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  • Protocol for CRISPR-Cas9 modification of glycosylation in 3D organotypic skin models.

Protocol for CRISPR-Cas9 modification of glycosylation in 3D organotypic skin models.

STAR protocols (2021-09-07)
Irina N Marinova, Hans H Wandall, Sally Dabelsteen
ABSTRACT

Glycosylation is one of the most common protein modifications in living organisms and has important regulatory roles in animal tissue development and homeostasis. Here, we present a protocol for generation of 3D organotypic skin models using CRISPR-Cas9 genetically engineered human keratinocytes (N/TERT-1) to study the role of glycans in epithelial tissue formation. This strategy is also applicable to other gene targets and organotypic tissue models. Careful handling of the cell cultures is critical for the successful formation of the organoids. For complete details on the use and execution of this protocol, please refer to Dabelsteen et al. (2020).

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Hydrocortisone, BioReagent, suitable for cell culture
Sigma-Aldrich
Adenine, ≥99%
Sigma-Aldrich
Insulin from bovine pancreas, powder, BioReagent, suitable for cell culture
Sigma-Aldrich
Dimethyl sulfoxide, Hybri-Max, sterile-filtered, BioReagent, suitable for hybridoma, ≥99.7%