Three little pigs worth the huff and puff?
URL: http://www.nature.com/cgi-taf/DynaPage.taf?file=/nbt/journal/v18/n11/full/nbt1100_1144.html
Nature Biotech, November 2000 Volume 18 Number 11 pp 1144 - 1145
Mark Westhusin & Jorge Piedrahita
Mark Westhusin (e-mail: m-westhusin@tamu.edu) is an associate professor at the Veterinary Physiology and Pharmacology , and Jorge Piedrahita (e-mail: jpiedrahita@tamu.edu) is an associate professor at the Department of Veterinary Anatomy and Public Health, both in the Center for Animal Biotechnology and Genomics, Texas A&M University, College Station, TX 77843.
Cloning pigs by nuclear transfer has been—and continues to
be—a significant challenge. A major difficulty has been that
assisted reproductive technologies representing key steps in
the nuclear transfer process (e.g., in vitro oocyte maturation,
oocyte activation, and embryo culture) are not as well
developed in pigs as in other species. In addition, the female
pig uniquely requires multiple viable fetuses to maintain
pregnancy. Recently, three reports1-3 have employed variations
on cloning methods to overcome these problems. A
comparison of these different approaches provides clues for
improving the efficiency of nuclear transfer in pigs and other
recalcitrant animals. (Table 1.)
Methods for nuclear transplantation involve several key steps,
each of which provide targets for optimizing the efficiency of
animal cloning. These include (1) acquisition of mature ova (2)
removal of the chromosomes contained within the ova
(enucleation), (3) transfer of cell nuclei obtained from the animal
to be cloned into enucleated ova, (4) activation of the newly
formed oocyte to initiate embryonic development, (5) embryo
culture in vitro, and (6) transfer of the cloned embryo into a
surrogate mother. Variations on this basic approach have been
used by all three groups1-3 that have now successfully cloned
pigs.
In the first published report of cloned pigs by Polejaeva et al1,
the authors collected in vivo matured ova from superovulated
gilts (young females that haven't farrowed) by surgically flushing
the oviducts. Micromanipulation was used to enucleate the ova,
then nuclei derived from granulosa cells were transferred into
the recipient ova by electrofusion. The fused embryos were then
activated, also by electrofusion, and placed into culture. The
following day, a second round of nuclear transfer was
performed by removing karyoplasts from the day old nuclear
transfer embryos, and transferring these into in vivo derived pig
zygotes (naturally fertilized embryos also collected surgically) in
which the two pronuclei had been removed. Fused couplets
were electrically activated and then transferred as soon as
possible into synchronized recipient gilts, and eventually five
cloned piglets were produced.
In the second report of cloned pigs (actually just one pig was
produced) by Onishi et al.2, in vivo matured oocytes were again
used as recipient ova, but techniques involving direct injection
of fetal fibroblast nuclei were used, similar to those previously
described for producing cloned mice4, 5. Development was
induced by electroactivation followed by short-term embryo
culture and transfer of the embryos into recipient females.
In the report published in last month's issue, Betthouser et al.3
use techniques more similar to those employed for cloning
other animals. These included in vitro oocyte maturation,
electrofusion of enucleated oocytes with fetal cells, chemical
activation, and in vitro culture prior to embryo transfer. A large
number of cloned embryos (>100) at various stages of
development were produced. These were then transferred into
a recipient female, resulting in two cloned male pigs.
Polejaeva et al. and Onishi et al. used vivo derived oocytes as
recipient ova as a way of bypassing the in vitro oocyte
maturation step to increase the competency of ova for
supporting embryonic development. The obvious disadvantage
of this approach is the additional costs in time and labor
required. Therefore, given the overall inefficiency of the cloning
procedure and the need to transfer hundreds of embryos per
recipient, the use of in vitro matured oocytes, as employed by
Betthouser et al. could represent a significant advancement.
The main rational for a second round of nuclear transfer, used
by Polejaeva et al., was to take advantage of the environment
provided by enucleated zygotes that had undergone a more
natural ooctye activation by fertilization. Although this work does
represent the first successful attempt at cloning pigs, this
method is cumbersome and very labor intensive requiring
multiple surgical procedures on multiple animals. Additional
research will have to be completed to determine whether this
approach has any advantage. If it turns out that two rounds of
nuclear transfer significantly improve the efficiency of producing
live offspring, the extra costs in time and labor may be justified.
However, given the fact that both Onishi et al. and Betthouser et
al. successfully cloned pigs using only one round of nuclear
transfer, it is doubtful this approach will be used in the long term.
Although unproven, there may be some advantage to injecting
nuclei2 compared with using cell fusion for nuclear transfer. With
electrofusion, both the cytoplasm and nucleus of the donor cell
are transferred into recipient ova. Factors contained within the
cytoplasm such as proteins and mRNA transcript could
theoretically interfere with reprogramming and/or early
development of cloned embryos. As with two rounds of nuclear
transfer, it remains to be seen whether this method will prove
more effective.
Undoubtedly, the potential of using pigs for xenotransplantation
is the major driving force behind most cloning efforts. The
genetic modification of pigs to enhance important agricultural
traits such as increased feed efficiency and growth, resistance
to disease and parasites would also be extremely beneficial.
Other applications are more questionable. For example, it is
difficult to imagine that a significant number of pigs would ever
be cloned as part of a strategy for producing pharmaceutical
proteins when other species such as goats or cows are
available. And what of the broad scale use of cloning for pig
production? This seems unlikely, given the fact that US farmers
were trying to give their pigs away due to overproduction, low
prices and the high cost of feeding them last year.
We are just beginning to glimpse the benefits of animal cloning
alone, or in combination with genetic engineering. Techniques
such as homologous recombination have proved invaluable for
identifying gene function, increasing our understanding of
human and animal diseases, and for developing animal models
of human diseases. Unfortunately, this technique has not been
applied to any species other than mice because of the lack of
embryonic stem cells. Cloning of somatic cells, however, has
allowed the targeting of genes in nonmurine species5. Now,
with the reports of cloning in swine, the door is open for
development of genetically modified pigs for
xenotransplantation, as well as to provide an alternate animal
model to mice for studying human diseases.
REFERENCES
1.Pokjaeva, I. A., et al. Cloned pigs produced by nuclear transfer from
adult somatic cells . Nature 407, 86–90 (2000). MEDLINE
2.Onishi, A., et al. Pig cloning by microinjection of fetal fibroblast
nuclei. Science 289, 1188–1190 ( 2000). MEDLINE
3.Betthauser, J. et al. Production of cloned pigs from in vitro systems.
Nat. Biotechnol. 18, 1055–1059. MEDLINE
4.McCreath, K.H., et al. Production of gene-targeted sheep by nuclear
transfer from somatic cells. Nature 405, 1066– 1069 (2000).
MEDLINE
5.Wakayama, T. et al. Full-term development of mice from enucleated
oofytes injected with cumulus cell nuclei. Nature 394, 369– 374
(1998). MEDLINE