Cloning endangered felids using heterospecific ... - CSIRO Publishing

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Reproduction, Fertility and Development, 2009, 21, 76–82

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Cloning endangered felids using heterospecific donor oocytes and interspecies embryo transfer Martha C. GómezA,D , C. Earle PopeA , David M. RicksA,B , Justine LyonsA , Cherie DumasA and Betsy L. DresserA,C A Audubon

Center for Research of Endangered Species, 14001 River Road, New Orleans, LA 70124, USA. B LSU Health Sciences Center, Department of Medicine, Gene Therapy Program, Louisiana State University, 533 Bolivar St, New Orleans, LA 70112, USA. C Department of Biological Sciences, University of New Orleans, 200 Lakeshore Drive, New Orleans, LA 70131, USA. D Corresponding author. Email: [email protected]

Abstract. Somatic cell nuclear transfer (SCNT) offers the possibility of preserving endangered species. It is one of the few technologies that avoids the loss of genetic variation and provides the prospect of species continuance, rather than extinction. Nonetheless, there has been a debate over the use of SCNT for preserving endangered species because of abnormal nuclear reprogramming, low efficiency and the involvement of extra mitochondrial DNA (mtDNA) of a different species in live offspring produced by interspecies SCNT. Despite these limitations, live endangered cloned animals have been produced. In the present paper, we describe recent research on the production of cloned embryos derived by fusion of wild felid fibroblast cells with heterospecific domestic cat cytoplasts and their viability after transfer into domestic cat recipients. In addition, we discuss epigenetic events that take place in donor cells and felid cloned embryos and mtDNA inheritance in wild felid clones and their offspring.

Introduction Somatic cell nuclear transfer (SCNT) represents an alternative for the production of genetically identical animals and offers the possibility of preventing the extinction of wild species. However, owing to the limited availability of oocytes from wild animals, the cloning of endangered species requires the use of donor oocytes from a related domestic species. Interspecies SCNT consists of the reconstruction of a cloned embryo by use of a donor cell and recipient oocyte from a different species, but from the same genus; whereas intergeneric SCNT consists of the reconstruction of a cloned embryo in which the donor nucleus and recipient cytoplast differ both in species and in genus. Several studies have demonstrated that it is possible to produce embryos from endangered species by interspecies or intergeneric SCNT; however; few live cloned wild mammals have been produced (Lanza et al. 2000; Loi et al. 2001; Gómez et al. 2004, 2008; Janssen et al. 2004; Kim et al. 2007) and these animals were derived from embryos reconstructed with donor oocytes of the same genus. Viable offspring from intergeneric SCNT have not been produced in any mammalian species, although pregnancies have been established with intergeneric cloned embryos after transfer into sheep or domestic cat recipients (Dominko et al. 1999; Chen et al. 2002; Yin et al. 2006). The successful development of interspecies and intergeneric cloned embryos is dependent on a variety of factors, which are similar to those reported for intraspecies SCNT, © IETS 2009

including source of oocyte cytoplasts, cell cycle synchronisation and genotype of the donor cells. Moreover, increasing evidence shows that aberrant epigenetic alterations that arise during SCNT (Dean et al. 2001; Santos-Rosa et al. 2002; Feil and Loi 2006) may be associated with perinatal and neonatal losses and the production of abnormal offspring (Wilmut and Paterson 2003; Gómez et al. 2004, 2008; Gómez and Pope 2006). This document is not an inclusive review of the state-of-theart for interspecies nuclear transfer; rather, it is an overview of our recent research on the production of wild felid cloned embryos and our efforts to overcome some of the hurdles encountered in the production of live offspring. We discuss the differences observed in in vitro and in vivo developmental competence of cloned embryos from four species of small wild felids that were reconstructed with heterospecific domestic cat cytoplasts and transferred into domestic cat recipients. In addition, we describe some epigenetic events that take place in donor cells and cloned felid embryos and mitochondrial (mt) DNA inheritance in wild felid clones and their offspring.

