Expression of temperature responsive genes in cell cultures derived from Bombyx mori

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  • ABSTRACT

    Insects are heterotherms that exhibit a close relationship between their ecology (especially temperature changes) and physiology. In the present study, selected genes associated with cell death and temperature were examined to determine gene expression in Bombyx mori in high and low temperature environments. We determined the amount of dsRNA, different concentrations of dsRNA, and different type of cells to set the conditions most efficient for RNAi. We then prepared dsRNA transcripts of the genes associated with cell death and temperature response. We analyzed cell damage via Trypan blue staining and found that cell viability was reduced after knockdown of these genes. The special transduced cell lines produced in the present study can be applied in various research fields. We also expect that these cell lines can be used as a research tool for the precise functional analysis of various genes.


  • KEYWORD

    Bombyx mori , BmN4 , BmN-SID-1 , RNAi , cell viability

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  • [Fig. 1.] Gel electrophoresis dsRNA that synthesize with specific primer. Identification of proper dsRNA synthesis through product size and sequencing.
    Gel electrophoresis dsRNA that synthesize with specific primer. Identification of proper dsRNA synthesis through product size and sequencing.
  • [Table 1.] Primers used in this study.
    Primers used in this study.
  • [Fig. 2.] (A) BmN-SID-1 cells demonstrate rapid response upon the depletion of IAP-1. It treated different volume of dsIAP-1 separately. At 24 h of soaking, cells were observed under microscope. Arrows indicate the apoptotic phenotype induced by the depletion of IAP-1. (B) MTS assay that treated only dsIAP-1 (n=6)
    (A) BmN-SID-1 cells demonstrate rapid response upon the depletion of IAP-1. It treated different volume of dsIAP-1 separately. At 24 h of soaking, cells were observed under microscope. Arrows indicate the apoptotic phenotype induced by the depletion of IAP-1. (B) MTS assay that treated only dsIAP-1 (n=6)
  • [Fig. 3.] (A) The cells socked dsIAP-1 was distributed with cell density (percentage). Arrows indicate the apoptotic phenotype induced by the depletion of IAP-1. (B) MTS assay that the cells socked dsIAP-1 were distributed with cell density (percentage) (n=6).
    (A) The cells socked dsIAP-1 was distributed with cell density (percentage). Arrows indicate the apoptotic phenotype induced by the depletion of IAP-1. (B) MTS assay that the cells socked dsIAP-1 were distributed with cell density (percentage) (n=6).
  • [Fig. 4.] (A) BmN4 cells and (B) BmN-SID-1 demonstrate rapid response upon the depletion of IAP-1. (A) At 24 h of transfection, cells were observed under microscope. Arrows indicate the apoptotic phenotype induced by the depletion of IAP-1. (I: control, II: mock, III: Fugene HD, IV: dsGFP, V: dsIAP-1, VI: Fugene HD+dsGFP, VII: Fugene HD+dsIAP-1). (B) At 24 h of transfection, cells were observed under microscope. Arrows indicate the apoptotic phenotype induced by the depletion of IAP-1. (I: control, II: mock, III: Fugene HD, IV: dsGFP, V: dsIAP-1, VI: Fugene HD+dsIAP-1). (C) MTS assay, VII cell viability is the lowest and consequence that induced by the depletion of IAP-1 (n=6). (D) MTS assay, VI cell viability is the lowest and consequence that induced by the depletion of IAP-1 (n=6).
    (A) BmN4 cells and (B) BmN-SID-1 demonstrate rapid response upon the depletion of IAP-1. (A) At 24 h of transfection, cells were observed under microscope. Arrows indicate the apoptotic phenotype induced by the depletion of IAP-1. (I: control, II: mock, III: Fugene HD, IV: dsGFP, V: dsIAP-1, VI: Fugene HD+dsGFP, VII: Fugene HD+dsIAP-1). (B) At 24 h of transfection, cells were observed under microscope. Arrows indicate the apoptotic phenotype induced by the depletion of IAP-1. (I: control, II: mock, III: Fugene HD, IV: dsGFP, V: dsIAP-1, VI: Fugene HD+dsIAP-1). (C) MTS assay, VII cell viability is the lowest and consequence that induced by the depletion of IAP-1 (n=6). (D) MTS assay, VI cell viability is the lowest and consequence that induced by the depletion of IAP-1 (n=6).
