Biology subjects

van der Kolk, K.-J.

Publications and source records attributed to van der Kolk, K.-J..

3 recordsLinked to original sources

bioRxiv2024

Automated microinjection for zebrafish xenograft models

Zebrafish xenograft models have been increasingly recognized for their ability to predict patient responses to cancer therapeutics, suggesting their potential as diagnostic tools in clinical settings. However, these models require the precise microinjection of cancer cell suspensions in many small and fragile zebrafish larvae. Manual injections are so challenging that, even after months of training, variability in experimental results persists among researchers. This limits the uptake and deployment of zebrafish xenograft models for clinical use and drug discovery. To address this challenge, we have designed, built, and validated an automated microinjection robot. Combined results of injections into the vasculature, perivitelline space, and hindbrain ventricle demonstrated an average injection success rate of approximately 60%, with a larvae survival rate exceeding 70%, comparable to manual injections using a traditional micromanipulator. Notably, the full automated mode was twice as fast as manual injections. This automation of the microinjection process significantly reduces the need for extensive personnel training while it enhances reproducibility, efficiency, and accuracy, paving the way for more extensive use of zebrafish xenograft models in drug discovery and patient diagnostics.

cancer biology↗

bioRxiv2023

Comparison of robotic automated and manual injection methods in zebrafish embryos for high throughput RNA silencing using CRISPR-CasRx

Recently, the CRISPR-RfxCas13d (CasRx) system was proven to induce efficient mRNA knockdown in animal embryos. Here we compared the efficiency of CasRx-based RNA depletion with that of Cas9-mediated DNA targeting under the same conditions, using automated robotic and manual injection methods. As a proof-of-principle target, we used the no tail (tbxta) gene in zebrafish embryos, for which knockdown and knockout embryonic phenotypes were easy to be scored. Both Cas9 and CasRx systems induced loss of function phenotypes of tbxta gene. Higher percentage of severe phenotype was observed using Cas9 protein compared to the mRNA while the efficiency was similar in terms of Cas13d protein and mRNA. In addition, both the robotic and manual injection approaches yielded similar percentages of phenotypes and mortality rates. Therefore, our study not only showcases the potential of RNA-targeting CRISPR effectors for precise and potent gene knockdown, but also emphasizes automated microinjection in zebrafish embryos as an excellent alternative to manual methods for achieving gene knockdown at a high throughput level.

bioengineering↗

bioRxiv2018

Deep learning image recognition enables efficient genome editing in zebrafish by automated injections

One of the most popular techniques in zebrafish research is microinjection, as it is a rapid and efficient way to genetically manipulate early developing embryos, and to introduce microbes or tracers at larval stages.\n\nHere we demonstrate the development of a machine learning software that allows for microinjection at a trained target site in zebrafish eggs at unprecedented speed. The software is based on the open-source deep-learning library Inception v3.\n\nIn a first step, the software distinguishes wells containing embryos at one-cell stage from wells to be skipped with an accuracy of 93%. A second step was developed to pinpoint the injection site. Deep learning allows to predict this location on average within 42 {micro}m to manually annotated sites. Using a Graphics Processing Unit (GPU), both steps together take less than 100 milliseconds. We first tested our system by injecting a morpholino into the middle of the yolk and found that the automated injection efficiency is as efficient as manual injection (~ 80%). Next, we tested both CRISPR/Cas9 and DNA construct injections into the zygote and obtained a comparable efficiency to that of an experienced experimentalist. Combined with a higher throughput, this results in a higher yield. Hence, the automated injection of CRISPR/Cas9 will allow high-throughput applications to knock out and knock in relevant genes to study their mechanisms or pathways of interest in diverse areas of biomedical research.

molecular biology↗