Authors:

Daniel Matthew Chambers

Date Created:

Not specified

Course Title:

Professor:

Not specified

About Paper:

Zebrafish are an attractive organism to perform in vivo cell biology experiments owing to their high productivity and transparency in early development stages. Visualization of proteins has been mostly conducted by overexpression under specific promoters. However, to bypass the artificial expression and function of genomic overexpression, our group aims to reveal the intrinsic expression of genes with a fluorescent tag knock-in (KI). Reported accurate KI methods utilizing homology directed repair method and ?C31 integrase-mediated transgenesis are known for their low efficiency and time-consuming nature. This project reports a highly efficient KI method with Non-Homologous End Joining DNA repair method. We used this mechanism to specifically tag actin fiber proteins at the c-terminus by inserting the designed DNA construct into the non- coding regions of the Zebrafish genome. Tagging actb1 with green fluorescent protein (GFP) enables visualization of actin dynamics in vivo. The reported method will pave the way for future endogenous protein tagging projects at different loci with a higher DNA integration efficiency. Keywords: Zebrafish; Non-Homologous End Joining; Actin; Intrinsic; Knock-In

Source:

Purdue University / 2024

Topics:

No topics listed

Co-authors:

Daniel Matthew Chambers