Step-by-Step Protocol for Making a Knock-In Xenopus laevis to Visualize Endogenous Gene Expression.

Area of Science:

• Molecular Biology
• Developmental Biology
• Genetics

Background:

• Studying endogenous gene expression in Xenopus laevis is crucial for understanding developmental processes.
• Existing methods may lack efficiency or the ability to recapitulate native regulatory elements.
• Reporter gene expression is a powerful tool for visualizing gene activity.

Purpose of the Study:

• To establish and validate a novel, efficient knock-in technique for Xenopus laevis.
• To enable the recapitulation of endogenous gene expression patterns using reporter genes.
• To provide a detailed protocol for the application of this new technique.

Main Methods:

• Development of a CRISPR-Cas9-based knock-in strategy named NEXTi (New and Easy Xenopus Targeted integration).
• Integration of a donor DNA with an eGFP reporter gene into the 5' untranslated region (UTR) of target genes.
• Microinjection of components into Xenopus embryos, followed by tissue-specific expression analysis and breeding for F1 generation confirmation.

Main Results:

• Successful generation of knock-in Xenopus embryos exhibiting tissue-specific eGFP expression (2-13% efficiency).
• Targeted integration into specific loci (krt.12.2.L, myod1.S, sox2.L, bcan.S) confirmed.
• Stable eGFP expression was achieved in F1 generation embryos derived from injected adult frogs.

Conclusions:

• The NEXTi technique provides an effective method for generating Xenopus models with reporter gene expression reflecting endogenous gene regulation.
• This technique facilitates the study of gene function and regulation during development in Xenopus.
• The described protocol allows for reproducible generation and validation of knock-in lines.