A. The detailed procedures for Steps 1 and 3–6 were previously described [36]. Briefly, poly(A)+ RNA derived from 0.5 µg of total RNA was absorbed onto 50 µg of oligo(dT) magnetic beads Dynabeads Oligo(dT)25 (Dynal, Oslo, Norway), and was subjected to first-strand and second-strand cDNA synthesis (Fig. 1A, Step 1). Trimming of the resultant double-stranded cDNA to an estimated average of 1,024 bp lengths from the 3′-termini (Step 2) was performed by dividing the bead-fixed cDNA into three aliquots, each of which was digested with one of three restriction enzymes [BanI, EcoO109I, and HincII (0.5 units)] in 20 µL of the buffer solution consisting of 20 mM Tris-HCl (pH 7.9), 10 mM MgCl2, 50 mM KCl, and 1 mM DTT at 37°C for 1 h. The reaction was stopped by adding 0.8 µL of 0.5 M EDTA-Na (pH 8.0), and the enzymes were inactivated by heating the mixture at 65°C for 20 min. The three 3′-trimmed cDNA-bead suspensions were combined, and the beads were washed three times with 50 µL of 10 mM Tris-HCl (pH 8.0)/1 mM EDTA (Tris-EDTA buffer, TE). The bead-fixed 3′-trimmed cDNA was blunt-ended with 0.5 units of T4 DNA polymerase (Roche Diagnostics, Tokyo, Japan) in 20 µL of a mixture containing 50 mM Tris·HCl (pH 8.8), 15 mM (NH4)2SO4, 7 mM MgCl2, 0.1 mM EDTA, 10 mM 2-mercaptoethanol, 0.02 mg/mL bovine serum albumin, and 0.1 mM each of dATP, dCTP, dGTP, and dTTP at 16°C for 10 min. The reaction was stopped by adding 0.8 µL of 0.5 M EDTA-Na (pH 8.0), and the beads were washed three times with 50 µL of TE. The subsequent reactions were performed essentially as previously described [36]. The bead-fixed blunt-ended cDNA was ligated with a linker containing the T7 promoter sequence (Step 3). The sense-strand cDNA was liberated by heat-denaturation (Step 4), and was again converted to the double-stranded form using the oligo(dT) primer containing the SP6 promoter sequence (Step 5). The double-stranded cDNA was amplified by PCR using known sequences at both ends as primers (Step 6).


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)

Schematic illustration of cDNA amplification (A) and subsequent subtraction (B).See the text for explanation.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0079236-g001: Schematic illustration of cDNA amplification (A) and subsequent subtraction (B).See the text for explanation.
Mentions: Total RNA was prepared from the stimulated (LPS and IFNγ) and non-stimulated primary-cultured neuronal/glial cells using the acid-guanidine-phenol-chloroform method [38], and was processed to amplify the total cDNA using PCR as schematically illustrated in Fig. 1A. The detailed procedures for Steps 1 and 3–6 were previously described [36]. Briefly, poly(A)+ RNA derived from 0.5 µg of total RNA was absorbed onto 50 µg of oligo(dT) magnetic beads Dynabeads Oligo(dT)25 (Dynal, Oslo, Norway), and was subjected to first-strand and second-strand cDNA synthesis (Fig. 1A, Step 1). Trimming of the resultant double-stranded cDNA to an estimated average of 1,024 bp lengths from the 3′-termini (Step 2) was performed by dividing the bead-fixed cDNA into three aliquots, each of which was digested with one of three restriction enzymes [BanI, EcoO109I, and HincII (0.5 units)] in 20 µL of the buffer solution consisting of 20 mM Tris-HCl (pH 7.9), 10 mM MgCl2, 50 mM KCl, and 1 mM DTT at 37°C for 1 h. The reaction was stopped by adding 0.8 µL of 0.5 M EDTA-Na (pH 8.0), and the enzymes were inactivated by heating the mixture at 65°C for 20 min. The three 3′-trimmed cDNA-bead suspensions were combined, and the beads were washed three times with 50 µL of 10 mM Tris-HCl (pH 8.0)/1 mM EDTA (Tris-EDTA buffer, TE). The bead-fixed 3′-trimmed cDNA was blunt-ended with 0.5 units of T4 DNA polymerase (Roche Diagnostics, Tokyo, Japan) in 20 µL of a mixture containing 50 mM Tris·HCl (pH 8.8), 15 mM (NH4)2SO4, 7 mM MgCl2, 0.1 mM EDTA, 10 mM 2-mercaptoethanol, 0.02 mg/mL bovine serum albumin, and 0.1 mM each of dATP, dCTP, dGTP, and dTTP at 16°C for 10 min. The reaction was stopped by adding 0.8 µL of 0.5 M EDTA-Na (pH 8.0), and the beads were washed three times with 50 µL of TE. The subsequent reactions were performed essentially as previously described [36]. The bead-fixed blunt-ended cDNA was ligated with a linker containing the T7 promoter sequence (Step 3). The sense-strand cDNA was liberated by heat-denaturation (Step 4), and was again converted to the double-stranded form using the oligo(dT) primer containing the SP6 promoter sequence (Step 5). The double-stranded cDNA was amplified by PCR using known sequences at both ends as primers (Step 6).

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