Breakthrough in Interspecies Pig Cloning: A*STAR and NUS Lead the Way

In a groundbreaking study published today in Scientific Reports, researchers from Singapore's Agency for Science, Technology and Research (A*STAR) and the National University of Singapore (NUS) have uncovered critical insights into improving interspecies somatic cell nuclear transfer (iSCNT) for pig embryos. iSCNT, a advanced cloning technique where the nucleus from a somatic cell of one species is transferred into the enucleated oocyte of another, holds immense promise for conserving endangered species by producing viable embryos when species-specific oocytes are scarce.

The study focused on species within the Suidae family, particularly the vulnerable bearded pig (Sus barbatus), which roams Southeast Asian rainforests and is housed at Singapore's Mandai Wildlife Reserve. By examining the effects of taxonomical distance—the evolutionary divergence between donor nucleus and recipient oocyte species—and the histone deacetylase inhibitor (HDACi) Scriptaid, the team demonstrated tangible advances in embryo development rates. This collaboration exemplifies Singapore's rising stature in biotechnology higher education and research.

Understanding iSCNT: A Tool for Endangered Species Revival

Somatic cell nuclear transfer (SCNT) revolutionized cloning since Dolly the sheep in 1996, but interspecies SCNT (iSCNT) extends this to cross-species applications. In iSCNT, the process involves isolating a somatic cell nucleus from the endangered animal, injecting it into a compatible oocyte from a related species (like domestic pig), activating it chemically or electrically, and culturing the reconstructed embryo to the blastocyst stage—the key milestone for implantation potential.

Challenges include nuclear-cytoplasmic incompatibilities, epigenetic reprogramming failures, and mitochondrial mismatches, leading to low blastocyst rates (often below 10%). For pigs, vital in xenotransplantation and agriculture, iSCNT could preserve genetic diversity in wild relatives threatened by habitat loss and disease.

Scriptaid, a reversible HDACi, facilitates epigenetic remodeling by increasing histone acetylation (e.g., H3K9ac), promoting gene activation essential for totipotency restoration. Prior studies showed Scriptaid boosting porcine SCNT success by 20-50%.

The Bearded Pig: Singapore's Link to Southeast Asian Wildlife Conservation

The bearded pig, with its distinctive facial whiskers up to 15 cm and tusks reaching 25 cm, is a keystone species in Bornean and Sumatran ecosystems, aiding seed dispersal and forest regeneration through migration—the only pig known for long-distance travels between islands. Classified as Vulnerable by IUCN, populations face deforestation, hunting, and African Swine Fever threats.

At Mandai Wildlife Reserve's Rainforest Wild ASIA, bearded pigs thrive under ex-situ conservation, providing somatic cells for research. This study marks a pioneering effort to clone them using domestic pig oocytes, bridging captive breeding gaps.

Methodology: Probing Taxonomic Barriers in Suidae iSCNT

Led by Hai-Jun Liu and Yuin-Han Loh from A*STAR's Institute of Molecular and Cell Biology (IMCB) Endangered Species Conservation via Assisted Reproduction (ESCAR) Lab, with NUS and Mandai collaborators, the experiment spanned intra-species (domestic pig), inter-species (domestic vs. bearded pig), and inter-subfamily transfers.

• Somatic cells: Fibroblasts from ear biopsies of domestic and bearded pigs.
• Oocytes: Collected post-slaughter (domestic) or aspirated (bearded).
• Reconstruction: Micromanipulation, fusion with Sendai virus, activation (ionomycin + DMAP).
• Scriptaid: 500 nM for 14-16 hours post-activation.
• Outcomes: Cleavage/blastocyst rates, division kinetics, immunofluorescence for H3K9ac, RNA-seq for transcriptomics (DEGs, GO/KEGG).

Data deposited in Genome Sequence Archive (CRA028400).

Taxonomical Distance: The 'Taxonomic Wall' Exposed

As taxonomical distance widened, blastocyst formation plummeted: 27.8% intra-species, 12.4% inter-species (bearded nucleus/domestic oocyte), 0% inter-subfamily. Transcriptomics revealed mitochondrial dysfunction as culprit—downregulated nuclear-encoded NDUF genes (NADH dehydrogenase) in distant transfers impair oxidative phosphorylation, starving embryos of ATP.

"These findings suggest that taxonomical distance... is closely associated with development fate of iSCNT embryos in Suidae through regulating the expression of mitochondrion-related genes."

Scriptaid's Rescue Effect: Epigenetic Key to Success

Scriptaid dramatically rescued inter-species iSCNT: blastocyst rates rose significantly for bearded pig embryos, division accelerated (earlier 2-4 cell transitions), and H3K9ac acetylation surged early, aiding reprogramming. RNA-seq showed upregulated mitochondrially-encoded ND genes, restoring oxidative phosphorylation balance.

This partial bypass of the 'taxonomic wall' via HDAC inhibition offers a protocol tweak for broader iSCNT applications, potentially doubling success in closely related species.Read the full study

Molecular Mechanisms: Mitochondria at the Core

GO enrichments consistently highlighted mitochondrion structure/function, with KEGG top-hit oxidative phosphorylation. This pinpoints mt-nuclear uncoupling as iSCNT's Achilles heel, Scriptaid as antidote.

Implications for Wildlife Conservation and Xenotransplantation

For bearded pigs and similar Suidae, iSCNT enables banking viable embryos for reintroduction, bypassing oocyte shortages. Broader: advances pig-to-human organ cloning for xenotransplants, where porcine models dominate. Singapore positions as biotech hub, leveraging A*STAR-NUS synergies.

Stakeholders: Mandai for sourcing, conservationists for revival tools, industry for scalable cloning.

Singapore's Biotech Powerhouse: A*STAR, NUS, and Beyond

A*STAR's IMCB ESCAR Lab pioneers assisted reproduction, NUS provides academic rigor (e.g., Loh's stem cell expertise). Collaborations like Flagship Pioneering-A*STAR-NUS fuel Singapore's RIE2030 biotech push, with S$37B investment.Explore Singapore higher ed opportunities. This study underscores multidisciplinary higher education driving global solutions.

Future Directions: Scaling iSCNT for Global Impact

Next: Test in vivo implantation, gene editing (CRISPR) for disease resistance, expand to felids/primates. Challenges: Ethical oversight, funding, regulatory harmonization. Outlook: Routine endangered cloning by 2030s, Singapore leading Asia.Mandai Wildlife bearded pig page

• Optimize Scriptaid dosing/combos (e.g., TSA).
• mtDNA matching strategies.
• AI for embryo selection.

Careers in Singapore's Cutting-Edge Biotech Research

This iSCNT advance highlights booming opportunities. A*STAR/NUS seek research fellows in stem cells, cloning, regenerative med—salaries S$70K+, fellowships like NRF. Aspiring scientists: pursue PhDs at NUS, join IMCB. Browse research jobs; university jobs; academic CV tips.

Photo by Melody Ayres-Griffiths on Unsplash

Conclusion: A Leap for Science and Conservation

The A*STAR-NUS study not only demystifies iSCNT barriers but propels endangered species cloning forward. For educators, researchers, students: Singapore's ecosystem offers unparalleled platforms. Explore Rate My Professor, higher ed jobs, career advice, university jobs. Share thoughts below—how might this reshape biotech?