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A cloned horse born to its dam twin
A birth announcement calls for a rethink on the immunological demands of
pregnancy.
Cesare Galli, Irina Lagutina, Gabriella Crotti, Silvia Colleoni, Paola
Turini,Nunzia Ponderato,
Roberto Duchi, Giovanna Lazzari Laboratorio di Tecnologie della
Riproduzione,
CIZ srl, Istituto Sperimentale Italiano Lazzaro Spallanzani, 26100 Cremona,
Italy
e-mail: cesare@galli2.191.it


Several animal species, including sheep, mice, cattle, goats, rabbits, cats,
pigs and, more recently, mules (1) have been
reproduced by somatic cell cloning, with the offspring being a genetic copy
of the animal donor of the nuclear material used for transfer into an
enucleated oocyte. Here we use this technology to clone an adult horse and
show that it is possible to establish a viable, full-term pregnancy in which
the surrogate mother is also the nuclear donor. The cloned offspring is
therefore genetically identical to the mare who carried it, challenging the
idea that maternal immunological recognition of fetal antigens influences
the well-being of the fetus and the outcome of the pregnancy. We derived
equine fibroblast cell lines from skin biopsies taken from one Arabian
thoroughbred male and one Haflinger female. Oocytes were obtained by in
vitro maturation of follicular oocytes recovered from equine ovaries
retrieved from abbatoirs. After removal of the nucleus and zona material,
embryos were reconstructed by cell fusion with the fibroblasts. Only fused
embryos (97%) were activated and cultured in vitro to the blastocyst stage;
these were then transferred into recipient mares (see supplementary
information).
We cultured 513 and 328 successfully fused reconstructed embryos from the
male and the female cell lines, respectively. In
total, 22 blastocysts developed, 8 from the male and 14 from the female cell
line. Of these, eight blastocysts from the male cell line and nine from the
female line were transferred non-surgically into nine recipients. Two of the
cloned embryos that were reconstructed with female cells were transferred
into the Haflinger mare from which the cell line was derived. Overall, four
single pregnancies were diagnosed by ultrasonography after 21 days of
gestation. Two were lost soon afterwards and one aborted at 187 days of
gestation; the fourth, however, was carried to term (336 days) and a healthy
female foal was born naturally on 28 May 2003 (birth weight, 36 kg).
Remarkably, this cloned foal was born from the same Haflinger mare who was
the original cell-line donor (Fig. 1). To confirm the genetic identity of
the foal and her mother, we analysed DNA from blood leukocytes from both
animals and from the placenta. Comparison of 12 equine-specific
microsatellite loci (Stock- Marks kit, Perkin-Elmer, Applied Biosystems)
confirmed that DNA from the foal and from the placenta was identical to that
of the recipient mare.

Our cloning procedure was relatively efficient, as one live foal was
produced from four pregnancies, although there was high developmental
failure from the cleavage stage to blastocyst (8 of 467 and 14 of 286
developed in the male and female cell lines, respectively) and early
implantation. This success was aided by advances in assisted reproduction in
the horse (2), particularly at the oocyteactivation stage, when protein
synthesis and phosphorylation must both be inhibited (3), and in the
refinement of the zona-free manipulation technique (4,5). The remarkable
birth of a live foal from its genetically identical recipient is at odds
with the idea that the maintenance of gestation depends on immunological
recognition of the pregnancy by the mother, based on evidence that abortion
can result from inadequate recognition of fetal antigens6. Furthermore, our
result adds the horse to the list of mammals that have so far been
successfully cloned from an adult somatic cell. In principle, cloning could
enable gelding champions to contribute their genotype to future generations,
as well as opening up an opportunity to verify the reproducibility of traits
such as character and sporting performance.

1. Woods, G. L. et al. Science doi:10.1126/science.1086743 (2003).
2. Galli, C. et al. Theriogenology 58, 705-708 (2002).
3. Lazzari, G.,Mari, G. & Galli, C. J. Reprod. Fertil. Abstr. Ser. 28,73
(2002).
4. Lagutina, I. et al. Theriogenology 59, 269 (2003).
5. Wilmut, I. Cloning and Stem Cells 5, 1-2 (2003).
6. Szekeres-Bartho, J. Int. Rev. Immunol. 21, 471-495 (2002).

Supplementary information accompanies this communication on Nature's
website.

Figure 1 The cloned foal, named Prometea, with its genetically identical
mother seven days after birth. The cells used for cloning were derived from
a skin biopsy of the mare. The enucleated oocytes were obtained by in vitro
maturation and removal of the nucleus from oocytes recovered from horse
ovaries from an abattoir. After embryo reconstruction by micromanipulation
and cell fusion, the cleaved embryos were cultured in vitro to the
blastocyst stage. Two of the derived embryos were transferred into the mare
donor of the cell line. A single pregnancy was established and was carried
to term with the birth of a female foal who is genetically identical to her
surrogate mother.


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