Your browser doesn't support javascript.
loading
A robust knock-in approach using a minimal promoter and a minicircle.
Keating, Margaret; Hagle, Ryan; Osorio-Méndez, Daniel; Rodriguez-Parks, Anjelica; Almutawa, Sarah I; Kang, Junsu.
Afiliación
  • Keating M; Department of Cell and Regenerative Biology, School of Medicine and Public Health, University of Wisconsin - Madison, Madison, WI, 53705, USA.
  • Hagle R; Department of Cell and Regenerative Biology, School of Medicine and Public Health, University of Wisconsin - Madison, Madison, WI, 53705, USA.
  • Osorio-Méndez D; Department of Cell and Regenerative Biology, School of Medicine and Public Health, University of Wisconsin - Madison, Madison, WI, 53705, USA.
  • Rodriguez-Parks A; Department of Cell and Regenerative Biology, School of Medicine and Public Health, University of Wisconsin - Madison, Madison, WI, 53705, USA.
  • Almutawa SI; Department of Cell and Regenerative Biology, School of Medicine and Public Health, University of Wisconsin - Madison, Madison, WI, 53705, USA.
  • Kang J; Department of Cell and Regenerative Biology, School of Medicine and Public Health, University of Wisconsin - Madison, Madison, WI, 53705, USA; UW Carbone Cancer Center, School of Medicine and Public Health, University of Wisconsin - Madison, Madison, WI, 53705, USA. Electronic address: junsu.kang@wi
Dev Biol ; 505: 24-33, 2024 Jan.
Article en En | MEDLINE | ID: mdl-37839785
ABSTRACT
Knock-in reporter (KI) animals are essential tools in biomedical research to study gene expression impacting diverse biological events. While CRISPR/Cas9-mediated genome editing allows for the successful generation of KI animals, several factors should be considered, such as low expression of the target gene, prevention of bacterial DNA integration, and in-frame editing. To circumvent these challenges, we developed a new strategy that utilizes minicircle technology and introduces a minimal promoter. We demonstrated that minicircles serve as an efficient donor DNA in zebrafish, significantly enhancing KI events compared to plasmids containing bacterial backbones. In an attempt to generate a KI reporter for scn8ab, we precisely integrated a fluorescence gene at the start codon. However, the seamlessly integrated reporter was unable to direct expression that recapitulates endogenous scn8ab expression. To overcome this obstacle, we introduced the hsp70 minimal promoter to provide an ectopic transcription initiation site and succeeded in establishing stable KI transgenic reporters for scn8ab. This strategy also created a fgf20b KI reporter line with a high success rate. Furthermore, our data revealed that an unexpectedly edited genome can inappropriately influence the integrated reporter gene expression, highlighting the importance of selecting a proper KI line. Overall, our approach utilizing a minicircle and an ectopic promoter establishes a robust and efficient strategy for KI generation, expanding our capacity to create KI animals.
Asunto(s)
Palabras clave

Texto completo: 1 Base de datos: MEDLINE Asunto principal: Pez Cebra / Sistemas CRISPR-Cas Idioma: En Revista: Dev Biol Año: 2024 Tipo del documento: Article

Texto completo: 1 Base de datos: MEDLINE Asunto principal: Pez Cebra / Sistemas CRISPR-Cas Idioma: En Revista: Dev Biol Año: 2024 Tipo del documento: Article