Laser-Assisted Gene Delivery

Laser-Assisted Gene Delivery: Revolutionizing Mouse Embryo Transgenesis

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Lena Kashyap in Science & Nature September 2025 • 4 min read.

"A novel method using lasers to enhance lentiviral gene delivery to mouse embryos, offering a simpler, more accessible approach to creating transgenic animal models."


Lentiviruses stand out as highly effective tools for delivering genes into mammalian cells. Once a cell is transduced, the lentiviral genome integrates permanently into the host's DNA, ensuring that the new genetic information is passed down through generations. This makes lentiviruses ideal for creating stable cell lines, delivering indicators in vivo, and producing transgenic animals by transducing single-cell fertilized eggs.

However, mouse fertilized eggs and early-stage embryos possess a natural defense: the zona pellucida. This glycoprotein matrix acts as a protective barrier, preventing lentiviruses from reaching the embryonic cells. Because lentiviruses are too large to penetrate this barrier, traditional methods rely on microinjection—a technique that requires injecting viral particles directly into the perivitelline space between the zona and the embryonic cells.

The need for skilled technicians and specialized equipment has limited the widespread use of lentiviruses in mouse embryo gene delivery. This article introduces a detailed protocol for permeabilizing the zona pellucida using a laser to create tiny perforations. This laser-perforation method doesn't harm the embryo and allows lentiviruses to access the embryonic cells easily. Embryos transduced in this manner can develop into blastocysts in vitro and, when implanted into pseudopregnant mice, can develop into transgenic pups. The laser system described is both effective and user-friendly. This method provides a straightforward alternative for creating transgenic mice without micromanipulation or microinjection.

How Does Laser-Assisted Lentiviral Delivery Work?

Laser-Assisted Gene Delivery

The method detailed here provides step-by-step instructions for permeabilizing the zona pellucida of mouse fertilized eggs, making the embryonic cells accessible for gene delivery via lentiviruses. Lentiviruses are naturally adapted for efficient gene delivery into mammalian cells, capable of infecting both dividing and non-dividing cells. They ensure stable integration of their genetic payload into the host chromosomes.

Researchers commonly expand the range of lentiviral host cells by pseudotyping the recombinant lentivirus with the vesicular stomatitis virus glycoprotein (VSV-G), which broadens the virus's tropism. Once transduction occurs, the lentiviral genes become a stable, expressed component of the host's chromosomes, making this an ideal tool for creating transgenic animals. When delivered to early-stage embryonic cells, the lentiviral genome is replicated and expressed throughout the entire organism.

  • Overcoming the Zona Pellucida: Traditional methods require skilled technicians and specialized equipment to bypass the zona pellucida.
  • Laser Perforation: The described method uses a laser to permeabilize the zona, facilitating lentiviral gene delivery without harming the embryo.
  • Simplified Transgenesis: This approach simplifies the creation of transgenic mice by eliminating the need for micromanipulation and microinjection.
The XYClone laser system, initially designed to aid in in vitro fertilization and embryonic stem cell cultivation, is a compact, user-friendly device. Once mounted on a microscope, it functions like an objective lens. The accompanying software enables precise aiming of the laser while observing through the microscope eyepieces. Once the zona pellucida is perforated by the XYClone laser, lentiviruses can be introduced into the culture media for gene delivery. This method also opens the possibility of simultaneously delivering multiple lentiviruses to incorporate several genes into the chromosomes.

The Future of Transgenesis

Laser-assisted perforation of the mouse fertilized egg zona holds promise for application to other species, potentially facilitating the entry of viruses or transfection reagents. The method simplifies the process, reduces the need for specialized skills and equipment, and enhances the efficiency of gene delivery, marking a significant step forward in the field of transgenesis.

About this Article -

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Everything You Need To Know

1

What are lentiviruses and why are they important for creating transgenic animal models?

Lentiviruses are a really effective way to get genes into mammalian cells. After a cell has been transduced, the lentiviral genome becomes a permanent part of the host's DNA, making sure that the new genetic information is passed on to future generations. This is why lentiviruses are useful for making stable cell lines, delivering indicators in vivo, and creating transgenic animals by transducing single-cell fertilized eggs. This contrasts with transient transfection methods where the genetic material is not integrated and is eventually lost.

2

What is the zona pellucida and why does it pose a challenge for gene delivery into mouse embryos?

The zona pellucida is a glycoprotein matrix that acts as a protective barrier around mouse fertilized eggs and early-stage embryos, preventing lentiviruses from reaching the embryonic cells. Because lentiviruses are too large to penetrate this barrier, traditional methods rely on microinjection—a technique that requires injecting viral particles directly into the perivitelline space between the zona and the embryonic cells. The zona pellucida is crucial in early development but presents a hurdle for gene delivery.

3

How does laser-assisted lentiviral delivery work to create transgenic mice?

Laser-assisted lentiviral delivery involves using a laser to create tiny perforations in the zona pellucida of mouse fertilized eggs. This allows lentiviruses to easily access the embryonic cells without harming the embryo. After the zona pellucida is perforated by a laser system like the XYClone laser, lentiviruses can be introduced into the culture media for gene delivery. This method simplifies the creation of transgenic mice because it eliminates the need for micromanipulation or microinjection. This technique is also applicable to other species.

4

What is the XYClone laser system and how does it facilitate gene delivery?

The XYClone laser system is a user-friendly device designed to aid in in vitro fertilization and embryonic stem cell cultivation. It can be mounted on a microscope and controlled via software to precisely aim the laser. This allows researchers to perforate the zona pellucida without needing extensive training in micromanipulation techniques. The system's ease of use and precision make it a valuable tool for gene delivery.

5

What are the advantages of using laser-assisted perforation for gene delivery in terms of multiple gene insertions and cell targeting?

By permeabilizing the zona pellucida using a laser, multiple lentiviruses can be simultaneously delivered to incorporate several genes into the chromosomes. This allows for the creation of more complex transgenic models with multiple genetic modifications. Moreover, the use of VSV-G pseudotyping expands the range of cells that lentiviruses can infect, enhancing the efficiency and scope of gene delivery.

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