Domestic cat cytoplasm can support reprogramming of non-domestic cat nuclei from the same or different genus after SCNT Domestic cat oocytes have been used to produce cloned embryos of endangered felids and we have demonstrated that domestic cat 10.1071/RD08222

1031-3613/09/010076

Cloning endangered felids

oocytes can reprogramme (as measured by in vitro development to the blastocyst stage and/or production of live offspring) the nuclei of several endangered felids (Gómez et al. 2006a, 2008). Other research groups have also demonstrated that oocytes from different non-felid species can reprogramme nuclei of an endangered felid, namely the marbled cat (Pardofelis marmorata; Wen et al. 2003; Thongphakdee et al. 2006, 2008). Rabbit oocytes, which are phylogenetically distant to the marbled cat, support the development of embryos reconstructed with marble cat or domestic cat donor cells to the blastocyst stage at higher rates than embryos reconstructed on domestic cat oocytes (Thongphakdee et al. 2006), but domestic cat embryos reconstructed with bovine oocytes did not develop beyond the eight-cell stage (Thongphakdee et al. 2008). Although the process(es) by which an oocyte reprogrammes the nucleus of a different species is not understood, the results outlined above suggest that certain transcription factors present in the cytoplasm of enucleated oocytes are conserved among mammalian species and, thus, are able to induce nuclear remodelling and reprogramming of nuclei from another species. Nonetheless, for interspecies or intergeneric SCNT, minimising the phylogenetic distance between the recipient cytoplast and the donor nucleus may enhance compatibility between the nuclear and mtDNAencoded proteins, thereby allowing efficient ATP production (Bowles et al. 2007, 2008). Considerable evidence indicates that the cell type, cell cycle phase, passage number and environmental conditions (such as in vitro culture or freezing–thawing procedure) of donor cells before use in SCNT strongly influence the proportion of embryos that develop to the blastocyst stage and the production of live offspring. All experiments in the present study were performed with African wild cat (AWC; n = 4; one male and three females), black-footed cat (BFC; n = 3; one male and two females), sand cat (SC; n = 2; two males) and rusty spotted cat (RSC; n = 2; one male and one female) donor cells (fibroblasts) that were generated from skin tissue collected by biopsy from the abdominal area. The tissue was processed and cultured in vitro under the same standard conditions using previously described protocols (Gómez et al. 2003) and cells were frozen at primary culture (PC) by resuspending fibroblasts in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS) and 10% (w/v) dimethylsulfoxide (DMSO) and cooled at 1.0◦ C min−1 to −80◦ C (Mr. Frosty; Nalgene, Rochester, NY, USA) before storage in liquid nitrogen. For further passages, frozen fibroblasts were thawed and cultured in flat-sided tissue culture tubes with DMEM. Using flow cytometry analysis, we had demonstrated previously that a higher percentage of AWC and domestic cat (DSH) fibroblast cells were in the G0 /G1 phase after cells were serum starved than after contact inhibition treatment; however, development to the blastocyst stage of reconstructed embryos was similar between both cell cycle synchronisation treatments (Gómez et al. 2003). Therefore, for cell synchronisation by serum starvation, AWC, BFC and RSC fibroblasts were cultured to 100% confluency, followed by replacement of culture medium with DMEM + 0.5% FBS and 5 additional days of culture. Sand cat cells were synchronised by contact inhibition, in which cells were cultured to 100% confluency, followed by an additional 5 days of culture,

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during which time the culture medium was replaced every other day. Chromosomal abnormalities in AWC and DSH fibroblast cells increased progressively with the number of passages and were correlated with the frequency at which abnormalities were observed in AWC and DSH cloned embryos (Gomez et al. 2006b). Accordingly, to avoid or reduce the incidence of chromosomal anomalies in fibroblast cells and reconstructed cloned embryos, we used AWC, BFC, RSC and SC at early passages (P1–P4), when the percentage of cells with chromosomal abnormalities is low. In addition, for all experiments, non-activated in vivo-matured domestic cat oocytes were used as recipient cytoplasts. To evaluate whether differences in species or genera between donor cells and recipient oocytes influence the in vitro and in vivo developmental competence of reconstructed embryos, we produced cloned embryos with AWC (Felis silvestris lybica), SC (Felis margarita) and BFC (Felis nigripes) donor cells that share the same genus with DSH (Felis silvestris catus) oocytes, as well as with RSC (Prionailurus rubiginosus) donor cells, which are from a different genus. The cleavage rate of reconstructed embryos was not affected by differences in species or genera between donor cells and recipient oocytes; however, development to the blastocyst stage was lower for interspecies cloned embryos reconstructed with BFC and SC fibroblast cells than that of the other species. Nevertheless, RSC–DSH embryos reconstructed by intergeneric SCNT developed to the blastocyst stage at similar percentages as interspecies AWC– DSH cloned embryos. Although fewer BFC–DSH and SC–DSH cloned embryos developed to the blastocyst stage, pregnancy rates and the number of embryos implanting after transfer into DSH recipients were similar to the rates observed after the transfer of AWC–DSH cloned embryos. Despite this, all implanted BFC–DSH cloned embryos were reabsorbed by Day 30 of gestation (Table 1). No pregnancies were established after transfer of intergeneric RSC–DSH cloned embryos into DSH recipients (Table 1). Previously, we found that DSH–DSH cloned embryos reconstructed with a female donor cell culture developed to the blastocyst stage at a lower frequency (3.3%) than did AWC– DSH cloned embryos reconstructed with a male cell culture (24.5%; Gómez et al. 2003). We concluded that the difference was due, in part, to the origin of the donor nucleus and was not a species difference. Subsequently, we evaluated the influence of the species and gender of the donor cell on in vitro development of interspecies or intergeneric cloned embryos reconstructed with fibroblast cells from four different species of small wild felids. No differences due to gender were observed, except that male RSC–DSH embryos developed in vitro at higher rates than did female RSC–DSH embryos (Table 2). When we compared embryo development between cell cultures within wild felid species, we observed that development to the blastocyst stage was affected by the genotype of the donor cells (Table 3). These results support our previous statement that development of interspecies and intergeneric cloned embryos is not affected by gender. In addition, we suggest that phylogenetic differences between the donor cell and the recipient oocyte does not affect the development of interspecies or intergeneric cloned embryos to the blastocyst stage; instead, development is affected,

