Safe harbor locus knock-in involves the insertion of transgene expression vectors into safe harbor sites, such as the Rosa26 and H11 loci. Studies have shown that these sites are actively expressed in all tissues and do not exhibit the transgene silencing phenomenon that occurs in random transgenesis. This method is commonly used to create mice for overexpression or conditional overexpression.

Conditional overexpression involves inserting a loxp-Stop-loxp sequence in front of the target gene to prevent its expression. When this model is crossed with a Cre model, the stop sequence is deleted in tissues where Cre is expressed, allowing the target gene to be expressed.

Overexpression mice directly obtain constitutive gene expression mice. Vitalstar has established a stable and efficient targeted transgenesis system at the Rosa26 and H11 loci, achieving targeted transgenesis of up to 10 kb.

Figure 1. Knocking in a transgene expression vector at the Rosa26 locus to prepare targeted transgenic mice.

1. Technical Process:

Design and construct gene-editing tools → Prepare editing tools and donor DNA → Microinjection → Embryo transfer → Mouse identification and screening → Expression detection → Line establishment

2. Technical Advantages:

(1) High specificity, targeted transgenesis techniques such as CRISPR-Cas9, TALEN, and ZFN can edit at specific locations in the genome, ensuring high specificity of gene modification.

(2) Precise insertion or deletion.

(3) High editing efficiency: Compared to traditional transgenic methods, targeted transgenesis technology has a higher editing efficiency, allowing for faster acquisition of transgenic mice.

(4) Genetic stability: Transgenic mice obtained through gene editing have genetic stability and can be stably inherited by offspring, facilitating long-term research.

(5) Stable expression: Targeted transgenesis ensures stable gene expression without variation in expression levels due to different insertion sites.

3. Successful Case:

Safe harbor site knock-in: Using CRISPR/Cas9 technology, the tdTomato sequence under the control of the 2C promoter was knocked into the H11 locus of mice, obtaining mice expressing tdTomato at the two-cell stage.