Recipient of the 2022 IETS Pioneer Award
In the intricate world of developmental biology, where scientists work to decipher the earliest beginnings of life, Dr. Carol L. Keefer stands out as a true pioneer.
Her groundbreaking work, spanning over four decades in clinical, industry, and academic settings, has fundamentally advanced our ability to understand and manipulate embryonic development. In recognition of her seminal contributions, Dr. Keefer was honored with the 2022 Pioneer Award from the International Embryo Technology Society (IETS)—a prestigious accolade that celebrates the earliest contributors to embryo transfer technology and industry 1 .
This award is more than just a personal honor; it is a testament to work that has pushed the boundaries of what is possible. Dr. Keefer's research has not only expanded scientific knowledge but also paved the way for practical applications in medicine and agriculture.
Celebrating the earliest contributors to embryo transfer technology and industry
To appreciate Dr. Keefer's work, it's essential to understand foundational concepts central to her research.
Often simply called "cloning," this is a technique where the nucleus of a somatic (body) cell is transferred into an egg cell that has had its own nucleus removed. The egg then reprograms the donated nucleus, and the resulting entity can develop into an embryo. This was the technique behind Dolly the sheep and was also used by Dr. Keefer to produce transgenic goats 1 .
This refers to the process of introducing an external gene (a transgene) into an organism so that the trait it codes for is expressed in the organism and its offspring. Dr. Keefer used this technology to create goats that could produce valuable proteins, like recombinant spider silk protein and human butylcholinesterase, in their milk 1 .
These are master cells that have the potential to become almost any cell type in the body. A major focus of Dr. Keefer's research at the University of Maryland has been on these cells, including work on inducing trophectoderm lineage differentiation in mouse embryonic stem cells—a crucial step in forming the placenta 1 .
This is the process of turning a specialized somatic cell back into a pluripotent state. Monitoring this complex process was a key challenge that Dr. Keefer's work helped to overcome 3 .
A prime example of Dr. Keefer's innovative research is a 2013 study focused on monitoring the reprogramming of bovine (cattle) cells.
For years, a major hurdle in creating stem cells from farm animals was the inability to easily identify when a cell had been successfully reprogrammed to a pluripotent state. Dr. Keefer and her team devised an elegant solution: a glowing reporter system that would light up when a key pluripotency gene was activated 3 .
The scientists started by identifying the promoter region of the bovine NANOG gene, a critical transcription factor that is essential for maintaining pluripotency in stem cells. They fused this promoter to a gene that codes for a Nuclear Localized Enhanced Green Fluorescent Protein (EGFP) 3 .
To ensure the bovine NANOG promoter would function correctly, they first tested it in mouse embryonic stem cells. Once confirmed, the construct was introduced into bovine fetal fibroblasts (bFFs)—common connective tissue cells from cow fetuses 3 .
The team then attempted to reprogram these transgenic bFFs into induced pluripotent stem cells (iPSCs) using two different methods:
The researchers then carefully monitored the cells and embryos. Successful activation of the NANOG gene would be signaled by the nucleus of the cell glowing green under a fluorescent microscope, indicating that the cell had become pluripotent 3 .
The experiment was a resounding success, providing a powerful new tool for the field.
The NANOG-EGFP reporter lit up in a stage- and location-appropriate manner in SCNT-derived embryos, confirming it accurately reflected the natural activity of the NANOG gene 3 .
Following the 10-day genetic reprogramming protocol, the bovine cells not only expressed the green fluorescent protein but also showed other markers of pluripotency, such as alkaline phosphatase. This confirmed that true iPSCs had been generated 3 .
The study demonstrated that this bovine NANOG reporter system could be reliably used to monitor nuclear reprogramming, distinguish cell allocation in cloned embryos, and aid in the establishment of validated stem cell lines for livestock 3 .
| Reprogramming Method | NANOG-EGFP Expression | Additional Pluripotency Markers | Conclusion |
|---|---|---|---|
| Cell Fusion (with NTERA2) | Yes, nuclear | Not specified | Demonced reprogramming capability |
| Genetic Reprogramming (4 factors) | Yes | Alkaline Phosphatase | Successful creation of bovine iPSCs |
| Somatic Cell Nuclear Transfer (SCNT) | Yes, stage-specific | Not specified | Validated reporter for embryonic development |
Dr. Keefer's work, and the field of embryo technology as a whole, relies on a suite of specialized research reagents.
| Reagent | Common Source | Function in Research |
|---|---|---|
| Fetal Bovine Serum (FBS) | Unborn calf fetuses | A complex mix of hormones, growth factors, and proteins added to culture media to support cell growth and proliferation . |
| Collagen | Rat tails, cow skin | Used to coat culture dishes to provide a natural surface (2D) or create a 3D matrix that helps cells attach and grow, mimicking the extracellular environment . |
| Matrigel™ | Mouse sarcomas | A hydrogel rich in extracellular matrix components; widely used to support the growth and differentiation of stem cells and for 3D cell culture . |
| Gelatin | Porcine skin & bones | Provides a coating on the surface of culture dishes to help cells adhere and grow . |
| Litmus Amebocyte Lysate (LAL) | Horseshoe crab blood | Used in a critical assay to screen for bacterial endotoxin contamination in reagents, ensuring they are safe for use with cells . |
These reagents are fundamental to maintaining cell cultures and conducting experiments in developmental biology. Without them, much of the progress in understanding embryonic development would not be possible.
The use of animal-derived reagents raises important ethical questions that researchers like Dr. Keefer must navigate. The field is increasingly moving toward synthetic alternatives where possible.
Dr. Carol Keefer's career is a powerful narrative of scientific curiosity coupled with practical application.
Her contributions have left an indelible mark on multiple fields. Her early work in microinjecting dead sperm to rescue genetics paved the way for advanced fertility treatments 1 . Her foray into industry led to the creation of "biofactory" goats that could produce valuable proteins, demonstrating the commercial potential of transgenic technology 1 .
Furthermore, her leadership extends beyond the lab. She served as the first female president of the IETS in 2003 and was selected as an external reviewer for the FDA's Risk Assessment of Animal Cloning, showcasing the high esteem in which she is held by the global scientific community 1 .
| Phase | Institution/Role | Key Achievement |
|---|---|---|
| Early Research & Academia | Johns Hopkins, University of Pennsylvania, University of Georgia | Contributed to successful rat cloning; discovered viable embryos could be obtained from microinjected dead sperm 1 . |
| Industry Application | American Breeder's Service, Nexia Biotechnologies | Made discoveries in embryo cloning; produced transgenic goats secreting spider silk protein in milk 1 . |
| Academic Leadership | University of Maryland (Professor) | Researched pluripotent stem cells; described induction of trophectoderm differentiation in mouse stem cells 1 . |
| Professional Service | IETS President, FDA Reviewer | First female IETS president (2003); provided expert risk assessment on animal cloning for the FDA 1 . |
Through her mentorship at the University of Maryland and her ongoing research into stem cells and reproductive technology, Dr. Keefer continues to shape the next generation of scientists.
Her receipt of the 2022 IETS Pioneer Award is a fitting recognition of a lifetime spent unlocking the secrets of life at its very earliest stages, turning what was once science fiction into tangible science that benefits us all 1 .