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Table 1. In vitro: Embryo cleavage (Day 2) and development to blastocyst stage (Day 8) of African wildcat, sand cat, black footed cat and rusty spotted cat embryos reconstructed into domestic cat enucleated oocytes AWC, African wild cat; BFC, black-footed cat; RSC, rusty spotted cat; SC, sand cat; DSH, domestic cat. In vivo: pregnancies and embryo implantations (Day 22), fetuses re-absorbed (Day 30–40) and live offspring of AWC, SC, BFC and RSC cloned embryos transferred into the oviduct of synchronised DSH recipients. a,b,c Different superscripts within the same column indicate significant differences (P < 0.05) Species

AWC SC BFC RSC ∗ Kittens

Cleavage/total fused (%)

No. blastocysts from total cleaved (%)

1017/1282 (79.3) 499/558 (89.4) 428/502 (85.2) 65/76 (85.5)

272 (26.7)a 83 (16.6)b 11 (2.6)c 17 (26.2)a

No. DSH recipients (%)

No. embryos transferred (%) Total

Total

Pregnant

40 45 20 5

17 (42.5) 14 (31.1) 9 (45) 0 (0)

1627 1600 698 201

Cell gender

No. blastocysts/cleaved embryos (%) AWC–DSH

BFC–DSH

RSC–DSH

254/944 (26.9)a 18/73 (24.6)a

6/181 (3.3)a 5/241 (2.0)a

16/45 (35.5)a 1/20 (5)b

Table 3. Development to the blastocyst stage (Day 8) of African wildcat, black footed cat, sand cat and rusty spotted cat cloned embryos reconstructed with different cell cultures AWC, African wild cat; BFC, black-footed cat; RSC, rusty spotted cat; SC, sand cat; DSH, domestic cat. Cell culture 1, 2, 3 and 4 were collected from different animals. AWC, BFC and RSC donor cells were at Passage 1–3, serum starved for 5 days, frozen and thawed immediately before somatic cell nuclear transfer (SCNT). SC donor cells were at Passage 2–4, at 100% confluence and were cultured for 5 additional days and dissociated before SCNT. a Different superscripts within the same column indicate significant differences (P < 0.05) Cell culture 1 2 3 4

Implanted

41 ± 10 35.5 ± 9.5 34.3 ± 9.2 40 ± 13

46 (2.8)a 28 (1.7)a 20 (2.9)a 0 (0)b

No. fetuses reabsorbed/ implanted embryos (%) 29 (63)a 14 (50)a 20 (100)b

No. kittens/total embryos transferred (%) Total at delivery

Live >36 h

Live >30 days

17 (1.0) 14 (0.9)

14 (0.9) 5 (0.3)

8 (0.5) 2 (0.1)∗

died 30 and 60 days after birth.

