THE POWER OF NATURE - CLASSICAL AND CONTEMPORARY OVERVIEW
Nuclear Potency Experimental Series: Nuclear Transfer
Developmental Biology is a multidisciplinary field of scholarship that encompasses as disparate endeavors as molecular biology, genetics, cell biology, biochemistry, biophysics, embryology and reproductive biology. It is, indeed, the holy grail of molecular biology; it facilitates the study of genetic networks in their fullest complexity. There are, however, a series of notable, landmark experiments that tend to punctuate the history of developmental biology. These experiments may be referred to as nuclear potency series as they all quite astutely, yet elegantly, highlight the role of nuclear potential, attendant genetic code and program in directing development. They also elucidate and unveil the repertoire of applications that may be tenable; they constitute a crossroads of medicine, engineering and ecology as will be shown.
The nuclear potency series, essentially manifested as nuclear transfer, has been employed with immense effect in model theory through an array of ever smart vectors, regulatory and structural genomic elements – particularly agile molecular tools - introduced into the genome of embryonic stem cells. Such systems as the Cre Lox for site-specific, temporal or conditional gene switch-off or knock-out, Flip-In for site-specific transgene integration; Promoter/Gene Trapping for Promoter/GeneTagging, discovery or identification, or the Zinc Finger Nuclease for gene editing, including foreign gene integration or knockout, including yet undiscovered systems, are fitting examples of smart, molecular, regulatory tools for characterization of gene architecture and utility – structure and function. Animal models are created, employing genetic engineering or stem cell engineering: the design as well as construction of such vector, regulatory and structural elements toward gene function assessment. The series has been suitably applied toward gene discovery using such molecular, tagging tools as promoter trapping and gene trapping. Gene Knockout technology, for example, has been invaluable in loss of gene function studies, including the elucidation of the role of tumor suppressor genes in cancer. Transgenes have similarly been spliced into nuclei of stem cells for careful spatial and temporal assessment of gain of gene function during development utilizing Gene Knockin technology.
The overall implication of such series of experiments may be far-reaching. One is, for example, able to discover new genes and create animal models toward the study of gene function, the modeling of basic gene function; create the versatile-cum-smart stem cells as well as attendant transgenic pharmaceuticals toward gene therapy, including proteins and metabolites for immune system dysfunction - production of transgenic antibodies for containment of pathogens. This may be deployed as pathogen-binding antibodies, complementing viral RNA binding therapy where this may be limiting during administration; creation of transgenic antigens as transgenic vaccines; transgenic hearts and tissue for heart disease; transgenic insulin or pancreas for diabetes; transgenic neuronal replacement in neuroscience and medicine - learning and memory; similarly, Parkinson’s or Alzheimer’s disease remedy. The nuclear potency series naturally entails the study and therapy of cancer or kidney disease.
Stem cells may be employed variously for reproductive cloning as well as therapeutic cloning. Stem cells may, as a result, be customized as specialized cells toward an array of approaches toward Regenerative Medicine with oocyte enucleation and nuclear transfer technology; utilizing essentially the primary totipotent stem cell (blastomere) carrying totipotent genes; pluripotent stem cell, derived from the blastocyst’s inner cell mass, ICM; adult somatic cell for genetic reprogramming, the creation of iPS, induced pluripotent stem cell [in fact, Somatic Resurrection]. Such cells may be similarly transferred to tetraploid blastocysts for self-creation of protein, cells, tissues, organs; whole organisms. Nuclear transfer, palpably, serves as an ingenious, versatile, multipurpose platform forembryo microsurgery and embryo reconstitution as genetic engineering. Dolly was, in fact, created using the induction of adult somatic cells as totipotent stem cells using nuclear transfer. Nuclear Transfer is, as a result, applied toward organogenesis and generation or resurrection of entire organisms - a naturally fitting application - under a socially sanctioned climate, with respect to law, polity and ethics. It actualizes gene therapy, organ transplantation or endangered species recovery. All the foregoing technologies also serve as ex situ gene preservation and evaluation - insurance against environmental collapse of a healthy, living planet.
