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ski2,a
a Department of Experimental Embryology, Institute of Genetics and Animal Breeding, Polish Academy of Sciences, Jastrzebiec, 05-551 Mroków, Poland
ABSTRACT
Blastomeres from eight-cell-stage rabbit embryos have been fused with enucleated metaphase II oocytes (ooplasts) or with ooplasts that were preactivated before fusion. Preactivation of ooplasts before nuclear transfer (NT) raises the rate of preimplantation development from 15% to 56%, which remains elevated in the next series of NT (48.6% and 47.2% in the second and third rounds, respectively). Transfer of eight-cell embryos from the third round to the recipient resulted in the birth of normal young. Synchronization of blastomere nuclei in the G1 phase with nocodazole before fusion results in 42% morula/blastocyst formation. However, in the second generation of NT embryos, the yield drops to as low as 17%, indicating deleterious effects of the second nocodazole treatment on blastomeres. The calculated number of clones per one round of cloning was 4.5, 3.9, and 3.8 in subsequent series; the highest number of morulae and blastocysts that developed from individual donor embryos after three rounds were 26 and 27, respectively.
developmental biology, ovum
INTRODUCTION
To improve the efficiency of serial cloning of rabbit embryos by nuclear transfer (NT) cell cycle, coordination between the oocyte and the blastomere must be refined. The coordination between donor nucleus and recipient cytoplasm requires that either the introduced nucleus be prevented from undergoing premature chromosome condensation (PCC) by reducing the level of meiosis promoting factor (MPF) in the recipient cytoplasm, or that donor nuclei be synchronized in the G1 phase of the cell cycle, which precludes their ability to re-replicate DNA (see [1] for review). Preactivation of recipient cytoplasm is one way to reduce the level of MPF so that nuclei introduced a few hours later are not influenced by it.
We examined whether preactivation of ooplasts with an electric field or synchronizing blastomeres in phase G1 improves preimplantation development in three series of NT in rabbit.
MATERIALS AND METHODS
Animals
Ninety-eight White New Zealand rabbits and 45 California rabbits were used. The experiments were carried out during the natural breeding season (i.e., from the end of January until the end of September); thus, the animals were exposed to a natural light cycle. Females were caged either separated or in pairs; males were caged separated. All animals were fed complete dry food with a metabolic energy of 2250 kcal/kg (Lapina II Extra, Dossche, Poland). Donor and recipient females and males that were subjected to vasectomy were anesthetized by i.m. injection of 2–2.5 ml (depending on animal weight) of 5% ketamine hydrochloride (Narkamon, Spofa, Prague, Czech Republic) and 0.5 ml of 2% xylazine (ScanVet, Poland). To obtain ovulated oocytes, mature females of the New Zealand breed aged 6–12 mo were given an i.m. injection of 100 IU eCG (Folligon, Intervet, Holland) and, after 72 h, they were given an i.v. injection of 100 IU hCG (Chorulon, Intervet, Holland). In addition, mating the females after hCG injection with vasectomized males provoked ovulation. To obtain four- and eight-cell embryos, mature females of the California breed aged 6–12 mo were hormonally stimulated in the same way and then mated twice with fertile males of the same breed.
Obtaining Oocytes and Blastomeres
Unfertilized oocytes were obtained 15 h after hCG injection by flushing the oviducts with warm (37°C) PB1 [2] supplemented with 10% fetal calf serum FCS (Biochrom, Budapest, Hungary). Follicular cells were removed by treatment with 150 IU/ml hyaluronidase (Sigma Chemical Company, St. Louis, MO) in PBS for 10 min at 37°C and then by delicate pipetting in PB1 with 10% FCS.
