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Pig Cloning by Microinjection of Fetal Fibroblast Nuclei

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Science  18 Aug 2000:
Vol. 289, Issue 5482, pp. 1188-1190
DOI: 10.1126/science.289.5482.1188

Abstract

Pig cloning will have a marked impact on the optimization of meat production and xenotransplantation. To clone pigs from differentiated cells, we microinjected the nuclei of porcine (Sus scrofa) fetal fibroblasts into enucleated oocytes, and development was induced by electroactivation. The transfer of 110 cloned embryos to four surrogate mothers produced an apparently normal female piglet. The clonal provenance of the piglet was indicated by her coat color and confirmed by DNA microsatellite analysis.

Live births of cloned sheep (1), cattle (2), and goats (3) have been achieved by somatic cell transfer, in which a nucleus donor cell is fused with an enucleated oocyte (4). The single report of pig cloning also used this method, with an undifferentiated early embryonic cell (the blastomere of a four-cell embryo) as the nucleus donor (5). Clonal propagation of selected porcine phenotypes is potentially important in meat production. In addition, genetic modification could be combined with cloning in the provision of potential donors for xenotransplantation to humans (6).

As a first step toward pig cloning from differentiated cells, we investigated parameters that might affect porcine embryogenesis in vitro. Mature oocytes were isolated from females of the Landrace breed (7) and were parthenogenetically stimulated to initiate embryonic development by one of two electroactivation protocols (8). Embryos were then exposed to the microfilament inhibitor cytochalasin B (to prevent chromosome loss by cytokinesis) and were incubated in one of three culture media under otherwise identical conditions (9) (Table 1). Fewer embryos developed after multiple pulses than after a single pulse at a slightly higher field strength (Table 1). In addition, development was influenced by the type of culture media, with the highest development to the blastocyst stage supported by culture in NCSU23 (Table 1).

Table 1

Effect of different electroactivation protocols and culture media on in vitro porcine embryo development. Oocytes were matured in vivo. Percentages are of oocytes surviving electroactivation.

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Subsequently, a single 100-μs activating pulse of 1.5 kV/cm was employed with culture in NCSU23. Under these conditions, we determined the developmental potential of mature versus in vitro–matured oocytes (10). Of 167 in vitro–matured oocytes subjected to this protocol, 116 (69.5%) cleaved within 48 hours, but only 4 (2.4%) developed to blastocysts. This value (2.4%) is significantly lower (P = 0.001, χ2 test) than the corresponding value for mature oocytes (31.2%) (Table 1). The subsequent experiments therefore used mature oocytes that had developed in vivo (7).

Several reports of livestock cloning describe the use of fetal fibroblasts as nucleus donors (11–13). Moreover, these cells can be genetically modified before cloning (11–13). We therefore evaluated the ability of porcine fetal fibroblasts to support development to term after nuclear transfer. Fetuses derived from a Meishan × Meishan (black coat) cross were killed at embryonic day 24 (E24) and used to establish primary cell cultures after dispersal by tryp-sinization (14). Cultures were established by plating cells at high density for two to six passages, after which they were allowed to reach confluence. Culture was continued for 16 days without media replenishment, producing cells at cell cycle phase G0(14, 15). Polymerase chain reaction (PCR) analysis (14) of the fibroblasts revealed the sex of the progenitor fetus in each case (16). Consistent with their fibroblast origin, the cultured cells were negative for cytokeratin and stage-specific embryonic antigen–1 but were strongly positive for the mesodermal marker vimentin (16).

Recently, mice were cloned from adult somatic cells by a distinctive nonfusion method in which donor nuclei were selectively introduced into enucleated oocytes by piezo-actuated microinjection (17). We adopted this method for porcine nuclear transfer. Oocytes from white (Landrace) or white-black spotted (Landrace × Large White × Duroc) gilts were depleted of metaphase II chromosomes and the first polar body by piezo-actuated micromanipulation in NCSU23 containing cytochalasin B (18). The nuclei of Meishan × Meishan (black coat) fetal fibroblasts were each introduced into a single enucleated oocyte by piezo-actuated microinjection (17,19). Reconstructed preembryos were incubated at 38.5°C for 3 to 4 hours before electroactivation and culture of the resultant nuclear transfer embryos (8, 9).

Although porcine embryos can develop well in vitro to the blastocyst stage, their subsequent development in utero after transfer to the uterine horns of surrogates is poor (20). Moreover, because pigs typically require at least four fetuses for a successful pregnancy, we reasoned that the presence of helper embryos produced by fertilization might assist the full development of cloned embryos.