Nuclear Transfer may be employed variously, contingent on design and goals, with (i) the naked, enucleated nucleus drawn from the zygote cell or (ii) the intact cell containing the nucleus using the embryonic stem cell - blastomere or embryonic stem cell from the inner cell mass as well as the sperm cell nucleus, for example. Transfer of the nucleus may be attained physically, chemically, biologically, or as a combination of the foregoing approaches. Choice procedures may be utilized, therefore, such as very fine micro-pipetting embryo microsurgery and reconstitution: zona evacuation and enucleation as well as nuclear transplantation; electroporation or electrofusion with an electrical field; laser assisted sperm cell or ES cell transfer into oocytes, morulae, or blastocysts; biochemical nuclear transfer such in vitro fertilization (IVF) as Assisted Reproductive Technology, ART. Organs such as heart, kidney, liver, or pancreas may be created in transgenic or genetically engineered animals or in the petri dish, to order, regenerated in situ with pertinent molecular agents. In the foreseeable future, if nuclear potency experiments are sustained, within an enabling legal and ethical framework, heart replacement, may become quite feasible, overriding an overall organ transplantation dilemma. The series may ultimately be useful in the field of conservation biology and ecology for endangered species recovery such as panda, bengal tiger, cheetah, the American mountain lion or rhino. All this effort should serve to realize urgent objectives toward ecosystem restoration, integrity and sustainability.
One of the main issues that evolved with transgenic technology was that it was largely limited by the random and non-specific integration or deletion of transgenes, alleles, or any sequence elements under study in the genome they were introduced.
Random gene introduction into genome implied that one particular transgene or allele deletion would result in different phenenotypes, making data inference such a headache, let alone problematic. This was true for both pronuclear and ES cell technologies.
This has, however, largely been solved using embryonic stem cells (ES Cells) with the advent of targeted ES cell technology while pronuclear micronjection has remained an untargeted, random approach for transgene introduction: potentially resulting in variable phenotypes for one particular transgene or DNA construct, much less inherent mosaicism arising whenever transgene integration occurs after the first zygotic division for pronuclear technology. There is currently, though, a much needed add-on to the range of transgenic technologies. This is Zinc Finger technology which would precisely target the transgene or sequence elements to a specific address along a stretch of DNA for both pronuclear or ES cells in (knock-in) mice. This should, in turn, serve to make transgene introduction more efficient using ES cells over pronuclear transgene introduction because of the following observation. One only needs to utilize a mere 20 blastocysts to generate an average of 2 transgenic pups with the targeted transgene as compared to 120 one -cell eggs in order to generate an essentially equal number of transgenic pups. This tends to save a significant amount of resources for investigators who would design their experiments accordingly, principally in times of shoe-string budgets: time, money, material, much less human in-put.
Granted, it should also be noted that Zinc Finger should be, overall, useful for targeted transgenesis for both pronuclear and ES cell technologies.
The evolution of developmental biology and nuclear potency series evolved as a result of a compendium of experiments and attendant data emanating primarily from the late 19th century Germany. This gave rise to a cascade of landmark experiments whose choice dateline is illustrated here; the list is far from exhaustive, of course.
i. Wilhelm Roux founded Entwicklungsmechanik - Developmental Mechanics. The "Father of Experimental Embryology" is, accordingly, an apt allusion for this endeavor.
Roux conducted the original landmark experiment in developmental biology in 1888 with frog eggs. He pioneered embryo microsurgery that has since evolved to modern transgenic technology. Roux's experiment was ingenious as it envisioned the fate of embryonic development by annulling the structure of one frog blastomere at the 2-cell stage. He cleverly utilized a tiny hot glass solid needle for this purpose. He noted that the remaining blastomere merely resulted in a half-embryo. Although Roux's interpretation of the experiment proved to be a function of artifact, it did open the door to a new age of experimental biology.
ii. Hans Driesch - Hans Driesch elegantly, yet quite carefully, redesigned Roux's experiment in order to elucidate the role of nuclear potency in development. He also was able to interpret the experimental data equally thoroughly. He separated early-stage sea urchin blastomeres, shaking the embryo vigorously during the procedure, at the 2-cell and 4-cell developmental stages. Each separated primary embryonic stem cell, the blastomere, much to his surprise, developed as a normal sea urchin. The earth was not flat! Hans Driesch termed this observation nuclear totipotency. One's all!
iii. Hans Spemann - Hans Spemann carried out equally elegant embryonic induction experiments using embryo microsurgery. Undeniably, this fine procedure is intimately associated with Hans Spemann. He observed and noted embryonic organizing regions that result in self-organization or self-differentiation of the embryo during development. In 1902, Hans Spemann attempted to settle the dichotomy of results between Roux's and Hans Driesch's landmark experiments. He replicated Hans Driesch's experiment with salamander eggs as they were easier to handle. He used a fine strand of his baby's [Margarett's] hair as a microsurgical tool to separate the 2-cell stage embryo into single blastomeres. Each separated primary embryonic stem cell, the blastomere, developed as a normal salamander, demonstrating the blastomere's nuclear totipotency. He further carried out an equally stunning experiment, the original nuclear transfer experiment using a fine strand of his baby's hair again in 1928. He was able to ligate a salamander embryo at the 1-cell stage into two halves; one half did not have any nucleus while the other had one nucleus. Only the nucleated half developed through a 16-cell embryo. He then carried a subsequent experiment; this time he loosened and untied the hair ligature, allowing one nucleus of the 16-cell embryo to travel to the other half of the embryo. He then ligated the embryo into two again, resulting in one side having one nucleus and the other side many nuclei, ie, multinucleated. The sun did not go around the earth! Each side developed into a normal salamander.