  • [Fig. 5.] (A) BmN4-3R, BmN-SID-1-3R cell lines demonstrate RNAi effect upon the proper synthesis of dsGFP. All treated dsGFP cells decrease the green fluorescent. (B) BmN-SID-1-3R cell line demonstrates RNAi effect upon the proper synthesis of dsGFP. Treated dsGFP cell decreases the green fluorescent.
    (A) BmN4-3R, BmN-SID-1-3R cell lines demonstrate RNAi effect upon the proper synthesis of dsGFP. All treated dsGFP cells decrease the green fluorescent. (B) BmN-SID-1-3R cell line demonstrates RNAi effect upon the proper synthesis of dsGFP. Treated dsGFP cell decreases the green fluorescent.
  • [Fig. 6.] (A) MTS assay after transfected dsHsp70 using Fugene HD with BmN-SID-1 cell line. control: untreated cell, I: incubated under low temperature 0℃ 3 d and RNA extract as soon as added dsHsp70 II: incubated under low temperature 0℃ 3 d and RNA extract after 4 d added dsHsp70, III: incubated under low temperature 0℃ 3 d and RNA extract as soon as added dsHsp70 2 times, IV: incubated under low temperature 0℃ 3 d and RNA extract after 4 d added dsHsp70 2 times (n=6). (B) shows MTS color change. (-): dsRNA not treated, (+): dsRNA treated. (C) Identification of mRNA expression levels of BmN-SID-1 cell lines that incubated under control and low temperature (0℃ 3 d).
    (A) MTS assay after transfected dsHsp70 using Fugene HD with BmN-SID-1 cell line. control: untreated cell, I: incubated under low temperature 0℃ 3 d and RNA extract as soon as added dsHsp70 II: incubated under low temperature 0℃ 3 d and RNA extract after 4 d added dsHsp70, III: incubated under low temperature 0℃ 3 d and RNA extract as soon as added dsHsp70 2 times, IV: incubated under low temperature 0℃ 3 d and RNA extract after 4 d added dsHsp70 2 times (n=6). (B) shows MTS color change. (-): dsRNA not treated, (+): dsRNA treated. (C) Identification of mRNA expression levels of BmN-SID-1 cell lines that incubated under control and low temperature (0℃ 3 d).
  • [Fig. 7.] (A) MTS assay after transfected ds3R using Fugene HD with BmN-SID-1 cell line. I: not treated dsRNA under high temperature, II: treated dsRNA under high temperature, III: not treated dsRNA under low temperature, IV: treated dsRNA under low temperature (n=6). (B) real-time PCR of BmN-SID-1 cell lines after treated ds3R that incubated under control and low temperature (0℃ 3 d). (C) Cells were observed under microscope. (D) shows MTS color change. (E) Identification of mRNA expression levels of BmN-SID-1 cell lines after treated ds3R that incubated under control and low temperature (0℃ 3 d).
    (A) MTS assay after transfected ds3R using Fugene HD with BmN-SID-1 cell line. I: not treated dsRNA under high temperature, II: treated dsRNA under high temperature, III: not treated dsRNA under low temperature, IV: treated dsRNA under low temperature (n=6). (B) real-time PCR of BmN-SID-1 cell lines after treated ds3R that incubated under control and low temperature (0℃ 3 d). (C) Cells were observed under microscope. (D) shows MTS color change. (E) Identification of mRNA expression levels of BmN-SID-1 cell lines after treated ds3R that incubated under control and low temperature (0℃ 3 d).
  • [Fig. 8.] (A) MTS assay after transfected ds using Fugene HD with BmN-SID-1 cell line. I: not treated dsRNA under high temperature, II: treated dsRNA under high temperature, III: not treated dsRNA under low temperature, IV: treated dsRNA under low temperature (n=6). (B) Cells were observed under microscope. (C) shows MTS color change. (-): dsRNA not treated, (+): dsRNA treated. (D) Identification of mRNA expression levels of BmN-SID-1 cell lines after treated dsCalreticulin that incubated under control and low temperature (0℃ 3 d).
    (A) MTS assay after transfected ds using Fugene HD with BmN-SID-1 cell line. I: not treated dsRNA under high temperature, II: treated dsRNA under high temperature, III: not treated dsRNA under low temperature, IV: treated dsRNA under low temperature (n=6). (B) Cells were observed under microscope. (C) shows MTS color change. (-): dsRNA not treated, (+): dsRNA treated. (D) Identification of mRNA expression levels of BmN-SID-1 cell lines after treated dsCalreticulin that incubated under control and low temperature (0℃ 3 d).