Generation of an in vitro model for peripheral neuropathy in Fabry disease using CRISPR-Cas9 in the nociceptive dorsal root ganglion cell line 50B11

Kaneski, Christine R.; Hanover, John A.; Hoffman, Ulrike H. Schueler

doi:10.1016/j.ymgmr.2022.100871

Cited by 7 publications

(2 citation statements)

References 27 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Importantly, our study extended the observation period up to 72 h post-differentiation, in contrast to many previous studies that typically incubated cells with forskolin for 20-36 h only followed by requisite treatments to stabilize the neuronal phenotype. [35,[37][38] We maintained the neuronal features of cells by replenishing the differentiation induction medium with fresh forskolin-containing medium every 24 h (Figure 2A, Forskolin++). By doing so, we ensured the continued development of neurites and axon elongation, along with the sustained elevation of cell body morphology, which was notably prominent at the 24 h mark post-differentiation.…”

Section: Neuronal Differentiation Leading To Neurite Outgrowth Coinci...mentioning

confidence: 99%

Label‐Free Visualization and Morphological Profiling of Neuronal Differentiation and Axonal Degeneration through Quantitative Phase Imaging

Kim,

Cetinkaya‐Fisgin,

Zahn

et al. 2024

Advanced Biology

View full text Add to dashboard Cite

Understanding the intricate processes of neuronal growth, degeneration, and neurotoxicity is paramount for unraveling nervous system function and holds significant promise in improving patient outcomes, especially in the context of chemotherapy‐induced peripheral neuropathy (CIPN). These processes are influenced by a broad range of entwined events facilitated by chemical, electrical, and mechanical signals. The progress of each process is inherently linked to phenotypic changes in cells. Currently, the primary means of demonstrating morphological changes rely on measurements of neurite outgrowth and axon length. However, conventional techniques for monitoring these processes often require extensive preparation to enable manual or semi‐automated measurements. Here, a label‐free and non‐invasive approach is employed for monitoring neuronal differentiation and degeneration using quantitative phase imaging (QPI). Operating on unlabeled specimens and offering little to no phototoxicity and photobleaching, QPI delivers quantitative maps of optical path length delays that provide an objective measure of cellular morphology and dynamics. This approach enables the visualization and quantification of axon length and other physical properties of dorsal root ganglion (DRG) neuronal cells, allowing greater understanding of neuronal responses to stimuli simulating CIPN conditions. This research paves new avenues for the development of more effective strategies in the clinical management of neurotoxicity.

show abstract

Section: Neuronal Differentiation Leading To Neurite Outgrowth Coinci...mentioning

confidence: 99%

Label‐Free Visualization and Morphological Profiling of Neuronal Differentiation and Axonal Degeneration through Quantitative Phase Imaging

Kim,

Cetinkaya‐Fisgin,

Zahn

et al. 2024

Advanced Biology

View full text Add to dashboard Cite

show abstract

“…Introducing mutations to the disease-related genes in the context of a normal cell line has been used as a powerful method to generate isogenic cellular models for the disease (9)(10)(11)(12)(13)(14).…”

Section: Introductionmentioning

confidence: 99%

CRISPR/Cas9-mediated Generation ofCOL7A1-deficient Keratinocyte Model of Recessive Dystrophic Epidermolysis Bullosa

Alipour,

Ahmadraji,

Yektadoust

et al. 2023

Preprint

View full text Add to dashboard Cite

Objective: Recessive dystrophic epidermolysis bullosa (RDEB) is a genetic skin fragility and ultimately lethal blistering disease caused by mutations in the COL7A1 gene which is responsible for coding type VII collagen. Investigating the pathological mechanisms and novel candidate therapies for RDEB could be fostered by new cellular models. Here, we developed multiple immortalized COL7A1-deficient keratinocyte cell lines using CRISPR/Cas9 technology as RDEB cellular model. Materials and Methods: In this experimental study, we used transient transfection to express COL7A1-targeting gRNA and Cas9 in HEK001 immortalized keratinocyte cell line followed by enrichment with fluorescent-activated cell sorting (FACS) via GFP expressing cells (GFP+ HEK001). Homogenous single-cell clones were then isolated, genotyped, and evaluated for type VII collagen expression. We performed a scratch assay to confirm the functional effect of COL7A1 knockout. Results: We achieved 46.1% (p < 0.001) efficiency of indel induction in the enriched transfected cell population. Except for 4% of single nucleotide insertions, the remaining indels were deletions of different sizes. Out of nine single clones expanded, two homozygous and two heterozygous COL7A1-deficient cell lines were obtained with defined mutation sequences. No off-target effect was detected in the knockout cell lines. Immunostaining and western blot analysis showed the lack of type VII collagen (COL7A1) protein expression in these cell lines. We also showed that COL7A1-deficient cells had higher motility compared with their wild-type counterparts. Conclusion: We reported the first isogenic immortalized COL7A1-deficient keratinocyte lines that provide a useful cell culture model to investigate aspects of RDEB biology and potential therapeutic options.

show abstract

Genome Editing Tools for Lysosomal Storage Disorders

Gonzalez

Nader

Siebert

et al. 2023

Advances in Experimental Medicine and Biology

View full text Add to dashboard Cite

Generation of an in vitro model for peripheral neuropathy in Fabry disease using CRISPR-Cas9 in the nociceptive dorsal root ganglion cell line 50B11

Cited by 7 publications

References 27 publications

Label‐Free Visualization and Morphological Profiling of Neuronal Differentiation and Axonal Degeneration through Quantitative Phase Imaging

Label‐Free Visualization and Morphological Profiling of Neuronal Differentiation and Axonal Degeneration through Quantitative Phase Imaging

CRISPR/Cas9-mediated Generation ofCOL7A1-deficient Keratinocyte Model of Recessive Dystrophic Epidermolysis Bullosa

Genome Editing Tools for Lysosomal Storage Disorders

Contact Info

Product

Resources

About