Development of nuclear transfer (cloning) technology in avian species
The first successful attempt of mammalian somatic cell Nuclear Transfer (NT)
came with the birth of two cloned lambs derived from cultured embryonic cells
(Campbell, et al. Nature 380: 64-6, 1996). Later, it was shown that a
differentiated somatic nucleus derived from an adult animal could be used to
produce a clone when fused to an enucleated oocyte, e.g., "Dolly" (Wilmut, et al.
Nature 385: 810-3, 1997). Following this pioneering work, the technology has
been applied successfully in a number of species. Transgenic sheep (Schnieke,
et al. Science 278: 2130-3, 1997) and cattle (Cibelli, et al. Science 280: 1256-8,
1998) have been produced using embryonic fibroblast cells that have been
genetically modified in culture. Several generations of mouse clones have been
produced from an adult female (Wakayama, et al. Nature 394: 369-74, 1998) and
more recently two cloned lambs were produced containing targeted genetic
modifications.
In this technology, the early embryo is reconstructed employing a somatic cell
nucleus (nuclear donor) injected into an enucleated egg (recipient cytoplast, also
described as cytoplast donor). To achieve normal development, the mitotic donor
nucleus must be reprogrammed by elements in the recipient cytoplast and
resume cell division.
To date, avian NT has not been possible due the technical difficulties associated
with accessing, visualizing and manipulating the early avian oocyte. Once the
required technological breakthroughs are accomplished, this technology has the
potential to add billions of dollars of incremental value to the food poultry and
biopharmaceuticals industries. For instance, while the U.S. poultry industry has
made significant advances with traditional breeding practices, it is expected that
NT will provide a powerful tool for breeders and growers. Birds with the very best
naturally occurring combination of agronomic traits can be cloned and
subsequently bred to produce flocks of ‘elite’ birds. Thus, the traditional breeding
schemes can be enhanced to result in a steep change in the value of the
industry. The production of biopharmaceuticals in chickens is an application of
NT with an impact equal to that in the poultry industry. Several companies have
taken cloning and combined it with transgenic methods to take advantage of the
powerful protein production capacity of farm animals, such as goats, sheep, and
cows. There are now several new and promising biopharmaceuticals, produced
from the milk of transgenic mammals, in the FDA approval process. Transgenic
poultry provides a cost effective and more rapid method than traditional
mammalian systems.
In this presentation, we will discuss our efforts to develop nuclear transfer
technology in avian species. We have divided the process into four separate
steps: visualization of nuclear structures within the early egg, laser-mediated
ablation of these structures, isolation and microinjection of a somatic cell nucleus
into an enucleated cytoplast and hatching of the reconstructed zygote.
1. Visualization of pronuclear structures within the egg
Enucleation of the fertilized egg requires rapid and reliable visualization of its
pronuclear structures. We have developed proprietary technology using laser
microscopy to visualize the early avian egg, which represents a novel approach
to resolve the inherent problems associated with the micromanipulation of the
avian egg. The major advantage of this method lies in its demonstrated ability to
generate images of living and optically dense structures for prolonged periods of
time, while not affecting their viability. This compatibility is a result of utilizing
biologically innocuous wavelengths of light that are able to penetrate much
deeper into scattering specimens. Hence, this method provides us with an
unprecedented capability for producing noninvasive, three-dimensional, real-time
images of the optically dense avian egg.
2. Laser-mediated enucleation of the egg
To remove the egg’s original DNA material, we have employed the precision of
lasers to selectively ablate the egg’s nuclear structures in order to create a
cytoplast donor.
3. Isolation and microinjection of a somatic cell nucleus
We have developed a microinjection unit to image somatic cells in suspension
and to isolate the nuclei by mechanical disruption of the cell’s cytoplasmic
membrane. After isolation of the nuclei, an injection pipette is loaded with the
isolated nucleus, which is injected into the germinal disk using a Piezo drill
system.
4. Hatching of the reconstructed zygote
In the last year, we have developed an effective protocol whereby the avian
ovum is surgically transferred into the oviduct of the recipient hen. Once
transferred, the embryo develops inside the recipient hen and travels through the
oviduct, where natural egg white proteins and a natural eggshell encapsulate it.
