An Optimized Preparation Method for Long ssDNA Donors to Facilitate Quick Knock-In Mouse Generation.

ABSTRACT: Fluorescent reporter mouse lines and Cre/Flp recombinase driver lines play essential roles in investigating various molecular functions in vivo. Now that applications of the CRISPR/Cas9 genome-editing system to mouse fertilized eggs have drastically accelerated these knock-in mouse generations, the next need is to establish easier, quicker, and cheaper methods for knock-in donor preparation. Here, we reverify and optimize the phospho-PCR method to obtain highly pure long single-stranded DNAs (ssDNAs) suitable for knock-in mouse generation via genome editing. The sophisticated sequential use of two exonucleases, in which double-stranded DNAs (dsDNAs) amplified by a pair of 5'-phosphorylated primer and normal primer are digested by Lambda exonuclease to yield ssDNA and the following Exonucle

SUBMITTER: Inoue YU 

PROVIDER: S-EPMC8147208 | biostudies-literature | 2021 Apr

REPOSITORIES: biostudies-literature