the community site for and by developmental biologists

Jon Moulton

Posts by Jon Moulton

Morpholinos with specificity controls please

Posted by , on 16 September 2020

Please, always run specificity controls with Morpholinos. Morpholinos can reveal gene functions that can be concealed by genetic compensation in a mutant, but rigorous specificity controls are essential to determining ...

Guidelines for morpholino use in zebrafish

Posted by , on 20 October 2017

If you work with zebrafish or Morpholinos, please read this. Guidelines for morpholino use in zebrafish. Stainier DYR, Raz E, Lawson ND, Ekker SC, Burdine RD, Eisen JS, Ingham PW, ...

Evo-devo music

Posted by , on 27 September 2017

It’s rare to see your working life captured in a music video.  This made me happy, I hope you enjoy it. Click the title above.

Open access video protocol: electroporating zebrafish ears

Posted by , on 10 January 2011

Open-access video protocol for electroporating into zebrafish ears

Zebrafish embryo pics

Posted by , on 20 August 2010

Worth 1000 words? http://blogs.nature.com/news/thegreatbeyond/2010/08/a_zebrafishs_first_minutes_of.html [update 31/8: I added the first of the videos (below) – Eva] (The original paper is in Science)

Recent comments by Jon Moulton

I should point out that I have been an employee of Gene Tools for over 15 years, offering technical support, attending conferences, developing new products and curating Morpholino citations among other tasks; therefore I have a fiduciary interest in the use of Morpholino technology as well as long familiarity with and enthusiasm for Morpholinos.
by Jon Moulton in Out with the old, in with the new: reassessing morpholino knockdowns in light of genome editing technology on February 11, 2015
I was concerned when Stefan Schulte-Merker and Didier Stainier' paper appeared in mid-2014, first in Development and then on The Node. Later in 2014 Nathan Lawson's paper in Developmental Cell was published. Prior to these papers I was intrigued and excited by CRISPR technology but felt that, while it might temporarily displace some use of Morpholinos, in the long run it would benefit Morpholino use by making the zebrafish model stronger. However, the tone of these publications concerned me as the authors suggested that no Morpholino data is strong unless it agrees with CRISPR data (that is, unless the Morpholinos have been validated by phenocopying CRISPR knockouts). I attended the Strategic Conference of Zebrafish Investigators meeting in January of 2015 at Asilomar in Pacific Grove, California. In the first morning sessions Dr. Nathan Lawson presented his data, followed by Dr. Didier Stainier. Dr. Lawson asserted that CRISPR knockouts were the gold standard to which all other expression-altering technologies should be compared. He tried to persuade the audience that if a Morpholino failed to phenocopy a CRISPR knockout then the data from the Morpholino were wrong, and that only "validated" (CRISPR-phenocopying) Morpholinos should be used. Dr. Lawson's presentation was followed by Dr. Stainier. Dr. Stainier presented array studies of CRISPR mutants which did not recapitulate the knockdown phenotypes of morphants. He showed that there were widespread changes in gene expression in the mutants relative to wild-type and suggested that the failure to phenocopy was due to compensation by the mutants; He suggested that in seeking homeostasis the mutants were obscuring the knockdown phenotype by altering expression of other genes. He suggested that while Morpholinos may sometimes present false positives, CRISPRs tend to present false negatives. After Dr. Stainier's presentation the community had a one-hour discussion titled "Mutants vs. Morphants". In general the community was very supportive of the existing Morpholino-based body of data and did not agree that future work with Morpholinos should only be accepted if the knockdowns are validated against CRISPR mutants. They pointed out that the problems with Morpholinos are well known and that with good experimental design and strong controls you can be confident of your results. They admonished Dr. Lawson's use of words like "rigor" and "gold standard" applied to CRISPRs, asserting that these terms were inflammatory and prejudicial and could inappropriately influence reviewers and study section leaders, especially those without strong bench experience with knockdowns and mutants. The last community member to speak said that it makes no sense to refer to "mutants" as if they are simply knockouts, that instead there is a range of alleles with different characteristics for every gene locus and that the CRISPR mutants would be expected to have a range of expression as do natural alleles. The community agreed that a paper should be produced stating that Morpholinos can stand on their own as a tool to interrogate gene function and delineating some community standards for use and comparison of mutants and morphants. The strong support of the zebrafish community has eased my concerns over the future acceptance of Morpholino data for grants and publications. With strong specificity controls (particularly the two-nonoverlapping-oligo approach) to eliminate false positives, the data produced by Morpholinos are not only as reliable as CRISPRs but can reveal functions of genes which a corresponding CRISPR mutant might obscure by compensation. Morpholinos are a fast method and, when lab worker costs are included, are as inexpensive as the mutation methods. There are many functions you can perform with a Morpholino which a CRISPR mutation cannot do: splice redirection, blocking poly-A signal sites, protecting miRNA target sites, blocking zipcode binding sequences, and the other molecular "masking tape" approaches available at the RNA level. I expect that the CRISPR-Morpholino controversy will still take some time to play out, but the reviewers and study section leaders will soon understand that Morpholinos are still the best available technology for RNA-level manipulations and are not expected to phenocopy all outcomes of DNA manipulations. Schulte-Merker S, Stainier DY. Out with the old, in with the new: reassessing morpholino knockdowns in light of genome editing technology. Development. 2014 Aug;141(16):3103-4. doi: 10.1242/dev.112003. Schulte-Merker and Stanier on The Node: https://thenode.biologists.com/out-with-the-old-in-with-the-new-reassessing-morpholino-knockdowns-in-light-of-genome-editing-technology/discussion/ Kok FO, Shin M, Ni C-W, Gupta A, Grosse AS, van Impel A, Kirchmaier BC, Peterson-Maduro J, Kourkoulis G, Male I, DeSantis DF, Sheppard-Tindell S, Ebarasi L, Betsholtz C, Schulte-Merker S, Wolfe SA, Lawson ND. Reverse Genetic Screening Reveals Poor Correlation between Morpholino-Induced and Mutant Phenotypes in Zebrafish. Dev Cell. 2014;[Epub ahead of print] doi:10.1016/j.devcel.2014.11.018.
by Jon Moulton in Out with the old, in with the new: reassessing morpholino knockdowns in light of genome editing technology on February 11, 2015
I'll miss the field reports form Xinjiang -- at least, 'till your next journey there. Thanks for posting!
by Jon Moulton in Homeward bound on May 11, 2012
Thanks Linda, I enjoyed reading your perspective on miRNA in Arabidopsis. Please keep them coming!
by Jon Moulton in Arrested Development in Plant miRNA Mutants on January 11, 2011