Improved Genome Editing via Oviductal Nucleic Acids Delivery-based In Vivo Electroporation Technique for Knockout Mice Generation

作者： Elena I. Leonova1, Angelina V. Chirinskaite1, Ilyas I. Akhmarov1, Polina S. Luganskaya1, Oleg A. Kirillov1, Daria A. Kandina1, Anna E. Romanovich2, Liubov A. Shkodenko3,4, Sergei A. Fedotov5, Aleksandr A. Rubel3, Yulia V. Sopova1 1Center of Transgenesis and Genome Editing,St. Petersburg State University, 2Resource Center Molecular and Cell Technologies,St. Petersburg State University, 3Amyloid Biology Laboratory,St. Petersburg State University, 4DNA-Nanosensoric laboratory,ITMO University, 5Laboratory of Toxinology and Molecular Systematics, L.A. Orbeli Institute of Physiology,National Academy of Sciences of the Republic of Armenia

简介：

Overview

This study presents the Improved Genome Editing via Oviductal Nucleic Acids Delivery (I-GONAD) method, which simplifies the generation of knockout mice. By using in utero electroporation, genome editing components are delivered directly into the oviducts of pregnant females, achieving efficiency comparable to traditional microinjection techniques.

Key Study Components

Area of Science

• Neuroscience
• Genetics
• Animal Models

Background

• Traditional microinjection requires zygote collection from donor females.
• Minimizing animal use is a priority in genetic research.
• The Nepa21 system allows for efficient electroporation of zygotes.
• CRISPR/Cas9 technology is utilized for targeted gene editing.

Purpose of Study

• To simplify the generation of knockout mice.
• To reduce the number of animals needed for genetic experiments.
• To evaluate the effectiveness of the I-GONAD method.

Methods Used

• In utero electroporation for delivering genome editing components.
• Computational analysis for identifying single guide RNA sequences.
• Genome browser tools for cross-referencing exome structure.
• CRISPR/Cas9-induced mutations targeting the ROSA26 locus.

Main Results

• The I-GONAD method shows comparable efficiency to traditional methods.
• Successful introduction of mutations into the ROSA26 locus on chromosome 6.
• Reduction in the number of animals required for knockout generation.
• Demonstration of a streamlined protocol for genetic editing.

Conclusions

• The I-GONAD method is an effective alternative to microinjection.
• This approach can facilitate more ethical research practices.
• Future studies may expand on the applications of this technique.

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