Morpholinos versus RNAi in Gallus gallus
Jon Moulton
Tuesday, 23 September 2008 15:23 UTC
Mende M, Christophorou NA, Streit A. Specific and effective gene knock-down in early chick embryos using morpholinos but not pRFPRNAi vectors.
Mech Dev. 2008 Sep 3. [Epub ahead of print]
In the chick embryo, two methods are now used for studying the developmental role of genes by loss-of-function approaches: vector-based shRNA and morpholino oligonucleotides. Both have the advantage that loss-of-function can be conducted in a spatially and temporally controlled way by focal electroporation. Here, we compare these two methods. We find that the shRNA expressing vectors pRFPRNAi, even when targeting a non-expressed protein like GFP, cause morphological phenotypes, mis-regulation of non-targeted genes and activation of the p53 pathway. These effects are highly reproducible, appear to be independent of the targeting sequence and are particularly severe at primitive streak and early somite stages. By contrast, morpholinos do not cause these effects. We propose that pRFPRNAi should only be used with considerable caution and that morpholinos are a preferable approach for gene knock-down during early chick development.
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Replies
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Thanks for posting this link! We had been using the pSilencer vector for similar studies, and this is a very useful warning. (Luckily for us, we decided to act also at the protein level by over-expressing a non-functional protein in hopes of inducing a kind of dominant-negative effect.)
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Hi Heather,
Now that I know you are a Gallus gallus fan, I’ll pass on this recent project. I wrote up a citation list for Morpholinos in chick embryos…and here it is.
Papanayotou C, Mey A, Birot AM, Saka Y, Boast S, Smith JC, Samarut J, Stern CD. A Mechanism Regulating the Onset of Sox2 Expression in the Embryonic Neural Plate. PLoS Biol. 2008 Jan 8;6(1):e2 [Epub ahead of print]
Voiculescu O, Papanayotou C, Stern CD. Spatially and temporally controlled electroporation of early chick embryos. Nat Protoc. 2008;3(3):419-26.
Shepard JB, Gliga DA, Morrow AP, Hoffman S, Capehart AA. Versican knock-down compromises chondrogenesis in the embryonic chick limb. Anat Rec (Hoboken). 2008 Jan;291(1):19-27.
Sauka-Spengler T, Barembaum M. Chapter 12 Gain- and Loss-of-Function Approaches in the Chick Embryo. Methods Cell Biol. 2008;87:237-56.
Shigetani Y, Howard S, Guidato S, Furushima K, Abe T, Itasaki N. Wise promotes coalescence of cells of neural crest and placode origins in the trigeminal region during head development. Dev Biol. 2008 Jul 15;319(2):346-58. Epub 2008 May 7.
Mende M, Christophorou NA, Streit A. Specific and effective gene knock-down in early chick embryos using morpholinos but not pRFPRNAi vectors. Mech Dev. 2008 Sep 3. [Epub ahead of print]
Tavares AT, Andrade S, Silva AC, Belo JA. Cerberus is a feedback inhibitor of Nodal asymmetric signaling in the chick embryo. Development. 2007 Jun;134(11):2051-60.
Hämmerle B, Tejedor FJ. A Novel Function of DELTA-NOTCH Signalling Mediates the Transition from Proliferation to Neurogenesis in Neural Progenitor Cells. PLoS ONE. 2007 Nov 14;2(11):e1169.
Matsson H, Eason J, Bookwalter CS, Klar J, Gustavsson P, Sunnegardh J, Enell H, Jonzon A, Vikkula M, Gutierrez I, Riveron JG, Pope M, Bu’lock F, Cox J, Robinson TE, Song F, Brook JD, Marston S, Trybus KM, Dahl N. Alpha-cardiac actin mutations produce atrial septal defects. Hum Mol Genet. 2008 Jan 15;17(2):256-65. Epub 2007 Oct 18
Taneyhill LA, Coles EG, Bronner-Fraser M. Snail2 directly represses cadherin6B during epithelial-to-mesenchymal transitions of the neural crest. Development. 2007 Apr;134(8):1481-90. Epub 2007 Mar 7.
Canto-Soler MV, Adler R. Optic cup and lens development requires Pax6 expression in the early optic vesicle during a narrow time window. Dev Biol. 2006 Jun 1;294(1):119-32. Epub 2006 Mar 27
Nakazawa F, Nagai H, Shin M, Sheng G. Negative regulation of primitive hematopoiesis by the FGF signaling pathway. Blood. 2006 Nov 15;108(10):3335-43. Epub 2006 Aug 3
Suzuki-Hirano A, Sato T, Nakamura H. Regulation of isthmic Fgf8 signal by sprouty2. Development. 2005 Jan;132(2):257-65. Epub 2004 Dec 8.