Table 2. Development to the blastocyst stage (Day 8) ofAfrican wildcat, black footed cat and Rusty spotted cat cloned embryos reconstructed with male or female donor cells AWC, African wild cat; BFC, black-footed cat; RSC, rusty spotted cat; DSH, domestic cat. AWC–DSH = male (one cell culture), female (three cell cultures); BFC–DSH = male (one cell culture), female (two cell cultures); RSC–DSH = male (one cell culture), female (one cell culture). a,b Different superscripts within the same column indicate significant differences (P < 0.05)

Male Female

Mean (± s.d.) per recipient

No. blastocysts (%) AWC–DSH (26.9)a

254/944 5/15 (33.3)a 11/33 (33.3)a 3/37 (8)b

BFC–DSH

SC–DSH

(3.3)a

(5.8)a

6/181 3/150 (2.0)a 2/97 (2.0)a –

5/86 22/51 (43.1)b 9/75 (12)a –

RSC–DSH 16/45 (35.5)a 1/20 (5)b – –

in part, by the genotype of the donor cell and, possibly, by other unknown factors. Donor cell preparation is an important step for the success of SCNT. Cloned embryos are frequently reconstructed with

donor cells that are freshly dissociated from a monolayer or are thawed and used immediately before SCNT. Cryopreservation of donor cells affects the in vitro developmental competence of pig cloned embryos (Zhang et al. 2006). Recently, we conducted studies in which SC–DSH cloned embryos reconstructed with donor cells that were thawed immediately before SCNT were reabsorbed after implantation at higher percentages and produced a lower incidence of live offspring than embryos reconstructed with donor cells that were cultured in vitro for a period of time after thawing (Gómez et al. 2008). We also observed that SC donor cells that were thawed and used immediately for SCNT had different epigenetic marks than donor cells cultured for a period of time before SCNT. Moreover, only 25% of the SC–DSH cloned embryos reconstructed with cells thawed before SCNT expressed the POU5F gene compared with 88% of cloned blastocysts reconstructed with cultured cells. Therefore, cryopreservation not only altered epigenetic events in donor cells, but may indirectly affect the number of embryos that implant and develop to term (Gómez et al. 2008). Therefore, we recommend that donor cells be allowed to undergo a period of in vitro culture after freezing–thawing to reduce negative effects on embryo viability of interspecies or intergeneric wild felid cloned embryos. Various strategies have been used to improve the efficiency of SCNT. One of the most recent methods is the treatment of cloned embryos with chemical agents that modify some epigenetic events and improve embryo viability. Treatment of intraspecies mouse, pig and rabbit cloned embryos with trichostatin A (TSA), an inhibitor of histone deacetylases, improved embryo development to the blastocyst stage (Kishigami et al. 2006, 2007; Zhang et al. 2007; Li et al. 2008; Shi et al. 2008) and increased the number of live offspring after transfer to foster mice (Rybouchkin et al. 2006; Wakayama 2007). In a previous study, we found that BFC–DSH cloned embryos implanted after transfer into the uterus of DSH recipients (Table 1; Gómez et al. 2006a); however, embryos were reabsorbed by Day 30 of gestation. Because nuclear reprogramming after SCNT may be improved by exposing cloned embryos to inhibitors of histone deacetylases, we have performed some initial studies to explore whether TSA enhances the in vitro and in vivo developmental competence of


BFC–DSH cloned embryos (M. C. Gómez, D. Ricks, C. E. Pope, J. Reiser, B. L. Dresser, unpubl. obs.). Preliminary data indicate that epigenetic marks in BFC–DSH cloned embryos are modified after exposure to two different TSA concentrations (50 and 100 nm). However, embryo cleavage, blastocyst development and in vivo viability of TSA-treated BFC–DSH cloned embryos were not enhanced; instead, development to the blastocyst stage was inhibited when BFC–DSH embryos were exposed to 100 nm TSA. Further experiments are required to determine the reason(s) for the detrimental effect of a higher dose of TSA on embryo development and to identify the lowest concentration that will induce histone hyperacetylation and improve embryo viability. Frequently, mammalian cloned embryos die at various preand postimplantation stages due to abnormal or faulty reprogramming of important early embryonic genes; however, we do not know the exact reason for the reabsorption of BFC–DSH cloned embryos. The possibility that factors other than abnormal nuclear reprogramming and/or faulty gene expression are involved in the loss of BFC–DSH cloned embryos during early pregnancy should be considered. The DSH has been shown to be a successful recipient of wild felid embryos. In fact, embryo transfer of wild felid embryos derived by IVF and SCNT has resulted in the birth of Indian desert cat (Felis silvestris ornata), AWC and SC kittens (Pope et al. 1993, 2000; Gómez et al. 2004, 2006a, 2008). Pregnancies have also been established in DSH recipients after the transfer of interspecies–BFC cloned embryos (Gómez et al. 2006a), intergeneric panda embryos reconstructed into enucleated rabbit oocytes (Chen et al. 2002) and leopard cat (Prionailurus bengalensis) embryos reconstructed into enucleated DSH oocytes (Yin et al. 2006). However, in all these cases, fetal development ceased during the first month of gestation. It is not known whether fetal losses were due to problems associated with SCNT and/or maternal–fetal incompatibility.Although some problems associated with SCNT will be overcome by better understanding of the molecular and biochemical events that take place during nuclear reprogramming (Hochedlinger and Jaenisch 2006), incompatibility between maternal and fetal interactions will be more difficult to resolve. One relatively straightforward approach would be to determine whether viable full-term fetuses can be produced by transfer of BFC or RSC embryos produced by IVF into DSH recipients. Such a study would clarify whether embryo or fetal incompatibilities are the main cause of fetal losses of BFC cloned embryos after transfer to DSH recipients and help refocus our efforts to produce live BFC offspring by SCNT. An additional approach proposed to enhance propagation of an endangered population by SCNT consists of producing cloned embryos that are more compatible with the recipient. The proposed technique involves transfer of the inner cell mass (ICM) of a cloned embryo into the trophoblastic vesicle of an embryo from the same species as the donor cytoplast and recipient (Loi et al. 2007). Thus, the placenta (trophoblast) of the resultant embryo would be more compatible with the recipient, which, consequently, would improve developmental capacity and the possibility of supporting a term pregnancy. Although, this proposal is interesting and innovative, no live offspring have been reported and incompatibility between the donor nucleus and


