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ATC Workshop Papers
From Cell to Production
Technical Challenges of Cloning Pigs for BioMedical
Research
SATACs and Transgenesis
Concerns About Gene Transfer and Nuclear Transfer
in Domestic Animals
Prospects and Hurdles in Optimizing the Vascular
Support of Engineered Tissues
ES Cells Make Neurons in a Dish
Nuclear Transfer and Gene Targeting in Domestic
Animals: Bioreactors of the Future
Application of Nuclear Transfer Technology
in the Generation of Pigs for Xenetransplantation
Genomics: Delivering Cell Culture Systems
for Tissue Therapy
Nuclear Transfer Technology
Gene Targeting in Domestic Species: Challenges
and Opportunities
Homologous Recombination and Genetic Engineering
of Transgenic Recombinant Animals
Nuclear Transplantation in the Cow: Future
Challenges
Enhancing Transgenics through Cloning
ES Cells Offer is a Power Tool for Understanding
the Genetic Control of Tissue Development and for Screening Potential
Therapeutic Drugs
Human Germline Engineering -- The Prospects
for Commercial Development
Mammalian Artificial Chromosomes for Animal
Transgenesis
Understanding Developmental Abnormalities
in Offspring Produced by Nuclear Transplantation
Role of Cell Cycle
Cloning and Other Reproductive Technologies
for Application in Transgenics
Cell Culturing Technology as a Major Hurdle
in the Commercialization of Genetically Altered Animals
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| ADVANCED TRANSGENESIS AND
CLONING: Genetic Manipulation in Animals
Electronic Workshop Presentation: Paper
No. 03
SOMATIC CELL NUCLEAR TRANSFER IN
MAMMALS
Participant:
Alan Colman
PPL Therapeutics, Inc.
Introduction
Cloning of multicellular animals and plants has been practiced for
tens (animals) to hundreds (plants) of years, although until recently
this always involved the propagation of groups of cells rather than
individual ones. Cloning using single somatic cells has only occurred
in the present century. So far, it is only in the plant kingdom that
success has been obtained with intact cells. In animals, success has
been obtained by transfering the nucleus from one somatic cell to
the cytoplasm of an enucleated egg—nuclear transplantation. This
difference between plant and animal cells has been widely thought
to reflect a more entrenched differentiated status in animal cells.
Whilst the molecular basis of this distinction is rather vague, there
is no disagreement that the interdependence of nucleus and cytoplasm
is so strong and self maintaining that it has proved almost impossible
to re-specify the differentiated status of a somatic cell. Whilst
it is now eminently possible to partially reprogramme the nucleus
of a somatic cell by the introduction of genes for a variety of transcription
factors, global reprogramming can only be either achieved or, more
importantly, demonstrated, by exposing the nucleus to a germ cell
cytoplasm. Dolly signifies the most spectacular demonstration of the
scope of this reprogramming since she is living proof that an adult
somatic cell retains the ability to be reprogrammed in the numerous
directions manifested by all the different cell types that comprise
a mammal. This work and the subsequent confirmation in cows and mice
presage a host of potential benefits for both biomedical research
and commercial applications in agriculture and medicine. I list below
what I see as the benefits and challenges in PPL Therapeutics' business.
The Benefits
My company, PPL Therapeutics (Scotland, UK; Blacksburg, USA; and New
Zealand) specialises in the production of therapeutic proteins in
the milk of transgenic livestock and genetically modified pig organs
for xenotransplantation. Because of its collaborative role with the
Roslin Institute in the Dolly project, PPL was the first company to
be able to broadcast the breadth of potential benefits that the nuclear
transfer techniques could bestow to our type of business. These are:
- When a transfected cell line is used as the nuclear donor, all
animals will be transgenic. Currently success rates in making
transgenic livestock are lower than 10% using DNA microinjection
of fertilised livestock embryos.
- It is possible that the best protein expression levels will
be obtained when multiple copies of a transgene integrate into
a particularly favourable chromosome locus. Gene targeting methods
may allow preselection of the ideal locus; e.g., a milk gene locus
in our case, but large (i.e., >2) multigene inserts may be
very difficult. An alternative strategy will be to obtain large
numbers of randomly transfected cell lines (e.g., fibroblasts)
and then attempt to activate the target gene in sub populations
of each line by addition of specific factors. This should allow
selection of the most productive cell line for conversion into
highly productive animals
- The ability to produce numbers of genetically identical transgenic
livestock animals in the first generation will greatly reduce
timelines to get therapeutic products to the market
- Nuclear transfer should facilitate a route to gene targeting
in livestock. The targeting could be used to remove/inactivate
unwanted genes (e.g., prion gene in sheep and cows), or alpha
galactosidase in pigs for xenotransplantation), or to add genes
to, or modify genes at specific loci
Nuclear transfer applications in areas such as agriculture, disease
models, and stem cell therapy will be covered by other contributors
but I would like to comment on the last category here, stem cell
therapy: a prevailing idea here is that adult human cells can be
converted into stem cell precursors by "passaging" via
nuclear transfer, through an enucleated egg (not necessarily a human
one). I would like to think that the plasticity in nuclear differentiation
revealed or unmasked in the Dolly experiments may ultimately be
further exploited to convert one adult cell type to another directly.
To even start on this quest, we need to understand the cellular
and molecular basis behind the recent successes. This brings me
to the challenges.
The Challenges
- The Dolly series of experiments and the earlier, pioneering
ones which produced the world’s first sheep using cultured
cells, used serum starved cells of which the majority were in
G0. G0 cells had not knowingly been used before so it is surely
no coincidence that this unexpected success should be attributed
to this step. This issue remains controversial, but it is uncontestable
that we need to know more about how to recognise the exact cells
which work since all the current nuclear transfer procedures are
inefficient. Having identified the desired cell class, we will
then need to understand how to optimise its formation and then
analyse its molecular and cellular signature. In order to do this,
it would be useful to have in cell populations, markers which
give a real time (and innocuous) read out of the cell cycle status
of the individual cells which then could be directly used for
nuclear transfer. The commercial goal of improved efficiency would
be achieved even if recognition alone were possible.
- Currently most people are using fibroblasts as the nuclear donors.
What about other cell types? Are they more efficient? The data
from the mouse experiments suggest that not all cells will work.
What are the rules?
- Gene targeting. How do we accomplish this? The fibroblast will
probably not show the homologous recombination frequency shown
by murine embryonic cell lines.
- There is an assumption that if a gene can be targeted to a
specific and active locus, then it too will be active. Will this
be true since we already know that the tandem repeats present
in many transgenic lines get silenced due to neighboring chromatin
effects?
Conclusion
Somatic cell nuclear transfer in mammals works. Further technology
refinement and improved understanding of the process are essential,
if the promise of nuclear transfer for commercial and basic research
applications is to be fulfilled.
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