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Mice born of two dads reveal hidden details of mammalian reproduction

Researchers at Shanghai Jiao Tong University have produced fertile adult mice from embryos constructed entirely with male genetic material. Mice carrying only paternal DNA developed into fertile adults following precision editing of seven imprinting control regions, revealing genomic imprinting as a core barrier to uniparental mammalian development.

Each mammalian embryo begins with the fusion of a maternal oocyte and a paternal sperm, creating a diploid zygote carrying one genome from each parent. Attempts to bypass this reproductive symmetry by generating embryos with only maternal or paternal genomes have repeatedly failed to produce viable offspring without extensive genetic or epigenetic intervention.

Although some success has been achieved in mice with two maternal genomes, previous attempts using only paternal DNA produced embryos that rarely survived past early gestation. Early work using haploid embryonic stem cells enabled partial progress, but the resulting mice survived only briefly after birth.

These failures have pointed to genomic imprinting, a mechanism of parent-of-origin–specific gene expression through epigenetic marks, as a probable developmental gatekeeper. The precise epigenetic signatures required to override this barrier have remained unresolved.

In the study, "Fertile androgenetic mice generated by targeted epigenetic editing of imprinting control regions," in the Proceedings of the National Academy of Sciences, researchers engineered androgenetic embryos to correct specific epigenetic marks using CRISPR-based editing, aiming to overcome developmental arrest associated with paternal-only genomes.

Researchers reconstructed 587 diploid androgenetic embryos by injecting pairs of sperm heads from two mice into enucleated oocytes, resulting in 277 blastocysts. These were then transferred into 18 pseudopregnant mice across multiple implantation experiments. Allele-specific targeting was accomplished by designing guide RNAs to recognize single-nucleotide polymorphisms between the parental strains.

Among 259 implanted blastocysts, three pups were born alive. Two survived to adulthood and displayed normal size and behavior, and fathered healthy offspring. Methylation analysis confirmed correction across all seven targeted imprinting control regions (ICRs) in the surviving adults. A third pup, which died shortly after birth, exhibited hypomethylation at the Igf2r ICR, consistent with known lethality patterns.

Restoration of imprinting patterns led to improved expression levels for most imprinted genes associated with the targeted ICRs, based on quantitative PCR in E13.5 embryos. Allele-specific expression analysis showed monoallelic expression for some imprinted genes in only a subset of embryos, suggesting that successful coediting of all ICRs remains infrequent.

Survival to late gestation correlated with more complete methylation remodeling. Of four E16.5 androgenetic fetuses recovered in a subsequent experiment, two displayed fully corrected ICRs, while the others had two aberrantly methylated regions each. No viable fetuses were found among embryos edited with non-targeting control gRNAs.

The ability to derive live, fertile mice from two paternal genomes opens new avenues in reproductive biology and developmental genetics. The low success rate hints at potential unknown imprinting elements and more research is needed to fully map the relevant regions.

The authors conclude that genomic imprinting represents a critical developmental checkpoint that can be bypassed through precise epigenetic engineering.