Principles of Gene Knock-in (KI) Technology
 

The core of gene knock-in technology lies in utilizing the cell's own DNA repair mechanisms to achieve precise integration of exogenous genes or specific mutations.
 

Its working principle involves an engineered nuclease recognizing and cutting a specific target DNA sequence, generating a DNA double-strand break (DSB). The cell then repairs this break via two main pathways:
 

Non-homologous end joining (NHEJ):​ The repair process is prone to introducing random small insertions or deletions at the break site, commonly used to achieve gene knockout.
 

Homology-directed repair (HDR):​ The cell's repair system uses an exogenously provided DNA fragment, homologous to the sequences flanking the DSB, as a template to synthesize complementary sequences at the break. This precisely "knocks in" the exogenous gene or mutation, achieving accurate gene editing.

 

KI efficiency is limited by multiple factors: HDR itself has a low probability, and the knock-in locus, fragment size, cell line choice, selection markers, etc., all impact the success rate. Common KI types include: Tag tag knock-in, reporter gene knock-in, and "safe harbor" site knock-in based on selection markers.
 

Common Gene Knock-in (KI) Mouse Models
 

Based on the characteristics of the knock-in site, they are mainly categorized into the following three types of models:
 

Conventional KI Mice:
 

The exogenous gene is inserted into an arbitrary site in the genome, which may be random integration or rely on simple homologous recombination targeting.
 

Characteristics:​ Low construction cost, short cycle, but the insertion site is uncertain. Gene expression is susceptible to "position effects" (unstable expression), and there is a risk of disrupting essential host genes.
 

Rosa26 KI Mice:
 

The exogenous gene is inserted into the Rosa26locus on mouse chromosome 6. This is a highly conserved non-coding genomic locus expressed widely in all cell types and developmental stages. Its promoter region exhibits high transcriptional activity, and its introns contain unique transcription start sites (ATG) that enable stable, reproducible expression of the transgene.
 

Characteristics:
 

1.Highly conserved non-coding region; insertion does not affect cellular function.

2.Open chromatin structure supports ubiquitous/ubiquitous expression of the transgene (e.g., whole-body expression tool models).
 

H11 KI Mice:
 

The exogenous gene is inserted into the H11locus on mouse chromosome 11. This locus is located near the immunoglobulin κ light chain gene cluster. The chromatin is naturally permissive, compatible with various promoters, supports high transgene expression in multiple tissues, and has low immunogenicity, making it suitable for heterologous protein expression.
 

Characteristics:
 

1.Located near the immunoglobulin κ light chain gene cluster; naturally permissive and compatible with various promoters.

2.Supports high transgene expression in multiple tissues (e.g., muscle, liver).
 

Application Scenario Comparison
 

Global/Ubiguitous Expression:​ Both Rosa26 KI and H11 KI mice can achieve whole-body expression. For example, Rosa26 KI mice utilize their ubiquitous expression characteristic to express a transgene (e.g., a fluorescent protein) in all tissues and cells of the animal. H11 KI mice can be used for whole-body expression of humanized antibodies. Conventional KI mice may have unstable expression due to uncertain insertion sites.
 

Conditional Knock-in:​ Rosa26 KI mice can be combined with the Cre system, using the Cre-loxP recombination system to achieve spatiotemporal and cell type-specific control of gene expression, enabling flexible conditional gene manipulation. H11 KI mice are not typically used for conditional knock-in. To achieve conditional knock-in with conventional KI, additional design of LoxP sites is required.
 

High Expression Demand:​ H11 KI mice, with their low immunogenicity and advantageous locus, are suitable for protein overexpression. Rosa26 KI mice may be limited by endogenous regulatory elements, making it difficult to meet ultra-high expression demands. Although conventional KI can also achieve high expression, optimization of elements like promoters is needed to enhance expression levels.
 

Avoiding Insertional Mutation Risk:​ Both Rosa26 and H11 KI sites are in intergenic regions without endogenous genes. Inserting a transgene does not disrupt essential host functional genes, reducing the risk of insertional mutations. Conventional KI, due to random insertion, carries the risk of disrupting genes within the genome.
 

Applications
 

Gene knock-in models are widely used in gene function research, disease model construction, and drug screening:
 

Gene Function Research:​ Introduce specific mutations in vivo to simulate human genetic mechanisms and explore pathogenesis.

Disease Model Construction:​ Establish animal models that precisely mimic human diseases for pathological studies and drug testing.

Drug Screening and Development:​ Construct humanized mouse models to accelerate antibody drug R&D and efficacy evaluation.

Cell Lineage Tracing and Analysis:​ Knock-in reporter genes (e.g., fluorescent proteins) to enable real-time tracking of specific cell or protein expression or to trace cell developmental lineages.
 

MingCeler Biotech Gene Knock-in
 

MingCeler Biotech’s TurboMice™ technology overcomes the challenges of long cycle times and low success rates for complex mouse models, enabling editing at virtually any target gene locus. In as little as two months, complete homozygous gene-edited mouse models can be prepared directly from embryonic stem cells. MingCeler Biotech can customize various gene knock-in mouse models according to client requirements. We welcome inquiries from all researchers.