Conventional (Constitutive) Gene Knockout by SMOC

Introduction

Tab 1 CRISPR Gene Editing
 

Advantages of CRISPR Gene Editing Technology

• Speed: Significantly faster than traditional methods.
• Versatility: Not limited by mouse genetic strains.
• Cost-Effectiveness: More efficient in terms of resources and time.

CRISPR/Cas9 Workflow

• Target Site Selection: Identify and design specific sgRNAs and optionally, a homologous recombination vector.
• Preparation: Synthesize sgRNA and Cas9 mRNA; optionally generate a homologous recombination vector.
• Microinjection: Introduce sgRNA and Cas9 mRNA (and optionally the vector) into mouse fertilized eggs.
• Transplantation: Transfer these eggs into the oviducts of pseudopregnant female mice.
• Screening: Generate and screen for positive chimeric mice (F0 generation).
• Breeding: Mate positive F0 mice with wild-type mice to obtain F1 heterozygous mice.

Timeframe for CRISPR/Cas9

• Typically takes 4-6 months to generate a conventional gene knockout mouse model using CRISPR technology

Mechanism of CRISPR/Cas9

• Synthetic sgRNAs and Cas9 mRNA are co-injected into fertilized mouse eggs, guiding the Cas9 enzyme to cut the target site in the genome. The repair mechanism, usually through the non-homologous end joining (NHEJ) pathway, often results in a frameshift mutation in the gene sequence.

Tab 2 Embryonic Stem Cell Targeting

Embryonic Stem Cell Targeting

• Process: Exchange of an endogenous allele with a mutated copy via homologous recombination in murine embryonic stem cells.
• Outcome: Targeted ES cells remain pluripotent and contribute to germ cell development in chimeric animals, ensuring germline transmission.
• Reliability: Remains the most classic and dependable method for creating genetically modified mouse models.

Types of Mouse Models Available

• Gene knockout
• Conditional gene knockout
• KO first
• Gene knock-in
• Point mutation
• Conditional point mutation
• Targeted gene overexpression
• Humanization

Project Selection Criteria by SMOC

• SMOC evaluates each project individually, considering project duration and technology risk, to select the most appropriate technology.

Timeframe for ES Cell Targeting

• Typically takes 9-12 months to generate a conventional gene knockout mouse model using ES cell targeting technology.

Detailed Procedure for ES Cell Targeting

• A homologous recombination vector is created, replacing one or more exons of the target gene with a neomycin (Neo) gene.
• The engineered ES cells are injected into blastocysts to produce a chimeric mouse.
• Breeding the chimeric mouse with a wild-type mouse yields heterozygous mice, which are then bred to obtain homozygous knockout mice.