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The Tribolium castaneum cell line TcA: a new tool kit for cell biology.

Silver K, Jiang H, Fu J, Phillips TW, Beeman RW, Park Y - Sci Rep (2014)

Bottom Line: Next generation transcriptome sequencing of TcA cells demonstrated expression of a wide variety of genes associated with specialized functions in chitin metabolism, immune responses and cellular and systemic RNAi pathways.TcA cells were highly sensitive to minute amounts of dsRNA, with a minimum effective dose of 100 pg/mL resulting in significant suppression of gene expression.Our results show that the TcA cell line, with its sensitivity to RNAi and functional TcA-specific promoters, is an invaluable resource for studying basic molecular and physiological questions.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Physiology, Kansas State University, Manhattan, KS 66506.

ABSTRACT
The red flour beetle, Tribolium castaneum, is an agriculturally important insect pest that has been widely used as a model organism. Recently, an adherent cell line (BCIRL-TcA-CLG1 or TcA) was developed from late pupae of the red flour beetle. Next generation transcriptome sequencing of TcA cells demonstrated expression of a wide variety of genes associated with specialized functions in chitin metabolism, immune responses and cellular and systemic RNAi pathways. Accordingly, we evaluated the sensitivity of TcA cells to dsRNA to initiate an RNAi response. TcA cells were highly sensitive to minute amounts of dsRNA, with a minimum effective dose of 100 pg/mL resulting in significant suppression of gene expression. We have also developed a plasmid containing two TcA-specific promoters, the promoter from the 40S ribosomal protein subunit (TC006550) and a bi-directional heat shock promoter (TcHS70) from the intergenic space between heat shock proteins 68a and b. These promoters have been employed to provide high levels of either constitutive (TC006550) or inducible (TcHS70) gene expression of the reporter proteins. Our results show that the TcA cell line, with its sensitivity to RNAi and functional TcA-specific promoters, is an invaluable resource for studying basic molecular and physiological questions.

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TcHS70 promoter structure and TcA response to heat shock for increased transcripts.(a) Genomic structure of the heat shock proteins 68a and 68b. (b) The promoter region containing the heat shock factor binding motifs, putative TATA boxes and transcription start sites. (c) Induction of expression of heat shock proteins 68a, b, and another homologous sequence TC10172 in TcA, determined by quantitative RT-PCR with rps3 normalization.
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f4: TcHS70 promoter structure and TcA response to heat shock for increased transcripts.(a) Genomic structure of the heat shock proteins 68a and 68b. (b) The promoter region containing the heat shock factor binding motifs, putative TATA boxes and transcription start sites. (c) Induction of expression of heat shock proteins 68a, b, and another homologous sequence TC10172 in TcA, determined by quantitative RT-PCR with rps3 normalization.

Mentions: The TcHS70 contained multiple putative heat shock sequence elements (Fig. 3a and b). Initially, we tested the TcHS70 activity after a heat shock in the endogenous cell. Immediately after a heat shock of TcA cells at 40°C for 30 min, total RNA was extracted to investigate the transcriptional responses of the hsp genes. We found ~300 to 500-fold increases in mRNA levels for each hsp68a, hsp68b, and another highly similar hsp TC010172 after normalization to rps3 transcript levels and comparison to the no heat shock control (Fig. 4c). We subsequently used the 1910bp intergenic region between the coding genes for hsp68a and hsp68b as the TcHS70 promoter (Fig. 4a and 5a). This sequence included the putative TATA boxes, transcription start sites located in each end, and numerous heat shock sequence elements (Fig. 4b).


The Tribolium castaneum cell line TcA: a new tool kit for cell biology.

Silver K, Jiang H, Fu J, Phillips TW, Beeman RW, Park Y - Sci Rep (2014)

TcHS70 promoter structure and TcA response to heat shock for increased transcripts.(a) Genomic structure of the heat shock proteins 68a and 68b. (b) The promoter region containing the heat shock factor binding motifs, putative TATA boxes and transcription start sites. (c) Induction of expression of heat shock proteins 68a, b, and another homologous sequence TC10172 in TcA, determined by quantitative RT-PCR with rps3 normalization.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4213800&req=5

f4: TcHS70 promoter structure and TcA response to heat shock for increased transcripts.(a) Genomic structure of the heat shock proteins 68a and 68b. (b) The promoter region containing the heat shock factor binding motifs, putative TATA boxes and transcription start sites. (c) Induction of expression of heat shock proteins 68a, b, and another homologous sequence TC10172 in TcA, determined by quantitative RT-PCR with rps3 normalization.
Mentions: The TcHS70 contained multiple putative heat shock sequence elements (Fig. 3a and b). Initially, we tested the TcHS70 activity after a heat shock in the endogenous cell. Immediately after a heat shock of TcA cells at 40°C for 30 min, total RNA was extracted to investigate the transcriptional responses of the hsp genes. We found ~300 to 500-fold increases in mRNA levels for each hsp68a, hsp68b, and another highly similar hsp TC010172 after normalization to rps3 transcript levels and comparison to the no heat shock control (Fig. 4c). We subsequently used the 1910bp intergenic region between the coding genes for hsp68a and hsp68b as the TcHS70 promoter (Fig. 4a and 5a). This sequence included the putative TATA boxes, transcription start sites located in each end, and numerous heat shock sequence elements (Fig. 4b).

Bottom Line: Next generation transcriptome sequencing of TcA cells demonstrated expression of a wide variety of genes associated with specialized functions in chitin metabolism, immune responses and cellular and systemic RNAi pathways.TcA cells were highly sensitive to minute amounts of dsRNA, with a minimum effective dose of 100 pg/mL resulting in significant suppression of gene expression.Our results show that the TcA cell line, with its sensitivity to RNAi and functional TcA-specific promoters, is an invaluable resource for studying basic molecular and physiological questions.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Physiology, Kansas State University, Manhattan, KS 66506.

ABSTRACT
The red flour beetle, Tribolium castaneum, is an agriculturally important insect pest that has been widely used as a model organism. Recently, an adherent cell line (BCIRL-TcA-CLG1 or TcA) was developed from late pupae of the red flour beetle. Next generation transcriptome sequencing of TcA cells demonstrated expression of a wide variety of genes associated with specialized functions in chitin metabolism, immune responses and cellular and systemic RNAi pathways. Accordingly, we evaluated the sensitivity of TcA cells to dsRNA to initiate an RNAi response. TcA cells were highly sensitive to minute amounts of dsRNA, with a minimum effective dose of 100 pg/mL resulting in significant suppression of gene expression. We have also developed a plasmid containing two TcA-specific promoters, the promoter from the 40S ribosomal protein subunit (TC006550) and a bi-directional heat shock promoter (TcHS70) from the intergenic space between heat shock proteins 68a and b. These promoters have been employed to provide high levels of either constitutive (TC006550) or inducible (TcHS70) gene expression of the reporter proteins. Our results show that the TcA cell line, with its sensitivity to RNAi and functional TcA-specific promoters, is an invaluable resource for studying basic molecular and physiological questions.

Show MeSH
Related in: MedlinePlus