Next-Generation Humanized Models Construction Technology: TurboKnockout®

There are typically two techniques used for the genetic modification of mice: embryonic steam (ES) targeting and CRISPR. ES targeting technology is precise and off-target effects are minimal, allowing for complex genetic modifications, but it is inefficient, time-consuming, labor-intensive, and costly. On the other hand, CRISPR technology is efficient and rapid, but it carries the risk of uncontrollable off-target effects.

Comparatively, ES targeting technology is accurate, has no off-target effects, and can perform complex genetic modifications, but it is inefficient, time-consuming, labor-intensive, and costly. CRISPR technology is efficient and fast, but there is an uncontrollable off-target risk.

Is there a technology that can both reduce costs and ensure precise targeting?

Cyagen has developed TurboKnockout^® gene editing technology to serve as an upgraded version of ES targeting by retaining the advantages of traditional ES targeting, such as maturity, precise modification, and stable results. Additionally, TurboKnockout^® reduces the breeding time by two generations, shortening the cycle to as little as 4 months. This makes it the preferred method for constructing complex models, such as those requiring large segment gene modifications. The optimized homozygous ES clone screening system, combined with blastocyst injection or tetraploid complementation technology, allows for the production of batches of homozygous gene-edited mice within just 5 months.

TurboKnockout^® Gene Knockout

Achieving 100% ESC-derived founder mice and self-deletion of Neo, reducing breeding time by two generations.

The TurboKnockout^® technology, based on ES targeting, employs unique lineage development and gene modification techniques to establish TurboKnockout^® ES cell lines with highly efficient genetic advantages. By employing microinjection at specific embryonic development stages, TurboKnockout^® ES cells completely replace endogenous ES cells, thus bypassing the "chimeric" stage and shortening the ES targeting production cycle to as short as 4 months. Moreover, TurboKnockout^® utilizes a unique self-deleting Neo cassette, allowing any TurboKnockout^® mouse to mate with any mouse strain and achieve 100% Neo deletion. This means TurboKnockout^® not only bypasses the "chimeric" stage but also truly self-deletes Neo upon mating, ensuring rapid generation of Neo-free heterozygous mice.

Advantages of TurboKnockout^® Technology

1. No patent disputes: TurboKnockout^® technology is free from patent disputes, making it a commonly used technique in new drug development projects.

2. Capable of large fragment gene editing: Suitable for complex genetic modifications, achieving knock-in and knockout of megabase-sized sequences. It can insert sequences of up to 350kb in a single event. ES cells can tolerate up to 7 rounds of targeting and still maintain germline transmission, enabling the construction of whole-genome humanized mice and humanized antibody mice.

3. No off-target effects: TurboKnockout^® technology is built upon traditional ES targeting technology and does not have off-target effects. Expression of the project can be detected at the cellular stage through PCR, chromosome karyotyping, Southern blot analysis, flow cytometry, RT-qPCR, WB, and other methods. Gene modification is accurate and stable.

4. Shorter cycle: By bypassing the "chimeric" stage and self-deleting Neo, breeding time is reduced by two generations, shortening the construction period to as little as four months.  Additionally, TurboKnockout^® can perform multi-step BAC recombination at the ES cell level, further reducing the service cycle.

5. Bulk Production of homozygous models: The optimized homozygous ES clone screening system, combined with pre-injection of embryos or tetraploid complementation technology, enables the bulk acquisition of homozygous gene-edited mice within 5 months.

6. Flexible strain selection: Cyagen offers a range of strain options including C57BL/6NCya, C57BL/6JCya, BALB/cAnCya, 129S2/SvPasCya, and more.

TurboKnockout^® Gene-edited Mouse Models

1. HUGO-GT™ Next-Generation Humanized Models

If a deeper understanding of pathogenic mechanisms is required, the use of long-fragment or even whole-genome humanized mice is required. Cyagen has launched the Humanized Genomic Ortholog for Gene Therapy (HUGO-GT^TM) Program, based on its independently developed TurboKnockout^® technology, to achieve in situ replacement of murine genes with human counterparts. This has successfully produced whole-genome humanized mice covering a more diverse range of intervention targets. These HUGO-GT^TM mice feature a more efficient large fragment vector fusion technology and can serve as universal templates for targeted mutation customization services. They represent the next-generation of preclinical research models that are closer to real-world biological mechanisms for drug development.

HUGO-GT™ Next-Generation Humanized Models Cases

Cyagen has replaced the mouse Smn1 gene with the human SMN2 gene, creating a Type I SMA model with SMN1 deficiency and expression of human SMN2 (hSMN2), known as B6-hSMN2 (SMA) mice (product code: C001504).

The impact of ASO molecules targeting the modulation of SMN2 splicing patterns on homozygous B6-hSMN2 (SMA) mice (hSMN2/hSMN2).