We therefore conducted two series of experiments to investigate the potential of helper (fertilized) embryos to assist in development in utero. Cloned embryos were transferred to surrogates after culture in vitro for 20 hours (series A, one-cell embryos) or 40 hours (series B, two- to four-cell embryos) (21). All offspring (9 from series A and 24 from series B) were white and therefore of nonclonal origin (Table 2). The failure of full-term development of cloned embryos among these four term pregnancies may suggest that if the ratio of nuclear transfer to helper (fertilized) embryos is important, it was clearly not optimized in these experiments.

Table 2

Development of cloned embryos in vitro and to term following transfer to surrogate mothers.

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A third experimental series (series C) was conducted in which 110 cloned embryos were transferred at the two- to eight-cell stage (Fig. 1A) between four surrogates that did not harbor helper (fertilized) embryos (Table 2). The cloned embryos were derived from fibroblast cultures at passages two to six. Three of the four surrogates returned to estrus 27, 35, or 61 days after transfer. The delayed resumption of estrus suggests that each surrogate had become pregnant; the porcine estrous cycle lasts 21 days. Because embryo-uterine attachment occurs at E13 to E14 in the pig, it is likely that development terminated after placentation in two of the pregnancies. We do not know the cause(s) of the terminations.

Figure 1

(A) Embryos clonally derived by microinjecting the nuclei of Meishan × Meishan (black coat) fetal fibroblasts into Landrace (white coat) enucleated oocytes. These embryos are at the four-cell stage after 40 hours of culture in vitro. Phase contrast microscopy is ×100. (B) Xena, the cloned piglet, at 24 hours, showing her black coat color; her white-colored Landrace surrogate mother is in the background.

A fourth surrogate in series C maintained pregnancy after the oviductal transfer of 36 cloned embryos that were derived from fibroblast cultures at passage two. One of the embryos developed to term, and the resulting piglet, named Xena, was delivered by natural birth on 2 July 2000, with birth and placental weights of 1.2 and 0.3 kg, respectively (both in the normal range for noncloned offspring). Her associated placenta was apparently anatomically normal. Some cloned cattle exhibit placental abnormalities (22, 23), and the placentas of mice cloned by nuclear microinjection are invariably larger than those of nonclones (24, 25).

Xena (Fig. 1B) is a healthy female with a black coat, as predicted by the clonal derivation of her Meishan × Meishan nuclear genome. To corroborate this derivation, we subjected genomic DNA from Xena, her Landrace surrogate mother, and the fibroblast cell culture from which Xena was derived to porcine strain-specific microsatellite analysis, with 23 marker sets. The analysis was performed “blind” in another laboratory (26). These analyses (Fig. 2) confirm that Xena shares the genome of her Meishan fibroblast progenitor, distinct from that of her Landrace surrogate.

Figure 2

Representative PCR analyses of microsatellite markers in genomic DNA from the Landrace surrogate mother (m), the cloned piglet Xena (c), and her progenitor fibroblast culture derived from a single female Meishan × Meishan fetus (f). Each panel shows data for a randomly selected microsatellite-specific primer pair: (A) SW1311, (B) SW1327, (C) SWR414, and (D) SW717. Traces were produced on a 373A Autosequencer (26). Sizes are in base pairs.

We have here shown that pigs can be cloned by microinjection of somatic cell nuclei into enucleated oocytes. We do not know all of the reasons for our success but speculate that it may derive in part from the rapidity of micromanipulation afforded by piezo-actuation of the micropipette. Furthermore, it is possible that success in porcine cloning is sensitive to contamination by nucleus donor cell cytoplasm. This would favor nuclear transfer by microinjection as opposed to fusion (cell transfer); unlike fusion, microinjection selectively removes much of the donor cell cytoplasm so that it is relatively dilute in the early embryo.

Random integration and gene targeting of cells in vitro, followed by clonal derivation, has been used to introduce germ line mutations in cattle (12) and sheep (11, 13). These findings raise the prospect that this approach can also be applied to pigs. Such an approach is particularly promising because pig embryonic stem cells have not been cultured. Porcine genome manipulation will also be assisted by the replication of individuals harboring desired genotypes, occurring either through conventional breeding or transgenesis. Our results, together with the recent report of intracytoplasmic sperm injection in the pig (27), indicate the potential of microinjection to facilitate porcine cloning.

  • * To whom correspondence should be addressed. E-mail: onishi{at}niai.affrc.go.jp

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