Exploring the "Jumpers" of the Genome (Part 1): The Application of Tol2 Transposon in Gene Function Research

(2). The principle of the Tol2 transposon system can be summarized in the following steps:

• Construction of the Transposon Vector: The target gene is inserted into the Tol2 transposon vector, creating a recombinant transposon.

• Transposase Expression: The Tol2 transposase is co-introduced into the host cells along with the recombinant transposon.

• Transposition: The Tol2 transposase recognizes the inverted repeat sequences at both ends of the recombinant transposon, excises it from the vector, and inserts it into the host genome.
• Gene Expression: The target gene inserted into the genome is stably expressed in the host cell, achieving gene overexpression.

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(3).  Advantages of the Tol2 Transposon System:

• Large Carrying Capacity: The Tol2 transposon can carry exogenous gene fragments of up to 11 kb, meeting the needs for overexpression of various genes.
• Efficient Integration: The technology is simple, and exogenous genes can be efficiently integrated into the host genome using plasmid transfection or electroporation. This allows for the long-term stable expression of the exogenous gene in host cells, making it suitable for studying long-term gene regulation mechanisms.
• No Insertion Site Preference: The Tol2 transposon integrates randomly into the host genome with no significant base preference.
• Broad Host Range: The Tol2 transposon can function in various vertebrate cells, including those from mammals, fish, birds, and more.
• Relatively Simple Operation: By using plasmid transfection (non-viral transduction), the target gene can be permanently integrated into the host genome without the need for a complex viral packaging process, which is often required in some viral vector systems.
• Short Screening Time: By introducing a resistance gene (such as neomycin resistance), cells with stable transfection can be quickly selected.

(4) Disadvantages of the Tol2 Transposon System:

• Potential Insertion Mutation Risks: Due to the random nature of its integration sites, the Tol2 transposon may insert into critical regions of the host genome, leading to gene mutations or abnormal regulation.
• Limited Vector Capacity: Although the Tol2 transposon can carry relatively large exogenous DNA fragments, there are still capacity limits. For some complex gene expression systems, the vector capacity may not be sufficient.
• Dependence on Transfection Efficiency: The transposase and the gene of interest are present on separate vector systems and need to be co-transfected into target cells. The efficiency of this process depends on the plasmid transfection efficiency of the cells. For primary cells or cells that are difficult to transfect, low transfection efficiency may affect the construction of stable cell lines.
• Potential Cytotoxicity: The transposition process may have certain effects on cells, such as inducing cellular stress responses, gene mutations, and in some cases, leading to abnormal cell growth, apoptosis, or other undesirable phenotypes. This may interfere with the normal physiological function of cells and affect subsequent experimental results.
• Need for Screening and Identification: Due to the randomness and uncertainty of insertion, various integration patterns and expression levels may result in different cell clones. A significant amount of screening and identification work is required to obtain the ideal stable transfected cell line.

3. Applications of the Tol2 Transposon System

The Tol2 transposon system has broad potential applications in gene function research, gene overexpression cell line construction, gene therapy, and the creation of transgenic animal models.

(1) Gene Function Research: The Tol2 transposon system can efficiently construct gene overexpression or knockout cell lines, which are used to study the function and regulatory mechanisms of genes.

Example 1: In 2018, the research group led by Li Yulong at the Peking University-Tsinghua University Life Sciences Joint Center published a paper titled "A genetically-encoded fluorescent sensor enables rapid and specific detection of dopamine in flies, fish, and mice" in Cell [2]. They successfully developed a new genetically-encoded dopamine fluorescent sensor, DA1h and DA1m, and applied it to detect the dynamic changes of endogenous dopamine in fruit flies, zebrafish, and mice. In the experiment, the researchers used the Tol2 transposon system to efficiently integrate the fluorescent probe DA1m into the zebrafish genome, creating a DA1m transgenic zebrafish model. The results demonstrated that long-term expression of the probe did not significantly affect the growth of the model organism and could detect dopamine release triggered by chemogenetic activation and visual stimuli. Additionally, the probe expression was confirmed in cells, mouse brain slices, and live fruit flies and mice via transfection and viral injection (AAV), supporting these findings.

Figure 3: Construction of the DA1m Transgenic Zebrafish Line Based on the Tol2 Transposon System
 

(2) Transgenic Animal Model Construction: The Tol2 transposon system can be used to construct transgenic animal models, such as zebrafish, medaka fish, Xenopus tropicalis, and mice, for studying the mechanisms of human disease development and drug screening.