ASO molecules increase the survival rate and delay tissue pathology in homozygous B6-hSMN2 (SMA) mice (hSMN2/hSMN2).

2. HUGO-Ab™ Mouse Models

Fully humanized antibody drugs have overcome many shortcomings of animal-derived antibodies and chimeric antibodies due to their high affinity, high specificity, and low toxicity and side effects, making them an inevitable trend in the development of therapeutic antibody drugs. Fully human antibodies produced in genomically humanized mice exhibit features such as low immunogenicity and high affinity. Additionally, the production is flexible due to the in vivo antibody maturation process.

Driven by the demand for innovative fully human antibody drug research and development, Cyagen has leveraged its solid technological innovation capabilities to independently develop the HUGO-Ab^® fully humanized antibody mouse. Based on the TurboKnockout^® technology, HUGO-Ab^® mouse models can produce fully humanized antibodies with high affinity and low immunogenicity in vivo, significantly accelerating the process of antibody discovery and new drug development. This achievement has been validated by numerous multinational pharmaceutical companies, biopharmaceutical firms, and academic research institutions.

Diagram illustrating the construction strategy of HUGO-Ab™ Mouse Models

3. Knockout Mice with Complete Gene Deletion

Complete gene knockout involves using gene knockout technology to remove all exons, several important exons, or functional regions of the target gene. This results in a mouse model in which the gene is not expressed in any tissues or cells throughout the body. This method is primarily used to study the physiological and pathological functions of a gene, provided that the gene is not essential for embryonic viability.

Selected cases of complete gene knockout mouse services

Reducing Hypothalamic Stem Cell Senescence Protects against Aging-Associated Physiological Decline.
Cell Metabolism (2020)
Gm31629 gene knockout mice

Legumain promotes tubular ferroptosis by facilitating chaperone-mediated autophagy of GPX4 in AKI.
Cell Death & Disease (2021)
lgmn gene knockout mice

4. Conditional Gene Knockout Mice

Conditional gene knockout involves inserting two LoxP sites into the flanking regions of one or more critical exons of the target gene to create TurboKnockout^®-flox mice with two loxP sites. These mice have a fully normal expression of the target gene before mating with mice expressing Cre recombinase. When TurboKnockout^®-flox mice are bred with mice expressing tissue-specific Cre recombinase, the target gene can be knocked out in specific tissues or cells while remaining expressed normally in other tissues or cells.

The Conditional Gene Knockout method is primarily used for:

• Research on genes that are embryonically lethal.
• Studying the physiological and pathological functions of genes in specific tissues or cells.
• Achieving temporal and spatial regulation of gene expression by combining with other inducible systems that control Cre expression.

Selected cases of conditional gene knockout mouse services

Transcriptional Repression of Aerobic Glycolysis by OVOL2 in Breast Cancer.
Advanced Science (2022)
OVOL2 conditional gene knockout mice

Hepatocyte TMEM16A Deletion Retards NAFLD Progression by Ameliorating Hepatic Glucose Metabolic Disorder.
Advanced Science (2020)
TMEM16A conditional gene knockout mice

5. Gene Knock-in Mice

Gene knock-in involves introducing specific mutations or exogenous genes at the target gene locus. For example, point mutations can be introduced into the target gene to simulate human genetic disease models. Alternatively, reporter genes (such as EGFP, mRFP, mCherry, mYFP, or LacZ) can be inserted at specific sites of the target gene through homologous recombination, allowing the expression of the target gene to be tracked using the reporter gene. Reporter genes can also replace the mouse's own genes, enabling simultaneous knockout (KO) and knock-in (KI). This technique is mainly used for drug screening, signal pathway studies, and tracking-related research.

Selected cases of gene knock-in mouse services

Leptin receptor-expressing neuron Sh2b1 supports sympathetic nervous system and protects against obesity and metabolic disease.
Nature Communications (2020)
 Sh2b1-Cre gene knock-in mice

An Orally Active Galectin-3 Antagonist Inhibits Lung Adenocarcinoma Growth and Augments Response to PD-L1 Blockade.
Cancer Research (2020)
LGALS3 conditional gene knock-in mice

6. KO-First Technology for Generating Gene Knockout Mice

The KO-First technology achieves gene knockout by inserting a gene disruption cassette flanked by FRT sites into an intron. This disruption cassette contains a splice acceptor, reporter gene, and Neo. In addition, loxP sites are inserted on both sides of the exon(s) to be knocked out in the target gene. Therefore, the resulting mice have the target gene knocked out and the reporter and resistance genes knocked in. By using the Flp/FRT and Cre/LoxP recombination systems, researchers can achieve both complete and conditional knockouts. When these mice are bred with Flp mice, the expression of the knocked-out gene can be restored, resulting in conditional gene knockout mice. Breeding these conditional knockout mice with Cre mice can then generate mice with the target gene knocked out.