Granata A, Savery D, Hazan J, Cheung BM, Lumsden A, Quaderi NA. Evidence of functional redundancy between MID proteins: implications for the presentation of Opitz syndrome. Dev Biol. 2005 Jan 15;277(2):417-24.
Ching YH, Ghosh TK, Cross SJ, Packham EA, Honeyman L, Loughna S, Robinson TE, Dearlove AM, Ribas G, Bonser AJ, Thomas NR, Scotter AJ, Caves LS, Tyrrell GP, Newbury-Ecob RA, Munnich A, Bonnet D, Brook JD. Mutation in myosin heavy chain 6 causes atrial septal defect. Nat Genet. 2005 Apr;37(4):423-8. Epub 2005 Feb 27.
Duong TD, Erickson CA. MMP-2 plays an essential role in producing epithelial-mesenchymal transformations in the avian embryo. Dev Dyn. 2004 Jan;229(1):42-53.
Gerlach-Bank LM, Cleveland AR, Barald KF. DAN directs endolymphatic sac and duct outgrowth in the avian inner ear. Dev Dyn. 2004 Feb; 229(2): 219-30.
Tucker RP. Antisense knockdown of the beta1 integrin subunit in the chicken embryo results in abnormal neural crest cell development. Int J Biochem Cell Biol. 2004 Jun;36(6):1135-9.
Nakamura H, Katahira T, Sato T, Watanabe Y, Funahashi J. Gain- and loss-of-function in chick embryos by electroporation. Mech Dev. 2004 Sep;121(9):1137-43. Review.
Krull CE. A primer on using in ovo electroporation to analyze gene function. Dev Dyn. 2004 Mar;229(3):433-9.
Sugiyama S, Nakamura H. The role of Grg4 in tectal laminar formation. Development. 2003 Feb 1;130(3):451-462.
Kos R, Tucker RP, Hall R, Duong TD, Erickson CA. Methods for introducing morpholinos into the chicken embryo. Dev Dyn. 2003 Mar;226(3):470-7.
Hall RJ, Erickson CA. ADAM 10: an active metalloprotease expressed during avian epithelial morphogenesis. Dev Biol. 2003 Apr 1;256(1):147-60.
Granata A. Quaderi N.A. The Opitz syndrome gene MID1 is essential for establishing asymmetric gene expression in Hensen’s node. Dev Biol. 2003 Jun 15;258(2):397-405.
Sheng G, dos Reis M, Stern CD. Churchill, a zinc finger transcriptional activator, regulates the transition between gastrulation and neurulation. Cell. 2003 Nov 26;115(5):603-13.
Moftah MZ, Downie SA, Bronstein NB, Mezentseva N, Pu J, Maher PA, Newman SA. Ectodermal FGFs induce perinodular inhibition of limb chondrogenesis in vitro and in vivo via FGF receptor 2. Dev Biol. 2002 Sep 15;249(2):270-82.
Tucker RP. Using Antisense Morpholino Oligos to Knockdown Gene Expression in the Chicken Embryo. Acta Histochemica et Cytochemica, 2002, vol. 35, no. 5, pp. 361-366
Tucker RP. Abnormal neural crest cell migration after the in vivo knockdown of tenascin-C expression with morpholino antisense oligonucleotides. Dev Dyn 2001 Sep;222(1):115-9
Kos R, Reedy MV, Johnson RL, Erickson CA. The winged-helix transcription factor FoxD3 is important for establishing the neural crest lineage and repressing melanogenesis in avian embryos. Development 2001 128(8):1467-1479
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Thanks! Looks like a good reading list, and all the 2008 references are new for me. Isn’t there some clever way to share it all whole from within Connotea? I haven’t been using it long enough to know.
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Hi Heather,
I’ve not been using Connotea, though I probably should go learn it. Meanwhile, I promise I won’t keep doing this, but two more came out today for chick.
Weisinger K, Wilkinson DG, Sela-Donenfeld D. Inhibition of BMPs by follistatin is required for FGF3 expression and segmental patterning of the hindbrain. Dev Biol. 2008 Sep 18. [Epub ahead of print]
Tucker RP, Tran H, Gong Q. Neurogenesis and neurite outgrowth in the spinal cord of chicken embryos and in primary cultures of spinal neurons following knockdown of Class III beta tubulin with antisense morpholinos. Protoplasma. 2008 Sep 30. [Epub ahead of print]
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