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the recipient oocyte is possibly the primary factor affecting the viability of interspecies or intergeneric cloned embryos. Mitochondrial DNA of interspecies AWC cloned kittens and their offspring Normal embryogenesis requires apposite interaction between the nucleus and the ooplasm, a process that involves the interaction of many nucleus-encoded proteins with the displacement loop (D-loop) of the mtDNA genome to mediate replication and transcription efficiently (Taanman 1999), regulation of total mtDNA copy numbers (Bowles et al. 2007) and supply of ATP for energy requiring activities (for a review, see Berridge et al. 1998). The interdependence between nuclear DNA and mtDNA is believed to be species specific (Kenyon and Moraes 1997). Therefore, the phylogenic distance between the recipient cytoplast and donor cells must be minimal to ensure that the nucleus is capable of regulating the mtDNA of the recipient cytoplast (Bowles et al. 2007). During fertilisation, sperm mitochondria are degraded by ubiquitination (Sutovsky et al. 2000), leaving the offspring with a single mtDNA population transmitted by the oocyte, which is denoted as homoplasmy. Cloned embryos can be designated as either homoplasmic (if they exhibit mtDNA derived solely from the recipient oocyte or derived from the donor cell) or as heteroplasmic (if they show evidence of mtDNA derived from both the recipient oocyte and donor cell). It has been suggested that mitochondrial heteroplasmy may induce incompatibility between the nucleus and the cytoplasm, which inhibits the development of cloned embryos (Chen et al. 2002; Bowles et al. 2007, 2008) at the time of genomic activation (Sansinena et al. 2005). In fact, a recent study reported that the copy number of cat mtDNA of cloned cat embryos reconstructed with bovine oocytes was stable from the one-cell to the eight-cell stage, whereas the bovine mtDNA copy number declined at the eight-cell stage and embryos did not develop to the blastocyst stage. A developmental block of cat–bovine cloned embryos was related to the presence of foreign mtDNA (Thongphakdee et al. 2008). In contrast with the developmental block observed in cat–bovine cloned embryos, we observed successful in vitro development to the blastocyst stage of interspecies and intergeneric wild felid embryos reconstructed with DSH oocytes (Table 1). Healthy homoplasmic (mtDNA derived entirely from recipient oocytes) wild cloned animals derived from interspecies SCNT have been produced (Loi et al. 2001; Kim et al. 2007). We examined mtDNA genotypes in male and female AWC clones and mtDNA transmission to their offspring. To investigate the mtDNA genetic relationships among AWC cell donors, DSH oocyte donors, clones and offspring of clones, we collected blood, and in one case bone, from seven female DSH (oocyte donors) and 16 AWC (eight female, eight male). Of the AWC, two were somatic cell donors (Jazz and Nancy), seven were clones (Ditteaux, Miles, Katie, Madge, Evangeline, Tillie and Emily) and seven were offspring produced after natural breeding between the male clone Ditteaux and the female clones Madge and Katie. DNA was extracted from blood using a PureGene DNA Isolation Kit (Qiagen, Germantown, MD, USA) and from bone marrow using a protocol modified from