For the first round of cloning, the embryos were flushed from the oviducts with PB1 supplemented with 10% FCS. Four-cell embryos (to be synchronized with nocodazole) were recovered 28–30 h after hCG injection; eight-cell embryos were obtained 42–44 h after hCG injection. For the second and third round of cloning, embryos were used that had developed in vitro from reconstituted zygotes of the previous round of cloning. Mucin and zona pellucida were digested with 0.5% pronase (Protease, Sigma) in Ringer solution at 38.5°C for 3–4 min. After rinsing the embryos in three changes of M2 medium (Sigma) they were transferred to Dulbecco salt solution (Gibco, Paisley, Scotland), which was devoid of calcium and magnesium ions, for 15–20 min. After this treatment, the embryos were pipetted with a flame-polished narrow-bore pipette in M2 medium to disaggregate them into single blastomeres.
Synchronization of Four-Cell Stage Blastomeres in G1
Single blastomeres from the four-cell stage were placed in a solution of 0.4 µg/ml nocodazole (Sigma) [3] in Menezo-B2 medium (Laboratoire CCD, Paris, France) with 10% FCS and cultured for 11 h at 38.5°C in an atmosphere of 5% CO2. After thorough rinsing, single blastomeres were individually cultured in 1.5-µl drops of Menezo-B2 with 10% FCS. Immediately after cleavage, individual 1/8 blastomeres were microsurgically placed into the perivitelline space of enucleated metaphase II oocytes.
Enucleation of Oocytes
Before enucleation, oocytes were preincubated for 30 min in M2 medium with 1 µg/ml cytochalasin D (CD, Sigma), at 38.5°C. For enucleation, the oocytes were placed in a micromanipulation chamber in a drop of the same medium under paraffin oil at room temperature. Enucleation was performed according to the method of McGrath and Solter [4], modified by Modliski and Smor
g [5]. Briefly, an oocyte that was to undergo surgery was oriented and fixed in a holding pipette with its first polar body positioned at 3 o'clock. An enucleation pipette with an internal diameter of 20 µm was introduced through the zona pellucida into the perivitelline space, and the first polar body with adjacent cytoplasm that contained a spindle of metaphase II was delicately sucked into the pipette and pulled out from the perivitelline space. To enucleate 25–30 oocytes took 1.5 h or less.
Preactivation of Ooplasts
Immediately after enucleation, ooplasts were rinsed of CD in M2 medium and transferred to a chamber that was filled with a dielectric solution in which they were activated. The solution consisted of 0.3 M glucose with 0.05 mM calcium and 0.1 mM magnesium ions, and about 10 mg/ml BSA (fraction V, Sigma).The distance between the electrodes in the chamber was 0.75 mm. The Electrocell Manipulator 2001 (BTX Gentronics, San Diego, CA) pulse generator was used. To activate ooplasts, each received two direct current (DC) impulses of 100 volts (V) each for 20 µsec. After treatment, the ooplasts were rinsed in M2 medium, transferred to Menezo-B2 with 10% FCS, and were cultured for 4.5–5 h at 38.5°C in an atmosphere of 5% CO2.
Introducing Blastomeres into the Perivitelline Space of Ooplasts
Blastomeres were preincubated for 20 min in M2 medium with 1 µg/ml CD at 38.5°C before being placed with ooplasts in a drop of the same medium under paraffin oil in a micromanipulation chamber. Single blastomeres were sucked into a 25-µm pipette, the tip of the pipette was introduced into the perivitelline space through the opening in the zona that had remained after enucleation, and the blastomere was deposited near the ooplast membrane. After a group of blastomeres had been placed in contact with ooplasts, the resulting pairs of cells were transferred to Menezo-B2 medium with 10% FCS at 38.5°C for about 15 min before they were used for cell fusion or ooplast activation/cell fusion.
Ooplast-Blastomere Fusion
Pairs of preactivated ooplasts and unsynchronized blastomeres were placed in the same dielectric solution-filled chamber that had been used for preactivation and were given three 20-µsec 100-V DC impulses. After treatment, the pairs were rinsed three times with Menezo-B2 with 10% FCS, placed onto a warm stage (38.5°C) in the same medium under paraffin oil, and examined every 10–15 min for the appearance of cell fusion.
Ooplast Activation and Ooplast-Blastomere Fusion
Pairs of nonpreactivated ooplasts and either synchronized or unsynchronized blastomeres were placed into a dielectric solution-filled chamber where they were treated with two 170-V DC pulses of 35 µsec each. Rinsing and observation were the same as described earlier.
