RNAi-Mediated Silencing of FOXO Factors

  • Priyankar Sanphui
  • Akash Saha
  • Subhas Chandra BiswasEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1890)


RNAi-mediated silencing of a particular gene is an important tool in the field of biology. Knocking down experiments has revealed the role of different proteins and their downstream targets. Here, we describe the procedure of silencing transcription factor of Forkhead box class “O” (FOXO) using shRNA.

Key words

FOXO shRNA Competent cell Transformation Transfection 



The work was supported by one of the 12th Five Year Plan Projects, miND (BSC0115) of CSIR, Govt. of India.


  1. 1.
    Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391(6669):806–811CrossRefGoogle Scholar
  2. 2.
    Kutter C, Svoboda P (2008) miRNA, siRNA, piRNA: Knowns of the unknown. RNA Biol 5(4):181–188CrossRefGoogle Scholar
  3. 3.
    Galili N, Davis RJ, Fredericks WJ, Mukhopadhyay S, Rauscher FJ 3rd, Emanuel BS et al (1993) Fusion of a fork head domain gene to PAX3 in the solid tumour alveolar rhabdomyosarcoma. Nat Genet 5(3):230–235CrossRefGoogle Scholar
  4. 4.
    Hillion J, Le Coniat M, Jonveaux P, Berger R, Bernard OA (1997) AF6q21, a novel partner of the MLL gene in t(6;11)(q21;q23), defines a forkhead transcriptional factor subfamily. Blood 90(9):3714–3719PubMedGoogle Scholar
  5. 5.
    Borkhardt A, Repp R, Haas OA, Leis T, Harbott J, Kreuder J et al (1997) Cloning and characterization of AFX, the gene that fuses to MLL in acute leukemias with a t(X;11)(q13;q23). Oncogene 14(2):195–202CrossRefGoogle Scholar
  6. 6.
    Jacobs FM, van der Heide LP, Wijchers PJ, Burbach JP, Hoekman MF, Smidt MP (2003) FoxO6, a novel member of the FoxO class of transcription factors with distinct shuttling dynamics. J Biol Chem 278(38):35959–35967CrossRefGoogle Scholar
  7. 7.
    Schmidt M, Fernandez de Mattos S, van der Horst A, Klompmaker R, Kops GJ, Lam EW et al (2002) Cell cycle inhibition by FoxO forkhead transcription factors involves downregulation of cyclin D. Mol Cell Biol 22(22):7842–7852CrossRefGoogle Scholar
  8. 8.
    Tran H, Brunet A, Grenier JM, Datta SR, Fornace AJ Jr, DiStefano PS et al (2002) DNA repair pathway stimulated by the forkhead transcription factor FOXO3a through the Gadd45 protein. Science 296(5567):530–534CrossRefGoogle Scholar
  9. 9.
    Zhang X, Tang N, Hadden TJ, Rishi AK (2011) Akt, FoxO and regulation of apoptosis. Biochim Biophys Acta 1813(11):1978–1986CrossRefGoogle Scholar
  10. 10.
    Gross DN, van den Heuvel AP, Birnbaum MJ (2008) The role of FoxO in the regulation of metabolism. Oncogene 27(16):2320–2336CrossRefGoogle Scholar
  11. 11.
    Sengupta A, Molkentin JD, Yutzey KE (2009) FoxO transcription factors promote autophagy in cardiomyocytes. J Biol Chem 284(41):28319–28331CrossRefGoogle Scholar
  12. 12.
    Akhter R, Sanphui P, Biswas SC (2014) The essential role of p53-up-regulated modulator of apoptosis (Puma) and its regulation by FoxO3a transcription factor in beta-amyloid-induced neuron death. J Biol Chem 289(15):10812–10822CrossRefGoogle Scholar
  13. 13.
    Sanphui P, Biswas SC (2013) FoxO3a is activated and executes neuron death via Bim in response to beta-amyloid. Cell Death Dis 4:e625CrossRefGoogle Scholar
  14. 14.
    Kyoung Kim H, Kyoung Kim Y, Song IH, Baek SH, Lee SR, Hye Kim J et al (2005) Down-regulation of a forkhead transcription factor, FOXO3a, accelerates cellular senescence in human dermal fibroblasts. J Gerontol A Biol Sci Med Sci 60(1):4–9CrossRefGoogle Scholar
  15. 15.
    Ui-Tei K, Naito Y, Takahashi F, Haraguchi T, Ohki-Hamazaki H, Juni A et al (2004) Guidelines for the selection of highly effective siRNA sequences for mammalian and chick RNA interference. Nucleic Acids Res 32(3):936–948CrossRefGoogle Scholar
  16. 16.
    Taxman DJ, Livingstone LR, Zhang J, Conti BJ, Iocca HA, Williams KL et al (2006) Criteria for effective design, construction, and gene knockdown by shRNA vectors. BMC Biotechnol 6:7CrossRefGoogle Scholar
  17. 17.
    Reynolds A, Leake D, Boese Q, Scaringe S, Marshall WS, Khvorova A (2004) Rational siRNA design for RNA interference. Nat Biotechnol 22(3):326–330CrossRefGoogle Scholar
  18. 18.
    Biswas SC, Shi Y, Sproul A, Greene LA (2007) Pro-apoptotic Bim induction in response to nerve growth factor deprivation requires simultaneous activation of three different death signaling pathways. J Biol Chem 282(40):29368–29374CrossRefGoogle Scholar
  19. 19.
    Amarzguioui M, Prydz H (2004) An algorithm for selection of functional siRNA sequences. Biochem Biophys Res Commun 316(4):1050–1058CrossRefGoogle Scholar
  20. 20.
    Hribal ML, Nakae J, Kitamura T, Shutter JR, Accili D (2003) Regulation of insulin-like growth factor-dependent myoblast differentiation by Foxo forkhead transcription factors. J Cell Biol 162(4):535–541CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Priyankar Sanphui
    • 1
    • 2
  • Akash Saha
    • 1
  • Subhas Chandra Biswas
    • 1
    Email author
  1. 1.Cell Biology and Physiology DivisionCSIR-Indian Institute of Chemical BiologyKolkataIndia
  2. 2.Department of ZoologySree Chaitanya CollegeHabraIndia

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