In a groundbreaking experiment, researchers in China have successfully created mice with DNA from two fathers—marking a major step in genetic science and our understanding of a curious biological phenomenon called imprinting. This achievement, led by Zhi-Kun Li and his team at the Chinese Academy of Sciences in Beijing, utilized CRISPR gene-editing technology to bypass the usual genetic limitations of having one father and one mother. While this approach holds promise for advancing the study of imprinting, humans are not yet the focus of this research.

Imprinting refers to a genetic phenomenon where certain genes are expressed differently depending on whether they come from the mother or the father. For healthy development, animals need to inherit a “dose” of these genes from both parents, and both doses must work together. Without the proper balance, the expression of these genes can go awry, leading to abnormal embryos. In the case of creating mice with two fathers, previous experiments failed because both the paternal and maternal genomes contribute to proper gene expression, making the development of a healthy embryo difficult.

Unlike earlier attempts that involved injecting sperm DNA into egg cells, which failed to produce healthy mice, Li and his team took a different approach. They focused on the imprinted genes and used CRISPR to “knock out” these genes in order to remove the traditional limitations that come from the need for both maternal and paternal gene contributions. The team targeted 20 specific genes that are critical for embryo development, out of the 200 or so imprinted genes known in mice.

The experimental process was incredibly complex. The researchers first cultured sperm DNA in the lab to collect stem cells, then used CRISPR to edit the imprinted genes. These edited cells were then combined with sperm from another male and injected into egg cells that had been stripped of their own DNA. The resulting embryos contained DNA from two males and were then implanted into the wombs of female mice, using a special “embryo shell” to provide the necessary cells for placenta formation.

Incredibly, some of the embryos developed into live pups that survived to adulthood. This is the first time such an experiment has yielded viable offspring. However, the success was not without its challenges. Out of 164 embryos transferred, only seven live pups were born, and these pups were not entirely normal. They grew larger than typical mice, with enlarged organs, and they had shorter lifespans. Additionally, they were infertile.

“This is exciting,” says Kotaro Sasaki, a developmental biologist at the University of Pennsylvania who was not involved in the research. “The team has avoided imprinting defects and found a new way to create mice with two fathers, which builds upon earlier work by another research group.” In 2021, a team led by Katsuhiko Hayashi at Osaka University in Japan succeeded in using cells from adult male mice to create immature egg cells, which were fertilized with sperm to generate bi-paternal embryos. These mice grew to adulthood and were able to reproduce.

Li’s team, however, took a more complicated route to create their bi-paternal embryos, and the success rate was much lower. Despite the challenges, the results represent a major advancement in genetic manipulation and provide a deeper understanding of imprinting.

While this research offers intriguing insights, there are major ethical and technical hurdles that make it unfeasible for human applications—at least for the time being. “Editing 20 imprinted genes in humans would be unacceptable, as it could lead to individuals who are not healthy or viable,” says Li. Sasaki agrees, noting that many of the lab techniques used in the experiment are not yet established for human cells, and the potential consequences of such gene edits in humans could be catastrophic.

The research also raises questions about the role of paternal and maternal genes in development. Previous studies have shown that mice with two mothers tend to be smaller and live longer, while those with two fathers are overgrown and die more quickly. This suggests that paternal genes might promote growth, while maternal genes may limit it. These findings could offer crucial insights into how imprinted genes influence physical development and lifespan.

While human applications of this technology remain far off, the work of Li and his team adds a valuable piece to the puzzle of genetic imprinting. The experiment provides compelling evidence that paternal and maternal imprinted genes play distinct roles in growth regulation. As researchers continue to explore the complexities of imprinting, the findings may ultimately lead to new strategies for addressing genetic disorders and improving reproductive technologies.

For now, though, creating mice with two fathers is a milestone—one that helps to reveal the deep mysteries of gene expression, but also highlights the technical and ethical barriers that still exist in the world of genetic manipulation.

By Impact Lab