• Title, Summary, Keyword: RNAi

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Validation of Gene Silencing Using RNA Interference in Buffalo Granulosa Cells

  • Monga, Rachna;Datta, Tirtha Kumar;Singh, Dheer
    • Asian-Australasian Journal of Animal Sciences
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    • v.24 no.11
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    • pp.1529-1540
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    • 2011
  • Silencing of a specific gene using RNAi (RNA interference) is a valuable tool for functional analysis of a target gene. However, information on RNAi for analysis of gene function in farm animals is relatively nil. In the present study, we have validated the interfering effects of siRNA (small interfering RNA) using both quantitative and qualitative gene silencing in buffalo granulosa cells. Qualitative gene knockdown was validated using a fluorescent vector, enhanced green fluorescence protein (EGFP) and fluorescently labeled siRNA (Cy3) duplex. While quantitatively, siRNA targeted against the luciferase and CYP19 mRNA was used to validate the technique. CYP19 gene, a candidate fertility gene, was selected as a model to demonstrate the technique optimization. However, to sustain the expression of CYP19 gene in culture conditions using serum is difficult because granulosa cells have the tendency to luteinize in presence of serum. Therefore, serum free culture conditions were optimized for transfection and were found to be more suitable for the maintenance of CYP19 gene transcripts in comparison to culture conditions with serum. Decline in fluorescence intensity of green fluorescent protein (EGFP) was observed following co-transfection with plasmid generating siRNA targeted against EGFP gene. Quantitative decrease in luminescence was seen when co-transfected with siRNA against the luciferase gene. A significant suppressive effect on the mRNA levels of CYP19 gene at 100 nM siRNA concentration was observed. Also, measurement of estradiol levels using ELISA (enzyme-linked immunosorbent assay) showed a significant decline in comparison to control. In conclusion, the present study validated gene silencing using RNAi in cultured buffalo granulosa cells which can be used as an effective tool for functional analysis of target genes.

Silencing of Lysyl Oxidase Gene Expression by RNA Interference Suppresses Metastasis of Breast Cancer

  • Liu, Jian-Lun;Wei, Wei;Tang, Wei;Jiang, Yi;Yang, Hua-Wei;Li, Jing-Tao;Zhou, Xiao
    • Asian Pacific Journal of Cancer Prevention
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    • v.13 no.7
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    • pp.3507-3511
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    • 2012
  • Objective: The aim of this study was to investigate possible mechanisms of LOX gene effects on invasion and metastasis of breast cancer cells by RNA interference. Methods: LOX-RNAi-LV was designed, synthesized, and then transfected into a breast cancer cell line (MDA-MB-231). Expression of LOX, MMP-2 and MMP-9 was determined by real-time PCR, and protein expression of LOX by Western blotting. Cell migration and invasiveness were assessed with Transwell chambers. A total of 111 cases of breast cancer tissues, cancer-adjacent normal breast tissues, and 20 cases of benign lesion tissues were assessed by immunohistochemistry. Results: Expression of LOX mRNA and protein was suppressed, and the expression of MMP-2 and MMP-9 was significantly lower in the RNAi group than the control group (P<0.05), after LOX-RNAi-LV was transfection into MDA-MB-231 cells. Migration and invasion abilities were obviously inhibited. The expression of LOX protein in breast cancer, cancer-adjacent normal breast tissues and benign breast tumor were 48.6% (54/111), 26.1% (29/111), 20.0% (4/20), respectively, associations being noted with clinical stage, lymph node metastasis, tumor size and ER, PR, HER2, but not age. LOX protein was positively correlated with MMP-2 and MMP-9. Conclusion: LOX displayed an important role in invasion and metastasis of breast cancer by regulating MMP-2 and MMP-9 expression which probably exerted synergistic effects on the extracellular matrix (ECM).

Simultaneous and Systemic Knock-down of Big Defensin 1 and 2 gene Expression in the Pacific Oyster Crassostrea gigas using Long Double-stranded RNA-mediated RNA Interference

