Genetic manipulation tools: what’s the specificity?

In situ hybridization of an eye marker in embryos injected with morpholinos targeting Danio rerio Srsf5a (high) and uninjected embryos (low).

During the study of the functions of a specific gene, opting for an experimental approach may lead to the observation of very different results. It is thus advisable to remain cautious regarding their interpretation. This is the problem posed by a recent study initiated by Pr. Patrick Motte (InBioS) and Dr. Marc Muller (GIGA-R), and published in the journal Nucleic Acids Research(1).

T

he objective of this study, initiated as part of a collaboration between the Laboratories for Functional Genomics and Plant Molecular Imaging (InBioS) and Organogenesis and Regeneration (GIGA-R), was to evaluate the role of splicing factors of the SR family (Ser/Arg-rich; proteins thus defined because they contain a domain rich in serine/arginine) over the course of the development of the zebrafish Danio rerio (model species of the vertebrate group). These proteins, which regulate various stages of gene expression and contribute to the diversity of the proteome, have a physiological role during development that remains little known. Conceptually, the functions of a gene can only be suggested if the phenotypic consequences of the absence of the functional product of the gene can be observed...It is therefore necessary to induce a partial or total loss of function and analyse the physiological, metabolic, and developmental consequences of this loss of function.

To evaluate this process, the researchers used (i) the injection of morpholinos into the embryos (this technique is widely used to diminish gene expression in zebrafish) and (ii) a targeted approach through the TALEN and CRISPR/Cas9 techniques, which are truly revolutionary genetic tools and very effective in generating mutations.

Diagram illustrating two types of genetic manipulations that can cause drastic changes (top) or much more discreet (bottom). ©Patrick Motte

Once the results have been obtained, significant differences can be observed between these two approaches. Researchers therefore decided to investigate and analyse the causes of this discrepancy. They came to the conclusion that, on the one hand, the morpholinos could act unpredictably (unexpectedly) on a high number of RNAs if these presented 11 consecutive bases hybridizing to the morpholinos and that, on the other hand, the mutation of SR genes lead to the overexpression of other members of this family, which may compensate for the absence of the targeted gene.

Overall, these results raise the question of the choice of experimental approach in the study of the functions of a specific gene, they call for caution in the interpretation of results, and contribute to the international debate on this problem.

To better understand

Splicing is a mechanism which allows a transcribed RNA to rid itself of intron sequences, i.e. the non-coding portions of genes, in order to form messenger RNA which will be translated into protein.

The proteome is the set of proteins expressed by a genome, a cell, tissue, an organ, an organism...

Mutagenicity (or mutagenesis) is the process of mutation formation; it can be natural or artificial (through exposure of the DNA to a "mutagenic agent").

In molecular biology, a morpholino is a type of molecule used to modify genetic expression

Scientific reference

(1) Number of inadvertent RNA targets for morpholino knockdown in Danio rerio is largely underestimated: evidence from the study of Ser/Arg-rich splicing factors, Marine Joris, Marie Schloesser, Denis Baurain, Marc Hanikenne, Marc Muller, Patrick Motte, Nucleic Acids Research, Volume 45, Issue 16, 19 September 2017, Pages 9547–9557, https://doi.org/10.1093/nar/gkx638

Contact

Pr. Patrick MOTTE, Laboratoire de Génomique fonctionnelle et imagerie moléculaire végétale, UR InBioS
 +32 4 3663810 | Patrick.Motte@uliege.be | 

Dr. Marc MULLER  Laboratory for Organogenesis and Regeneration, URif GIGA-R
+32 4 3664437 | M.Muller@uliege.be |