iv. Briggs and King - In 1952, in Philadelphia, USA, Briggs and King conducted the first successful egg evacuation and nuclear transplantation experiment using the leopard frog, Rana Pipiens. Briggs and King noted that nuclei from undifferentiated embryos had more potency for self-directing normal development than differentiated nuclei. Several leopard frogs had just been cloned; indeed, created using the very fine procedure of embryo microsurgery.
v. Tarkowski - In 1961, Tarwoski carried out the first mammalian nuclear transfer experiment in Wales, the UK. This effort resulted in a mouse chimaera, to usher in a new era of investigation into cell fate and gene function during development with mammalian stem cells.
vi. Capecchi et al - In 1989, Capecchi collaborated with colleagues in the UK and US to develop mouse embryonic stem cell gene-targeted technology. This is a variant of a prior, random gene knock-out technology. Targeted technology ascertains non-random loss of gene function. It may also be modified for gain of gene function. This is a powerful and relatively fast tool in the creation of genetically engineered animal models employed for overall gene function evaluation.
vii. Wilmut et al: Of Black Face, Ewe, & Lamb: In 1999 at Roslin Institute, University Edinburgh, Scotland, Wilmut and colleagues stunned the world with the announcement of Dolly's birth. Dolly was the first mammal created with nuclear transfer technology, employing adult stem cells: Hans Spemman's conception of the ultimate "Fantastic Experiment" which was technically untenable in his era. Ian Wilmut and colleagues cleverly employed electrofusion: fusion of the nucleus and the evacuated embryo at the 1-cell stage whose nucleus had been microsurgically removed. A version of this experiment utilises electrofusion of diploid 2-cell mouse embryos in order to create tetraploid embryos. The introduction of (i) mouse blastomeres into enucleated zona of 1-cell embryos or (ii) embryonic stem cells at the tetraploid blastocyst stage result in viable, normal embryos or offspring that are entirely derived from nuclei of transferred primary embryonic stem cells, blastomeres or the blastocyst's Inner Cell Mass embryonic stem cells as practiced by the author of this article toward research and development of ES Cell technology; recognizing reproducibility of technology as the principal premise for assessment of technology development and success. The experiments were carried out in collaboration with, at least, 30 laboratories at Tufts University School of Medicine and Veterinary Medicine, Harvard Medical School and MIT since 1985 through 2010. This family of experiments also undescores the strategic importance of employing efficiency of experimental design, mathematical models and optimization of transgenic technology as research concepts, methods and tools: selection of research paradigms.
The nucleus used for fusion in the creation of Dolly was derived from an adult stem cell whose genes had been reprogrammed to self-sustain normal development. Adult stem cells may, therefore, be used with this technology upon proper induction of totipotency through introduction of totipotent genes or their epigenetic activation or modulation. The procedure creates iPS, induced pluripotent somatic cells whose function is similar to embryonic stem cells. In a way, this was the culmination of a brilliant series of nuclear potency experiments, pioneered by Roux, Hans Driesch as well as Hans Spemman in the late 19th century. This was also a landmark experiment that was variously referred to by mass media as the equivalence of the human moon landing. In truly practical, tangible terms, the experiment represented a huge leap to a new era of developmental biology. It would strategically serve to expedite the study of gene function and attendant application toward drug discovery; cell, tissue or organ generation. It compliments a suite of sustainable technologies that tend to actualize the equally nascent, scholarly discipline of planetary medicine: endangered species recovery, ecosystem preservation and attendant health and integrity of the ecosphere – comprised of the cardinal life-support systems network: atmosphere, hydrosphere, geosphere and biosphere. This effort would inevitably result in Sustainability, the temporal and spatial integrity of nature and human civilization premised on nature’s own self-sustaining and self-regulating energy networks of the sublime, sustainable universe.
Humankind has had another moon landing!
Author: Nom de plume: Sam Chire
Regular name: John Mkandawire