Overview of gene editing technology

Since 2000, we have established a reliable and efficient service platform for genetically engineered mice and we are committed to providing the best-in-class solutions for genetically engineered animal models. To achieve that, we have:

• Dedicated project management team 

• AI-driven automated design platform SmartEddi 

• Team of highly experienced research staff

The following three types of genetic engineering technologies are used to generate genetically engineered mouse(GEM) models：

• Embryonic stem cell targeting technology

• CRISPR gene editing technology

• Transgenic technology

Comparison

Embryonic Stem Cell Targeting

In murine embryonic stem cells, the exchange of an endogenous allele of a target gene for a mutated copy via homologous recombination allows the generation of  targeted ES cells with defined mutations. Genetically engineered ES cells remain pluripotent and are able to develop into germ cells in the chimeric animals, thereby ensuring the germline transmission and the creation of a genetically modified line. As of today, embryonic stem cells targetng remains the most classic and reliable genetic engineering technology for mouse models.

Currently, SMOC provides mouse ES cells with three types of genetic background: C57BL/6, 129/S6, B6;129.

These mouse ES cells can be used to generate the following types of mouse models:

• Gene knockout

• Conditional gene knockout

• KO first

• Gene knock-in

• Point mutation

• Conditional point mutation

• Targeted gene overexpression

• Humanization

SMOC will select the most appropriate technology for each project on a case-by-case basis, by taking project duration and technology risk into account. 

Workflow of mouse genetic engineering with ES cell targeting 

• Design and generate homologous recombination vectors

• Transfer the homologous recombination vectors into murine ES cells

• Screen and validate the positive targeted ESC clones

• Microinject positive ESC clones into mouse blastocysts

• Transplant the injected mouse blastocysts into the uterus of pseudopregnant female mice

• Generate and screen positive chimeric mice F0

• Obtain F1 heterozygous mice by mating positive F0 with wild-type mice or FLP mice

CRISPR Gene Editing Technology

Advantages of CRISPR gene editing technology

• Faster compared to the traditional method.

• Not limited by the availability of mouse genetic strains

• More cost efficient 

Workflow of mouse genetic engineering with CRISPR/Cas9 technology

• Select target site and design sgRNA, design homologous recombination vector (optional)

• Prepare sgRNA and Cas9 mRNA, generate homologous recombination vector (optional)

• Microinject sgRNA and Cas9 mRNA (and homologous recombination vector (optional)) into mouse fertilized eggs

• Transplant fertilized eggs into the oviducts of pseudopregnant female mice

• Generate and screen positive chimeric mice F0

• Obtain F1 heterozygous mice by mating positive F0 with wild-type mice

Random Transgenes

A pre-designed gene (or genes) are randomly integrated into the mouse genome through pronuclear microinjection to generate transgenic mice. PiggyBac transposon system usually improves the efficiency. 

Workflow of mouse genetic engineering via random transgenes 

• Design and generate transgenic plasmid

• Inject linearized transgenic plasmid into mouse fertilized eggs

• Transplant fertilized eggs into the oviducts of pseudopregnant female mice

• Generate and screen positive chimeric mice F0

Workflow of mouse genetic engineering via piggBac transposon system 

• Design and generate PiggyBAC transposon plasmid

• Inject transposon plasmid and PiggyBAC transposase into mouse fertilized eggs

• Transplant fertilized eggs into the oviducts of pseudopregnant female mice

• Generate and screen positive chimeric mice F0