Knockdown of dre-miR-219
Jon Moulton
Wednesday, 14 January 2009 15:28 UTC
I can tell you a Morpholino was used to knock down miR-219 in zebrafish, but I’m afraid I found the text to be rough going and I didn’t finish the paper. This is not a failing of the authors, but my own. Nevertheless, I present the reference for those with better language skills.
Zhang MC, Lv Y, Qi YT, Zhang Z, Fu XN, Yuan CG, Lai LH. Knockdown and overexpression of miR-219 lead to embryonic defects in zebrafish development. Fen Zi Xi Bao Sheng Wu Xue Bao. 2008 Oct;41(5):341-8. Chinese.
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Replies
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Hi Jon,
thanks for some nice updates on the use of morpholinos. It should be stressed perhaps that there are also reports of miRNA inhibition by morpholinoes written in english – but I guess you know. In particular case we are testing different LNA inhibitors targeting miRNAs in juvenile fish – that is months after hatching. The problem is as allways delivery. Are there any experience on delivering morpholinoes in fish beyond the embryonic stages?
Perhaps it would be better to go the in vitro way with our fish cell cultures. In that case are there succefull reports or data on the use of morpholinoes in fish cell cultures?
Brian -
Good day Brian,
Yes, papers using miRNAs to block Morpholinos are published in English too. There is a partial reference list at the bottom of this page (I’m sure I’ve missed some):
http://www.gene-tools.com/node/31Besides microinjection, I know of two methods for delivery into fish that have been successful. Vivo-Morpholinos have worked in juvenile fish, though so far there have been no papers published reporting their use in fish. Electroporation has worked for delivery into small regions of the embryonic fish. In both of these cases the Morpholinos targeted splicing or translation but the RNA target doesn’t really matter for developing methods for cytosolic delivery. The delivery methods are applicable for miRNA inhibition. I’ll cite some electroporation papers below.
I know of this paper describing use of Special Delivery Morpholinos to knockdown a gene in cultured zebrafish cells:
Liu TX, Howlett NG, Deng M, Langenau DM, Hsu K, Rhodes J, Kanki JP, D’Andrea AD, Look AT. Knockdown of zebrafish Fancd2 causes developmental abnormalities via p53-dependent apoptosis. Dev Cell. 2003 Dec;5(6):903-14.
http://www.cell.com/developmental-cell/abstract/S1534-5807(03)00339-3
Special Delivery oligos are DNA-Morpholino heteroduplexes delivered with ethoxylated polyethylenimine (EPEI). The Special Delivery system is no longer available, though we post protocols for making your own Special Delivery oligos (See section D, http://www.gene-tools.com/node/17 ) and we are still supplying EPEI. That system has been supplanted by Endo-Porter delivery, which also uses endosomal uptake and escape but offers more experimental flexibility with less toxicity for most cell types.The electroporation study I was thinking of was on a poster from Chuck Kimmel’s lab at University of Oregon, but there are some other electroporation papers that might be useful, some in zebrafish and some in Xenopus.
Cerda GA, Thomas JE, Allende ML, Karlstrom RO, Palma V. Electroporation of DNA, RNA, and morpholinos into zebrafish embryos. Methods. 2006 Jul;39(3):207-11.
http://www.bio.umass.edu/biology/karlstrom/KarlstromPDFs/Cerdatal.2006.Electropor.pdfPaper on electroporation into the embryonic Xenopus CNS:
Falk J, Drinjakovic J, Leung KM, Dwivedy A, Regan AG, Piper M, Holt CE. Electroporation of cDNA/Morpholinos to targeted areas of embryonic CNS in Xenopus. BMC Developmental Biology 2007, 7:107 [epub ahead of print]
http://www.biomedcentral.com/content/pdf/1471-213X-7-107.pdfAbstract of a paper describing an electroporation protocol optimized for Xenopus tadpole brains:
Bestman JE, Ewald RC, Chiu SL, Cline HT. In vivo single-cell electroporation for transfer of DNA and macromolecules. Nat Protoc. 2006;1(3):1267-72.
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=17406410There is also a lot of electroporation work done in chick embryos. Here’s an example from that literature:
Voiculescu O, Papanayotou C, Stern CD. Spatially and temporally controlled electroporation of early chick embryos. Nat Protoc. 2008;3(3):419-26.
http://www.ncbi.nlm.nih.gov/pubmed/18323813Delivery can be a tough problem if you don’t have an established tool. We’ve wrestled with that for years and only recently developed a solution for systemic delivery into adult animals. Vivo-Morpholinos are mainly being administered i.v. in rodents, though there have been a few tests in fish. For more information about Vivo-Morpholinos, see:
http://www.gene-tools.com/vivomorpholinos
(citation list at bottom of linked age)
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