The transferred egg is laid next day and can then be incubated and hatched like
a normal chick.
We have shown proof of concept of each of the four steps described above. Our
current efforts are focused on optimizing each of the steps, as well as integrating
them into a reliable technology to produce the world’s first cloned bird.
50 _mAnnual National Breeders Roundtable
St. Louis, Missouri
May 3 - 4, 2001
Speaker: Dr. Leandro Christmann
Question 1
From: Dr. Jim Arthur
For cloning to be used in producing breeders, it must be cost effective. ARer the cloning technology
fully developed what is your estimated cost to produce a single cloned chick?
is
Response: Given that cloning methodology is still under development it is difficult to estimate the cost to
produce a single bird. However, as the technology develops and becomes an effective and routine
procedure, I believe that a team of five people could potentiality produce dozens of cloned birds every
week.
Question 2
From: David Harry
There are recent reports of developmental problems, premature aging, etc. in mammals. Are these
epigenetic or genetic problems? If genetic (and hence transmissible), it would create problems for
breeders.
Response: We are just beginning to comprehend the complexities underlying the developmental biology of
cloned animals. During next few years, we hope to better understand these mechanisms and devise
strategies to improve cloning efficiencies. Certainly some of the developmental problems associated with
cloning can be assigned to genetic causes. One example of ways to avoid these problems is routine
karyotyping of cells to be used as nuclear donors.
Question 3
From: Dr. Guy Barbado
Have you considered surgically implanting a 50mL conical centrifuge tube to collect (or insert) follicles
into the oviduct?
Response: There are a number of techniques to fistulate the hen's oviduct to be able to both collect and
transfer ova. We tried a number of these methodologies and found that fistulation of the hens oviduct was
benefcial for collection of ova. However, reinsertion of the ova was less successful. Given the high rates of
development of ova following our regular surgical transfer procedure described earlier we opted for using
this as a standard technology.
Question 4
From: Nico Buddiger
Is cloning going to help primary breeders improve their pure lines or is it more a tool to help us speed up
our gene flow towards our customers? (Cloning is reducing the variance and that is not our goal within our
pure lines)
i
Response:We are tryingto understandwhich kindof applicationwould be of greatervalue to the poultry
industryandwe are confident that the marketwill define this poinL Ourprimarygoal inthis meeting is to
informthe breedersof new technologies anddetermine their best potentialuse.
Question5
From:Dr. Douglas Rhoads
Yournuclearpreparationsrequire some cell culturing. Do you isolate particularcell types? Are you
relying on productionof stem cells?
Response:We are currentlyutilizingfetal fibroblasts,but a numberof differentcell types have been
successfullyused to producecloned mammals.I see no reason to believe that it would be differentin avian
species and anticipate selection of cells type will dependon the application.Forinstance,we may find
fibroblastcells are adequatefor cloning and for the productionof geneticallymodified birds through
randomintegrationofmmsgenes. Onthe other hand, embryonicstem or embryonicgerm cell lines may be
requiredif we do moresophisticatedgenetic manipulationsuch as gene targeting.
Question6
From:JimPetitte
With re-nucleationhave you seen cell division? Areyou concernedaboutnuclearreprogramming?
Response:To date we have independentlyshown proofof concept in each of the steps necessaryto clone a
bird,i.e. visualization,enucleation,renucleationandembryo transfer.We are initiatingthe process of
integratingthese steps into a technologypackageas well as optimizingeach of the stepsand should have
some preliminaryanswers to this questionin the next few weeks.
Question 7
From: Catherine Ricks
What percentage of nuclear transferredeggs actually hatch?
Response: As I mentioned before the firstexperiments integrating the four steps arejust beginning and we
should have some preliminaryresults in the next few weeks and months.
Question8
From:Dr. Joseph Darden
Can the hen stage be omitted, and tyansfergenetic material fi'om egg to egg? Can recipient hens be reused?
Response: I am not sure how that would be implemented. I suspect thateventually we would have to
produce birdsin the process. Recipient hens can be used a second time as egg donors.