Editing Xenopus (section)

=== Gene expression knockdown techniques ===
The expression of genes can be reduced by a variety of means, for example by using antisense oligonucleotides targeting specific mRNA molecules. DNA oligonucleotides complementary to specific mRNA molecules are often chemically modified to improve their stability ''in vivo''. The chemical modifications used for this purpose include phosphorothioate, 2'-O-methyl, morpholino, MEA phosphoramidate and DEED phosphoramidate.<ref name=":0">{{cite journal | vauthors = Dagle JM, Weeks DL | title = Oligonucleotide-based strategies to reduce gene expression | journal = Differentiation; Research in Biological Diversity | volume = 69 | issue = 2–3 | pages = 75–82 | date = December 2001 | pmid = 11798068 | doi = 10.1046/j.1432-0436.2001.690201.x }}</ref>

==== Morpholino oligonucleotides ====
{{Main|Morpholino}}

Morpholino oligos are used in both ''X. laevis'' and ''X. tropicalis'' to probe the function of a protein by observing the results of eliminating the protein's activity.<ref name=":0" /><ref name=":1">{{cite journal | vauthors = Blum M, De Robertis EM, Wallingford JB, Niehrs C | title = Morpholinos: Antisense and Sensibility | journal = Developmental Cell | volume = 35 | issue = 2 | pages = 145–149 | date = October 2015 | pmid = 26506304 | doi = 10.1016/j.devcel.2015.09.017 | doi-access = free }}</ref> For example, a set of ''X. tropicalis'' genes has been screened in this fashion.<ref>{{cite journal | vauthors = Rana AA, Collart C, Gilchrist MJ, Smith JC | title = Defining synphenotype groups in Xenopus tropicalis by use of antisense morpholino oligonucleotides | journal = PLOS Genetics | volume = 2 | issue = 11 | pages = e193 | date = November 2006 | pmid = 17112317 | pmc = 1636699 | doi = 10.1371/journal.pgen.0020193 | doi-access = free }}<br />{{cite web |url=http://www.gurdon.cam.ac.uk/~smithlab/screens/Xenopus-morpholino-pilot/ |title=A ''Xenopus tropicalis'' antisense morpholino screen |date=4 March 2014 |publisher=Gurdon Institute |access-date=17 January 2007 |archive-date=12 June 2018 |archive-url=https://web.archive.org/web/20180612164223/https://www.gurdon.cam.ac.uk/~smithlab/screens/Xenopus-morpholino-pilot/ |url-status=dead }}</ref>

Morpholino oligos (MOs) are short, antisense oligos made of modified nucleotides. MOs can knock down gene expression by inhibiting mRNA translation, blocking RNA splicing, or inhibiting miRNA activity and maturation. MOs have proven to be effective knockdown tools in developmental biology experiments and RNA-blocking reagents for cells in culture. MOs do not degrade their RNA targets, but instead act via a steric blocking mechanism RNAseH-independent manner. They remain stable in cells and do not induce immune responses. Microinjection of MOs in early ''Xenopus'' embryos can suppress gene expression in a targeted manner.

Like all antisense approaches, different MOs can have different efficacy, and may cause off-target, non-specific effects. Often, several MOs need to be tested to find an effective target sequence. Rigorous controls are used to demonstrate specificity,<ref name=":1" /> including:
* Phenocopy of genetic mutation
* Verification of reduced protein by western or immunostaining
* mRNA rescue by adding back a mRNA immune to the MO
* use of 2 different MOs (translation blocking and splice blocking) 
* injection of control MOs
[[Xenbase]] provides a searchable catalog of over 2000 MOs that have been specifically used in Xenopus'' research.'' The data is searchable via sequence, gene symbol and various synonyms (as used in different publications).<ref>[http://www.xenbase.org/reagents/morpholino.do Xenbase]</ref> Xenbase maps the MOs to the latest ''Xenopus'' genomes in GBrowse, predicts 'off-target' hits, and lists all ''Xenopus'' literature in which the morpholino has been published.