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Table 4. Extent of mitochondrial DNA homoplasmy or heteroplasmy in African wild cat clones, as calculated by the number of single nucleotide polymorphisms derived from one of the oocyte recipients or from the somatic cell donor AWC, African wild cat; OD, oocyte donor; CD, cell donor; SNP, single nucleotide polymorphism AWC clones

Sex

OD

Ditteaux Miles Katie Madge Tillie Emilie Evangeline

Male Male Female Female Female Female Female

CD

Cha-cha Kittie Friend Dierdre Dierdre Julie tte Juliette Juliette

No. SNPs

Jazz Jazz Nancy Nancy Nancy Nancy Nancy

Classification

Total

Derived from OD

Derived from CD

11 6 6 5 7 7 7

11 6 6 3 7 6 6

0 0 0 2 0 1 1

Homoplasmic Homoplasmic Homoplasmic Heteroplasmic Homoplasmic Heteroplasmic Heteroplasmic

Table 5. Mitochondrial DNA transmission from African wild cat (AWC) clones to AWC offspring produced by natural breeding The extent of heteroplasmy or homoplasmy was calculated by the number of maternal or paternal single nucleotide polymorphisms (SNPs) seen in the offspring AWC offspring

Nyla 15832 15833 15834 16E6 407E C1E6

Sex

Female Female Male Male Male Male Male

Mother

Madge Madge Madge Madge Katie Katie Katie

Father

Ditteaux Ditteaux Ditteaux Ditteaux Ditteaux Ditteaux Ditteaux

Aplenc et al. (2002). The D-loop of the mitochondrial control region (CR) was sequenced by Davis Sequencing (Davis, CA, USA) using custom primers. The feline CR spans approximately 1560 bp (Lopez et al. 1996), of which our primers amplified a 300-bp product. The polymerase chain reaction (PCR) products were successfully amplified from all samples and sequences were compared against the GenBank database using BLASTn (http://www.ncbi.nlm.nih.gov/BLAST/). The analysis showed that all amplified PCR products matched with >95% similarity to a partial sequence of the Felis catus control region of mitochondrial origin. Although it has been shown that cytoplasmic mitochondrial (cymt) DNA sequences may integrate into an organismal nuclear genome (giving rise to nuclear DNA sequences of mitochondrial origin, or numts; Antunes et al. 2007), we amplified the location of the overlapping homologous region between the cymt and the numts known to exist in the CR (Lopez et al. 1996; Antunes et al. 2007). Single nucleotide polymorphisms (SNPs) among the sequences were identified by aligning approximately 275 bp of the sequences of groups consisting of one somatic cell donor, two oocyte donors and one clone offspring.The approximate extent of homoplasmy (derived solely from recipient oocytes) or heteroplasmy was calculated for each clone by observing the number of SNPs derived from one of the oocyte recipients or from the somatic cell donor. Four of the seven clones were homoplasmic and three were heteroplasmic

No. SNPs

Classification

Total

Maternal

Paternal

14 8 4 5 8 8 8

14 8 4 5 8 8 8

0 0 0 0 0 0 0

Homoplasmic Homoplasmic Homoplasmic Homoplasmic Homoplasmic Homoplasmic Homoplasmic

(Table 4). Among seven offspring produced by natural breeding between two clones (Ditteaux and Katie or Ditteaux and Madge), all carry only maternally derived mtDNA (Table 5). To date, the heteroplasmic and homoplasmic AWC clones are healthy (at 4–5 years of age) and three of them have been reproductively tested. Conclusions We have demonstrated that wild felids can be produced by interspecies SCNT, that using donor cells thawed immediately before SCNT influences the viability of cloned embryos and that the coexistence of mtDNA of two different species, namely AWC and DSH, does not have detrimental effects on AWC clones. Our results indicate that the phylogenetic distances between DSH and AWC are within appropriate ranges to ensure an appropriate nucleocytoplasmic interaction and produce normal AWC clones. However, the coexistence of mtDNA populations in a cloned embryo reconstructed with a greater phylogenetic distance between the donor cell and recipient oocyte may have a greater impact on embryo development and, consequently, result in suboptimal function of the electron transfer chain and an increased possibility of mitochondrialrelated diseases (St. John et al. 2004; Bowles et al. 2007). Thus, a detailed study of the mtDNA genotype of BFC–DSH, RSC–DSH and SC–DSH cloned embryos and kittens should


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