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

Area of Science:

• Genetics
• Molecular Biology
• Developmental Biology

Background:

• Traditional knockout mouse generation involves multiple animal-intensive steps: embryo collection, ex vivo microinjection, and surgical transfer.
• Existing methods face challenges including high animal numbers, needle clogging, and potential embryo mortality during microinjection.
• There is a need for more efficient and accessible techniques for creating genetically modified mouse models.

Purpose of the Study:

• To present a detailed protocol for the Improved Genome Editing via Oviductal Nucleic Acids Delivery (I-GONAD) method.
• To offer a streamlined, single-step alternative for generating knockout mice.
• To highlight the advantages of I-GONAD over conventional knockout generation strategies.

Main Methods:

• The I-GONAD technique utilizes advanced electroporators (e.g., Nepa21) for in vivo gene editing.
• CRISPR-Cas components (Cas9 protein and guide RNA) are microinjected into the oviducts of pregnant mice (0.7 days post-conception).
• In vivo electroporation delivers the genetic material directly into zygotes within the oviduct, enabling targeted gene knockout.

Main Results:

• The I-GONAD procedure allows for the generation of knockout mice in a single step.
• This method bypasses the need for ex vivo embryo manipulation and surgical transfer.
• Successful generation of pups with targeted gene knockouts following the I-GONAD protocol.

Conclusions:

• I-GONAD offers a significantly more efficient and accessible approach to creating knockout mice.
• The technique reduces the number of animals required and overcomes limitations of traditional methods.
• This protocol provides a valuable tool for researchers seeking to generate genetically modified mouse models.