Example 2: In 2010, researchers from the National Institute of Genetics in Japan published an article titled A simple and highly efficient transgenesis method in mice with the Tol2 transposon system and cytoplasmic microinjection [3], which introduced a new transgenic mouse preparation method based on the Tol2 transposon system — Tol2-mediated cytoplasmic injection (Tol2:CI). The article discusses in detail its role in improving transgenic efficiency and constructing mouse models. The Tol2 transposon system, through transposase, recognizes the ITRs and efficiently inserts the exogenous gene into the genome. The study designed three experimental conditions:Pronuclear injection (PNI), Tol2 pronuclear + cytoplasmic injection (Tol2:PNI+CI), Tol2 cytoplasmic injection (Tol2:CI), The results showed that the Tol2:CI method achieved a total transgenic efficiency of over 20%, which is much higher than the traditional pronuclear injection method (about 2%–5%). Additionally, the cytoplasmic injection method caused less embryo damage, had a higher survival rate, and showed higher integration frequency with high DNA and mRNA concentrations. The Tol2 system is not only applicable to model organisms such as zebrafish but also works well for mammals such as mice. Transgenic mice generated by the Tol2:CI method express the exogenous gene correctly, and these genes are passed on to the next generation through the germline. The Tol2 system demonstrated efficiency, stability, and ease of use in constructing transgenic mouse models, providing new technical means for genetic research and functional genomics.

Figure 4: Schematic Diagram of Transgenic Mouse Model Construction Based on the Tol2 Transposon System (PNI: Pronuclear Injection; CI: Cytoplasmic Injection)
 

(3).Gene Therapy: The Tol2 transposon system can permanently integrate therapeutic genes into the target cell genome, providing a new tool for gene therapy of genetic diseases, tumors, and other conditions.

Example: Proteinuria is an important marker of kidney dysfunction and is typically assessed by measuring the protein content in urine, serving as a key clinical indicator of kidney disease. In 2022, a research team led by Rachel Lennon at the University of Manchester published an article online titled A novel nanoluciferase transgenic reporter to measure proteinuria in zebrafish [4], in which they used the Tol2 system to create a transgenic zebrafish line that stably expressed NL-D3. This system allows the quantification of proteinuria levels by measuring the luminescence intensity in the embryo culture medium using a fluorometer. In the experiment, the researchers successfully induced proteinuria by knocking down genes associated with kidney disease and treating zebrafish embryos with chemicals. They detected an increase in proteinuria using the NL-D3 reporter system. Additionally, the researchers used CRISPR-Cas9 technology to knock down the Col4a3 and Col4a4 genes, simulating the pathological features of Alport syndrome, and validated the potential of the NL-D3 system in drug screening. In conclusion, the Tol2 transposon system played a critical role in this study, enabling the stable integration of the NL-D3 reporter gene into the zebrafish genome and its specific expression in the liver, providing a reliable tool for detecting proteinuria, a key marker of kidney dysfunction.

Figure 5: Construction of the NL-D3 Transgenic Zebrafish Line Based on the Tol2 Transposon System
 

References
[1]Wells, J. N. & Feschotte, C. A Field Guide to Eukaryotic Transposable Elements.Annu. Rev. Genet.54, 539–561 (2020).
[2]Sun F, Zeng J, Jing M, Zhou J, Feng J, Owen SF, Luo Y, Li F, Wang H, Yamaguchi T, Yong Z, Gao Y, Peng W, Wang L, Zhang S, Du J, Lin D, Xu M, Kreitzer AC, Cui G, Li Y. A Genetically Encoded Fluorescent Sensor Enables Rapid and Specific Detection of Dopamine in Flies, Fish, and Mice. Cell. 2018 Jul 12;174(2):481-496.e19.
[3]Sumiyama K, Kawakami K, Yagita K. A simple and highly efficient transgenesis method in mice with the Tol2 transposon system and cytoplasmic microinjection. Genomics. 2010 May;95(5):306-11.
[4]Naylor RW, Lemarie E, Jackson-Crawford A, Davenport JB, Mironov A, Lowe M, Lennon R. A novel nanoluciferase transgenic reporter measures proteinuria in zebrafish. Kidney Int. 2022 Oct;102(4):815-827.
 

Continuous Updates – Stay Tuned!

After delving into the Tol2 transposon, are you curious about other "jumpers" in the genome? The transposon family is vast, with each member having unique transposition mechanisms and application advantages. In addition to the Tol2 transposon, other transposons like PiggyBac and Sleeping Beauty also play a crucial role in genetic engineering. In the upcoming articles, Brain Case will introduce these transposons in detail, exploring their principles, applications, and advantages. Stay tuned for more insights into the fascinating world of transposons!

The Tol2 transposon system, known for its efficiency and flexibility, has become an important tool in gene editing and gene therapy. Brain Case now offers Tol2 transposon system-related gene overexpression services, providing a one-stop solution from vector construction, cell transfection, to stable cell line selection. Researchers are welcome to consult and discuss Via Email: bd@ebraincase.com