Nature Genetics
25, 255 - 257 (2000)
doi:10.1038/77000
Mitochondrial DNA heteroplasmy in cloned cattle produced by fetal and
adult cell cloningRalf Steinborn1, 2, Pamela Schinogl1, Valeri Zakhartchenko4, Roland Achmann2, Wolfgang Schernthaner4, Miodrag Stojkovic4, Eckhard Wolf4, Mathias Müller1
& Gottfried Brem1, 2, 31 Institute of Animal Breeding Genetics, University of
Veterinary Medicine, Vienna, Austria 2 Ludwig-Boltzmann Institute for Immuno- Cyto- and Molecular
Genetic Research, Vienna, Austria 3 Department of Biotechnology in Animal Production, Institute
for Agrobiotechnology, Tulln, Austria 4 Department of Molecular Animal Breeding and Genetics,
Gene Center, Ludwig-Maximilian University, Munich,
Germany
Correspondence should be addressed to Ralf Steinborn Ralf.Steinborn@vu-wien.ac.atMammals have been cloned from adult donor cells1,
2,
3. Here
we report the first cases of mitochondrial DNA (mtDNA) heteroplasmy in adult
mammalian clones generated from fetal and adult donor cells. The heteroplasmic
clones included a healthy cattle equivalent of the sheep Dolly, for which
a lack of heteroplasmy was reported4.
In mammals cloned by nuclear transfer of embryonic cells, the mtDNA is
derived mainly from the recipient cytoplast5,
6,
7,
8, with
a minor contribution from the donor cell6,
7,
8. The recognition
and proper processing of foreign somatic mitochondria by the cytoplasmic machinery
after microinjection into a zygote has been previously demonstrated9,
but donor mtDNA was not found after nuclear transfer of differentiated somatic
donor cells such as fetal and adult cells4. We analysed heteroplasmy
in ten somatic cattle clones generated from three donor cell types: primary
cultures of fetal fibroblast (FF) cells10, adult epithelium-derived
(AE) cells of the mammary gland, and adult fibroblast (AF) cells derived from
ear skin11. We found that the ratios of donor cell to recipient
cytoplast mtDNA copy number were 0.6%, 0.4% and 0.8% for the FF-, AE- and
AF-cell types, respectively, before nuclear transfer. Ratios were inferred
from real-time quantitations6 in the donor and recipient cells.
We quantified the percentage of donor mtDNA in the cloned animals by allele-specific
real-time PCR (ref. 7) using single-nucleotide
polymorphisms in parental mtDNAs and mismatched allele-specific primers (Fig. 1 and Table 1). Overall, the
donor-to-recipient ratios of parental mtDNAs before fusion remained the same
throughout embryogenesis or development to term of 7 of our 10 cattle clones.
Tissues of the other three clones showed a significant reduction or absence
of donor mtDNA, due to an unknown mechanism. In detail, from the 6 FF cell
clones (Table 1), we observed heteroplasmy in four
clones at ratios ranging from 0.4% to 4%, including the transmission of heteroplasmy
to the male germ line. We detected donor mtDNA in all five fetal tissues of
CFI325-sFF at ratios of 1% in heart, 3% in cerebellum and kidney, and 4% in
liver and skin. In contrast, in the clones CFI324-sFF and CFII324-sFF, donor
mtDNA was either absent or at significantly lower ratios across the tissue
types. We observed presence and absence of heteroplasmy for the two AE-cell
clones and determined donor mtDNA ratios ranging from 0.7% to 0.9% in the
two AF-cell clones (Fig. 1 and Table
1).
 | | Figure 1. Quantitation of heteroplasmy. | | |  | a, Map of the main control region of bovine mtDNA. b,
Polymorphic sites. Allele-specific quantitation at sites underlined using
mismatched allele-specific primers with complementary 3' ends to bases
underlined (for oligonucleotide sequences, see
http://www.nature.com/ng/supplementary_info
). c, Representative plot obtained by allele-specific real-time
PCR (ref. 7; equal mtDNA copies per sample as
confirmed in parallel by real-time PCR(ref. 6))
showing heteroplasmy in clone CC367-sAF. Non-target allele: homoplasmic genomic
DNA. Note: late cycle amplification of non-target allele is inherent to allele-specific
PCR. Numbering according to reference (GenBank accession number, V00654).
CF, cloned fetuses; CC, cloned calves; ns, non-starved donor cells; s, serum-starved
donor cells; FF, AE and AF, donor cell type; −, identity with reference;
*, deletion; +1, 2 and 3, insertions.
 Full Figure and legend (130K) |
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Our two normal and healthy heteroplasmic cattle clones, CCI328-nsFF and
CC367-sAE, demonstrate that mtDNA heteroplasmy does not necessarily impede
normal development. It was suggested that mtDNA heteroplasmy could have been
involved in the high death rate and impaired fitness observed in mammalian
clones12. Although the reasons behind the failure to detect
donor mtDNA in Dolly, in nine other somatic sheep clones4, and
in three of our ten cattle clones remain unclear, several conclusions can
be drawn. At least three mechanisms are not responsible for the absence of
donor mtDNA: (i) a general mechanism similar to that proposed to exclude sperm-derived
mtDNA (ref. 12); (ii) maintaining donor cells
in G0 for four to eight days before nuclear transfer4,
10,
11;
and (iii) cryopreservation of donor cells before nuclear transfer, at least
for the material studied here. The cloned sheep analysed previously4
were generated by nuclear transfer of cryopreserved donor cells13;
hence, it cannot be excluded that this procedure causes a reduction of donor
mtDNA copy numbers or leads to an impairment of nucleo-mitochondrial interaction.
Cell culture conditions represent another factor that, potentially, can affect
the original donor mtDNA copy number. In our hands, extensive passaging of
cells led to a complete depletion of mtDNA. As for the cryopreservation, we
cannot exclude that prolonged in vitro culture can result in a reduction
of mtDNA copy number.
Somatic cell cloning may be an alternative to microinjection9,
14
for generating heteroplasmic animal models of mtDNA diseases15.
The implications of heteroplasmy generated by nuclear transfer may force a
re-evaluation of pro-creative strategies attempting to correct maternally
inherited mitochondrial genetic disorders.
Note: Supplementary information is available on the Nature Genetics web site (http://genetics.nature.com/supplementary_info/).
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Acknowledgments
We thank A. Pollack for use of the ABI PRISM 7700 Sequence Detection System;
M.A. Nowshari, G. Mö lacher, H. Füllgrabe, A. Range, A. Bergthaler
and P. Stojkovic for technical assistance; and S.J. Weiss for comments on
the manuscript. This work was supported in part by Agrobiogen and the Deutsche
Forschungsgemeinschaft (WO685/2-1 and WO685/3-1 to E.W.).
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