  • Jee, Bo Young;Kim, Min Sun;Cho, Mi Young;Lee, Soon Jeong;Park, Myung Ae;Kim, Jin Woo;Choi, Seung Hyuk;Jeong, Hyun Do;Kim, Ki Hong
    • Fisheries and aquatic sciences
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    • v.17 no.3
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    • pp.377-380
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    • 2014
  • RNA interference (RNAi)-mediated transcriptional knock-down of Crassostrea gigas big defensin 1 and 2 genes (Cg-BigDef1 and Cg-BigDef2) was investigated. The cDNA sequences of Cg-BigDef1 and Cg-BigDef2 were identical, excluding an additional fragment of 20 nucleotides in Cg-BigDef1; thus, a long double-stranded RNA (dsRNA) targeting the mRNA of Cg-BigDef2 effectively downregulated both Cg-BigDef2 and Cg-BigDef1. In addition, long dsRNA targeting green fluorescent protein (GFP) did not affect transcription of the two big defensin genes. These results suggest that the transcriptional downregulation of Cg-BigDef1 and Cg-BigDef2 was mediated by sequence-specific RNA interference (RNAi). Despite injection of long dsRNA targeting Cg-BigDef2 into only the adductor muscle, knock-down of Cg-BigDef1 and Cg-BigDef2 was observed in the adductor muscle, hemocytes, mantle, and gills, suggestive of systemic spread of RNAi in C. gigas. Furthermore, the inhibitory effect of dsRNA persisted until 72 h post-injection, indicative of a long-lasting RNAi-mediated knock-down of target genes.

Inhibition of mIGF-1 and mGHR Gene Expression using Tetracycline-Inducible RNAi System in Mouse Liver Cell (Tetracycline 유도적인 RNAi System을 이용한 생쥐 성장 관련 유전자의 발현 억제)

  • Son, Hye Jin;Koo, Bon Chul;Kwon, Mo Sun;Lee, Young Man;Kim, Teoan
    • Reproductive and Developmental Biology
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    • v.38 no.3
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    • pp.99-105
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    • 2014
  • In this study, to further understand the mechanism of animal growth and to develop a miniature transgenic animal model, we constructed and tested tetracycline-inducible RNAi system using shRNA targeting the mRNA of mouse insulin-like growth factor (mIGF-1) or mouse growth hormone receptor (mGHR) gene. Quantitative real-time PCR analysis of mouse liver cell (Hepa1c1c7) cells transfected with these vectors showed 85% or 90% of expression inhibition effect of IGF-1 or GHR, respectively. In ELISA analysis, the protein level of IGF-1 in the cells expressing the shRNA targeting IGF-1 mRNA was reduced to 26% of non-transformed control cells. Unexpectedly, in case of using shRNA targeting GHR, the IGF-1 protein level was decreased to 75% of control cells. Further experiments are needed to explain the lower interference effect of GHR shRNA in IGF-1 protein. Accumulated knowledge of this approach could be applicable to a variety of related biological area including gene function study, gene therapy, development of miniature animals, etc.

Expression of Gas6 Receptors, Tyro3, Axl, and Mertk, in Oocytes and Embryos and Effects of Mertk RNAi on the Oocyte Maturation

  • Kim, Kyeoung-Hwa;Lee, Sang-Eun;Lee, Kyung-Ah
    • Development and Reproduction
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    • v.16 no.3
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    • pp.195-204
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    • 2012
  • Recently, we reported growth arrest-specific gene 6 (Gas6) as a new maternal effect gene (MEG), that expressed in the oocytes but functioned principally during embryogenesis. Gas6 RNAi-treated oocytes developed to metaphase II (MII) stage but they have affected M-phase promoting factor (MPF) activity and incurred abnormal pronuclear (PN) formation during fertilization. Gas6 is a ligand of TAM family members (Tyro3, Axl and Mertk) of receptor tyrosine kinase (RTK). Purpose of the present study was to evaluate the expression of Tyro3, Axl and Mertk transcripts in oocytes and early embryos. Expression of Gas6 and Mertk mRNA was detectable in oocytes and follicular cells, while Tyro3 and Axl mRNA was expressed only in follicular cells. Expression of Mertk mRNA was relatively constant during oocytes maturation and embryogenesis, but the other receptors, Tyro3 and Axl, were not expressed in oocytes and PN stage of embryos at all. Knockdown of Mertk mRNA and protein by using sequence-specific Mertk double strand RNA (dsRNA) did not affect oocytes maturation. In this case, however, contrary to the ligand Gas6 RNA interference (RNAi), MPF activity had not been changed by Mertk RNAi. Therefore, we concluded that the Gas6-Mertk signaling is not directly related to the oocyte maturation. It is still required to study further regarding the function of Mertk as the receptor of Gas6 during preimplantational early embryogenesis.

