ZnUMBA – a live imaging method to detect local barrier breaches

Abstract

Epithelial barrier function is commonly analyzed using transepithelial electrical resistance, which measures ion flux across a monolayer, or by adding traceable macromolecules and monitoring their passage across the monolayer. While these methods measure changes in global barrier function, they lack the sensitivity needed to detect local or transient barrier breaches, and they do not reveal the location of barrier leaks. Therefore, we developed a method that we named Zinc-based Ultrasensitive Microscopic Barrier Assay (ZnUMBA), which overcomes these limitations, allowing for detection of local tight junction leaks with high spatiotemporal resolution (Chumki et al., 2022; Higashi et al., 2023; Stephenson et al., 2019; Varadarajan et al., 2022). Here, we present expanded applications for ZnUMBA. ZnUMBA can be used in Xenopus embryos to measure the dynamics of barrier restoration and actin accumulation following laser injury. ZnUMBA can also be effectively utilized in developing zebrafish embryos as well as cultured monolayers of MDCK II epithelial cells. ZnUMBA is a powerful and flexible method that, with minimal optimization, can be applied to multiple systems to measure dynamic changes in barrier function with spatiotemporal precision.

Protocol for establishing knockout cell clones by deletion of a large gene fragment using CRISPR-Cas9 with multiple guide RNAs

“… 1 We have also successfully deleted GC-rich regions that are challenging to edit. 11 The common method using a single type of gRNA relies on codon misalignment due to deletions or insertions of a few bases, which may result in the expression of fragmented proteins with initiation codons appearing later than their mutated sites. However, if the large deletions in this method are successful, complete loss of protein expression can be achieved, eliminating these concerns.…”