Morpholino injection in Xenopus.
The study of gene function in developmental biology has been significantly furthered by advances in antisense technology made in the early 2000s. This was achieved, in particular, by the introduction of morpholino (MO) oligonucleotides. The introduction of antisense MO oligonucleotides into cells enables researchers to readily reduce the levels of their protein of interest without investing huge financial or temporal resources, in both in vivo and in vitro model systems. Historically, the African clawed frog Xenopus has been used to study vertebrate embryological development, due to its ability to produce vast numbers of offspring that develop rapidly, in synchrony, and can be cultured in buffers with ease. The developmental progress of Xenopus embryos has been extensively characterized and this model organism is very easy to maintain. It is these attributes that enable MO-based knockdown strategies to be so effective in Xenopus. In this chapter, we will detail the methods of microinjecting MO oligonucleotides into early embryos of X. laevis and X. tropicalis. We will discuss how MOs can be used to prevent either pre-mRNA splicing or translation of the specific gene of interest resulting in abrogation of that gene's function and advise on what control experiments should be undertaken to verify their efficacy.
Department of Genetics, UNC McAllister Heart Institute, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.
Panna
P
Tandon
Chris
C
Showell
Kathleen
K
Christine
Frank L
FL
Conlon
D000818:Animals; D015153:Blotting, Western; D002457:Cell Extracts; D004625:Embryo, Nonmammalian / Q000166:cytology / Q000378:metabolism; D055785:Gene Knockdown Techniques; D006801:Humans; D008845:Microinjections / Q000295:instrumentation / Q000379:methods*; D060172:Morpholinos / Q000008:administration & dosage* / Q000235:genetics*; D014176:Protein Biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014982:Xenopus laevis / Q000196:embryology* / Q000378:metabolism*
D002457:Cell Extracts; D060172:Morpholinos