Table 2.

Summary of the strengths and limitations to guide the use of some common techniques used in kidney research

Gene Expression Manipulation TechniqueEffectStrengthsLimitationsWhen It Should Be Used
siRNA transfectionRNAi (post-transcriptional gene silencing)Inexpensive, rapid, and high-throughputThe transfection of synthetic siRNA usually leads to transient effects. Moderate risk of off-target effects. Can lead to variable and partial gene silencingTo perform a rapid experiment in organisms such as Chlamydomonas, C. elegans, or Drosophila, or in cell lines to assess the target gene function. It is good practice to use several siRNAs with different sequences that target the same gene to confirm the specificity of the observed effects
mRNA transfection/microinjectionGene (over)expressionInexpensive, rapid, and high throughputThe effects of mRNA transfection/microinjection strongly depend on the quantity of mRNA introduced into the organism. Careful interpretation of the results is warranted especially when the organism is forced to translate the exogenous mRNA at high, nonendogenous levelsOften used in rapid zebrafish and cell-based experiments to assess gene function or to perform rescue experiments (in combination with silencing of the same gene or of an interacting gene)
MO transfection/microinjectionPost-transcriptional gene silencingHigh RNA affinity with superior ability to invade RNA secondary structure, resulting in high targeting predictability. Resistance to nucleasesThe effects of transfection/microinjection of MO are transient. Moderate risk of off-target effects. Can lead to variable and partial gene silencingOften used in rapid zebrafish and cell-based experiments. It is good practice to use several MOs with different sequences that target the same gene to confirm the specificity of the effects observed
ZFNsGene editingCan target virtually any sequence in the genomeEngineering and design of ZFN is challenging and time consuming. Risk of off-target effectsWhen wanting to obtain a mutant organism or a stable cell line
TALENsGene editingDesign simplicityRisk of off-target effectsWhen wanting to obtain a mutant organism or a stable cell line
CRISPR/CasGene editingDesign simplicity and efficiency, which make this technique overall significantly less expensive and less time consuming than other gene editing systemsCRISPR/Cas system shares with other gene-editing systems the risk of off-target effects. Target sequence needs be adjacent to a specific protospacer adjacent motifWhen wanting to obtain a mutant organism or a stable cell line. Can also be used for rapid gene silencing experiments without selection of the mutated founder organism or cell clone (e.g., to produce zebrafish crispants or mixed mutant cell populations). Due to its design simplicity and efficiency, it is to be considered the technique of choice to achieve gene editing in most cases. Whole-genome sequencing to assess specificity of gene editing in a mutant organism or stable cell line, albeit expensive, should be performed
  • siRNA, short interfering RNA; RNAi, RNA interference; MO, morpholino; ZFNs, zinc finger nucleases; TALENs, transcription activator-like effector nucleases; CRISPR, clustered regularly interspaced short palindromic repeat.