Using Morpholinos to Control Gene Expression

Abstract

Morpholino oligonucleotides are stable, uncharged, water-soluble molecules used to block complementary sequences of RNA, preventing processing, read-through, or protein binding at those sites. Morpholinos are typically used to block translation of mRNA and to block splicing of pre-mRNA, though they can block other interactions between biological macromolecules and RNA. Morpholinos are effective, specific, and lack non-antisense effects. They work in any cell that transcribes and translates RNA, but must be delivered into the nuclear/cytosolic compartment to be effective. Morpholinos form stable base pairs with complementary nucleic acid sequences but apparently do not bind to proteins to a significant extent. They are not recognized by any proteins and do not undergo protein-mediated catalysis-nor do they mediate RNA cleavage by RNase H or the RISC complex. This work focuses on techniques and background for using Morpholinos. © 2017 by John Wiley & Sons, Inc.

Bicc1 and Dicer regulate left-right patterning through post-transcriptional control of the Nodal inhibitor Dand5

“…To test whether the proximal element of the dand5 3′-UTR was instrumental in mediating Bicc1-dependent translational repression, antisense target protector morpholino oligomers (tpMOs) were designed. tpMOs have been recently used to block specific sequences in UTRs, i.e., miRNA or protein-binding sites, to analyze post-transcriptional regulation 25 . We specifically targeted the distal 103–139 region (d-tpMO) and 5′ adjacent medial sequences (m-tpMO) of the identified minimal Bicc1 responsive 3′-UTR in S and L. The medial tpMOs were complementary to nucleotides 65–89 for the L and S alloalleles ( dand5 .L m-tpMO; dand5 .S m-tpMO).…”

Rho Guanine Nucleotide Exchange Factor ARHGEF17 Is a Risk Gene for Intracranial Aneurysms

“…We performed functional studies in the zebrafish model and showed that arhgef17 is abundantly expressed in the head region of zebrafish, including brain blood vessels. We have followed the technical guidelines for morpholino use in zebrafish 57,58 , and showed injection of two splicing-blocking MOs at low dosages (~3 ng) causes intracranial hemorrhage without overall embryonic abnormalities. The morpholinos-based knockdown leads to the perturbation of DH domain or results in nonsense-mediated mRNA decay.…”

Phosphorodiamidate morpholino targeting the 5′ untranslated region of the ZIKV RNA inhibits virus replication

“…The control oligo used in this study is a standard control oligo that targets a human beta-globin intron mutation that causes beta-thalassemia. This oligo, designated as Co DWK-1, causes little change in phenotype in any known test system except human beta-thalessemic hematopoetic cells and is appropriate negative control for custom vivo-morpholino oligos (Moulton, 2017). The sequence of Co DWK-1 is as follows: 5′-CCTCTTACCTCAGTTACAATTTATA-3′.…”