Table 1.
Technical specification for the manual microinjections.
Table 2.
Technical specifications for the automated microinjections.
Fig 1.
Morpholino knockdown efficiency with manual and automated injections.
(A) The survival and knockdown efficiency of slc45a2-MO manual and automated (auto) microinjections were measured as the number of larvae displaying an albino phenotype at 3 days post-fertilization (dpf). Control-MO injected larvae and uninjected larvae were processed in parallel and the resulting pigmented (wild-type) larvae were also counted at 3 dpf. “n =“ indicates the number of eggs used to obtain this cumulative result. (B) Efficiency comparison between the automated injection into the yolk and manual injections performed by two independent experimentalists (P1: experienced and P2: expert; not statistically significant). “n =“ indicates the number of experiments used to calculate the average and standard deviation. Each experiment refers to different technical and biological experiments.
Fig 2.
Imaging classification for injection.
Representative digital images measured from below of an agarose grid (“Empty”) that supports zebrafish eggs with the first cell visible (“Inject”) or not visible (“No cell”), eggs in a two- or higher cell stage (“Two cell”) or non-viable eggs (“Sick”). In the “Inject” image an injection location is indicated by a black dot with (x y) coordinates.
Fig 3.
Distance between annotated and computed injection location.
(A) Bar graph depicting the frequency of the distance between annotated and computed injection position (prediction). (B) Digital image with a circle around an annotated injection point to illustrate the average distance between annotation and prediction.
Fig 4.
Automated injections of CRISPR/Cas9.
(A) Survival and average efficiency of slc45a2 gRNA/Cas9 manual, click-to-inject (semi-auto) and automated (auto) microinjections both in the yolk and in the cell were measured as the number of larvae displaying an albino phenotype at 3 days post-fertilization (dpf). Uninjected larvae were processed as controls and the resulting pigmented (wild-type) larvae were also counted at 3 dpf. “n =“ indicates the number of eggs that were used to obtain the cumulative results. (B) Comparison of the average efficiency and standard deviation between the automated (auto), click-to-inject (semi-auto) and manual injections performed by three independent experimentalists (P1: experienced, P2: expert and P3: novice). “n =“ indicates the number of experiments that were used to calculate the average and standard deviation. Each experiment refers to different technical and biological experiments. * P<0.05.
Fig 5.
(A) Average survival and efficiency of DNA automated (auto) and manual injections as measured by the COPAS system. “n =“ indicates the number of eggs that were used to obtain the cumulative results. (B) Comparison of the average efficiency and standard deviation between the automated and manual cell injections. P4 indicates a different experienced experimentalist and “n =“ indicates the number of experiments that were used to calculate the average and standard deviation.
Fig 6.
Average injection time required to obtain one positive genetically modified larva.
Abbreviations: MO, slc45a2 morpholino; gRNA, slc45a2 gRNA/Cas9; DNA, Tol2 construct; Auto, automated injections; P1-4, four different experimentalists.