Juchaux, M. | Search bioRxiv

bioRxiv2026

I. Phantom: a multiplex CRISPR-Cas9 gRNA toolkit for efficient generation of fully unpigmented F0 zebrafish

Zebrafish are an increasingly popular model organism, valued for their natural transparency during embryonic stages. However, imaging at later developmental stages is affected by several types of pigments distributed over the body surface. These pigments partially or completely absorb or reflect light, and some also exhibit fluorescence, thereby interfering with imaging. Chemical approaches, such as phenylthiourea (PTU), are available to address this problem. However, PTU only inhibits melanin production and can affect development, making its use controversial. Many mutant zebrafish lines lacking single pigments have already been described, and combinations of melanin and iridophore deficient lines such as the casper and crystal fish greatly improved in vivo imaging at late stages. However, breeding multiple recessive mutations with a desired reporter transgene is laborious, and xanthophores remain in these lines. Here, we address these limitations by developing and optimising a multiplex CRISPR-Cas9 gRNA set that simultaneously targets the three major pigment types, while minimising mortality. This approach generates a new phenotype, termed phantom, characterised by the complete absence of visible pigmentation directly in F0 zebrafish larvae, while retaining the ability to survive to adulthood and reproduce.

molecular biology↗

bioRxiv2019

Experimental and modeling study of the formation of cell aggregates with differential substrate adhesion

The study of cell aggregation in vitro has a tremendous importance these days. In cancer biology, aggregates and spheroids serve as model systems and are considered as pseudo-tumors that are more realistic than 2D cell cultures. Recently, in the context of brain tumors (gliomas), we developed a new PEG-based hydrogel, with adhesive properties that can be controlled by the addition of poly(L-lysine) (PLL), and a stiffness close to the brains. This substrate allows the motion of individual cells and the formation of cell aggregates, and we showed that on a non-adhesive substrate (PEG without PLL is inert for cells), the aggregates are bigger and less numerous than on an adhesive substrate (with PLL).\n\nIn this article, we present new experimental results on the follow-up of the formation of aggregates on our hydrogels, from the early stages (individual cells) to the late stages (aggregate compaction), in order to compare or two cell lines (F98 and U87) the aggregation process on the adhesive and non-adhesive substrates.\n\nWe first show that a spaceless model of perikinetic aggregation can reproduce the experimental evolution of the number of aggregates, but not of the mean area of the aggregates. We thus develop a minimal off-lattice agent-based model, with a few simple rules reproducing the main processes that are at stack during aggregation. Our spatial model can reproduce very well the experimental temporal evolution of both the number of aggregates and their mean area, on adhesive and non-adhesive soft gels and for the two different cell lines. From the fit of the experimental data, we were able to infer the quantitative values of the speed of motion of each cell line, its rate of proliferation in aggregates and its ability to organize in 3D. We also found qualitative differences between the two cell lines regarding the ability of aggregates to compact. These parameters could be inferred for any cell line, and correlated with clinical properties such as aggressiveness and invasiveness.

biophysics↗