Cell-SELEX Based Identification of an RNA Aptamer for Escherichia coli and Its Use in Various Detection Formats

  • Dua, Pooja;Ren, Shuo;Lee, Sang Wook;Kim, Joon-Ki;Shin, Hye-su;Jeong, OK-Chan;Kim, Soyoun;Lee, Dong-Ki
    • Molecules and Cells
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    • v.39 no.11
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    • pp.807-813
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    • 2016
  • Escherichia coli are important indicator organisms, used routinely for the monitoring of water and food safety. For quick, sensitive and real-time detection of E. coli we developed a 2'F modified RNA aptamer Ec3, by Cell-SELEX. The 31 nucleotide truncated Ec3 demonstrated improved binding and low nano-molar affinity to E. coli. The aptamer developed by us out-performs the commercial antibody and aptamer used for E. coli detection. Ec3(31) aptamer based E. coli detection was done using three different detection formats and the assay sensitivities were determined. Conventional Ec3(31)-biotin-streptavidin magnetic separation could detect E. coli with a limit of detection of $1.3{\times}10^6CFU/ml$. Although, optical analytic technique, biolayer interferometry, did not improve the sensitivity of detection for whole cells, a very significant improvement in the detection was seen with the E. coli cell lysate ($5{\times}10^4CFU/ml$). Finally we developed Electrochemical Impedance Spectroscopy (EIS) gap capacitance biosensor that has detection limits of $2{\times}10^4CFU/mL$ of E. coli cells, without any labeling and signal amplification techniques. We believe that our developed method can step towards more complex and real sample application.

Genetic Modification of Coffee Plants

  • Shinjiro Ogita;Hirotaka Uefuji;Park, Yong-Eui;Tomoko Hatanaka;Mikihiro Ogawa;Yube Yamaguchi;Nozomu Koizumi;Hiroshi Sano
    • Journal of Plant Biotechnology
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    • v.4 no.3
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    • pp.91-94
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    • 2002
  • An efficient molecular breeding technique for coffee plants was developed. In order to produce transgenic coffee plants, we established a model transformation procedure via Agrobacterium method. We isolated a gene encoding a protein possessing 7-methylxanthine methyltransferase (theobromine synthase) activity, and it was designated as Coffea arabica 7-methylxanthine methyl transferase; CaMXMT. Using this clone, we produced transgenic coffee plants, in which the expression of CaMXMT is suppressed by double-stranded RNA interference (RNAi) andlor anti-sense methods. The expression pattern of CaMXMT was analyzed by reverse transcription-PCR method and we found that, in the transformed cell lines, the level of transcripts were obviously suppressed by RNAi. The endogenous level of caffeine in the transformed cells was dramatically reduced in comparison with non-transformed cells.

Functional Polymeric Materials for Biomedical Application (생체의료용 기능성 고분자 재료의 개발)

  • Sung, Yong-Kiel;Song, Dae-Kyung;Sung, Jung-Suk
    • Polymer Korea
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    • v.30 no.1
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    • pp.1-9
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    • 2006
  • The development of functional polymeric materials for biomedical application has progressed on the basis of functionality, biocompatibility and biodegradability. In this paper we review the functional polymeric biomaterialsbased systems and propose a range of biomedical applications in the near future. These systems include the functional biodegradable polymers synthesized in our research laboratory, biodegradable polymeric materials, thermosensitive polymeric materials, cationic polymeric materials, non-condensing polymeric biomaterials, bio-polymeric DNA matrix for tissue engineering, and polymeric biomaterials for RNA interference (RNAi) technology.

The Role of Transglutaminase in Double-stranded DNA-Triggered Antiviral Innate Immune Response

  • Yoo, Jae-Wook;Hong, Sun-Woo;Bose, Shambhunath;Kim, Ho-Jun;Kim, Soo-Youl;Kim, So-Youn;Lee, Dong-Ki
    • Bulletin of the Korean Chemical Society
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    • v.32 no.11
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    • pp.3893-3898
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    • 2011
  • Cellular uptake of double-stranded DNA (dsDNA) triggers strong innate immune responses via activation of NF-${\kappa}B$ transcription factor. However, the detailed mechanism of dsDNA-mediated innate immune response remains yet to be elucidated. Here, we show that the expression of tazarotene-induced gene 3 (TIG3) is dramatically induced by dsDNA stimulation, and the siRNA-mediated down-regulation of TIG3 mRNA results in significant suppression of dsDNA-triggered cytokine expression. Because TIG3 has been previously shown to physically interact with transglutaminase (TG) 1 to activate TG activity, and TG2 has been shown to induce NF-${\kappa}B$ activity by inducing $I{\kappa}B{\alpha}$ polymerization, we tested whether TG also plays a role in dsDNA-mediated innate immune response. Pre-treatment of TG inhibitors dramatically reduces dsDNA-triggered cytokine induction. We also show that, in HeLa cells, TG2 is the major TG, and TIG3 physically interacts with TG2. Combined together, our results suggest a novel mechanism of dsDNA-triggered innate immune response which is critically dependent on TIG3 and TG2.