Culture of Reconstituted Embryos and Embryo Transfer
The embryos were cultured in drops of 30–40 µl of Menezo-B2 medium with 10% FCS under paraffin oil in tissue-culture Petri dishes (Corning, Acton, MA) at 38.5°C in 5% CO2. Some eight-cell embryos developed from zygotes that had been reconstituted in the third round of NT into preactivated oocytes and were transferred to White New Zealand females. The recipients had been pretreated with 100 IU eCG followed by 50 IU hCG, and then were mated twice with vasectomized males. Embryo transfer was performed 45–46 h after hCG treatment. The embryos were sucked into the tip of an Eppendorf pipette in 1.5–2 µl of warm PB1 with BSA, and deposited into the oviduct of the recipient.
Control Experiments
Reliability of enucleation was tested using Hoechst-stained oocytes (Sigma). Of 60 pairs of ooplast+karyoplast, meiotic chromosomes were found in 54 (90%) karyoplasts. Another group of 76 pairs was fixed and stained with hematoxylin. In 63 pairs (83%), karyoplasts contained an intact meiotic spindle with chromosomes. In the remaining pairs, either the spindle was found in the ooplast or analysis was precluded because of poor staining.
Experimentally chosen parameters for ooplast preactivation (two 100-V pulses, 20 µsec each) and ooplast activation and ooplast/blastomere fusion (two 170-V pulses, 35 µsec each) were tested by activating control oocytes. Of 75 oocytes treated by the first method, 87% were found to be activated (i.e., they extruded the second polar body and developed a pronucleus). Forty-two oocytes developed into blastocysts (56%), the remaining ones were blocked in development between the 2-cell and 16-cell stage. Of 93 oocytes treated by the second method, 84% were found to be activated, 54 of which reached the blastocyst stage.
Experimental Groups
A schematic drawing of experimental groups appears in Figure 1. Groups were assigned the short names of Untreated, Preactivated, and Synchronized.
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RESULTS
Nuclear Transfer Efficiency
Table 1 shows the efficiency of ooplast-blastomere fusion in three series of NT for three experimental groups. In the untreated group, fusion efficiency decreased in the third round of cloning, whereas in the synchronized group, it decreased in the second round. In the preactivated group, fusion efficiency was the highest of all three experimental groups and was only slightly reduced in the third series. This reduced efficiency might be explained by the weaker electric field given to this group versus the other two (i.e., three 100-V DC pulses of 20 µsec each vs. two 170-V DC pulses of 35 µsec each); however, for preactivation, this group had been treated with two 100-V DC pulses of 20 µsec each about 4.5–5 h earlier.
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Preimplantation Development of Serially Cloned Embryos
Results are presented in Table 2. In the untreated group of embryos the rate of first cleavage decreased in the third round of cloning, whereas in the synchronized group, it had already sharply decreased in the second series. Indeed, none of the embryos reconstructed from three eight-cell embryos of the second series resumed first cleavage; therefore, the third round for the synchronized group is not included in Table 2.
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Preactivated ooplasts seem to rescue reconstructed embryos from the decline of the rate of first cleavage in the second and third round of cloning. Also, the rate of first cleavage is higher in preactivated ooplasts than in the other two groups in the first series. Subsequent cleavages until the eight-cell stage followed the pattern of the first cleavage (i.e., the rates decreased in the second series in the synchronized group, in the third series in the untreated group, and did not change in the preactivated group). Reduction in the rate of morula/blastocyst formation in subsequent cloning series occurred in all groups, including the preactivated group. However, reduction in the preactivated group was slight: from 56.3% in series 1 to 47.2% in series 3. Half the number of untreated embryos developed in series 3 compared with series 1. In the synchronized group, a reduction from 42.2% to 17.1% occurred in series 2. Moreover, out of six embryos that developed, there were five morulae, and only one was a blastocyst.
In the untreated group, the mean cell number of blastocysts in series 1 was 90.1 ± 6.3 (a total of 5 blastocysts); in series 2 the number was 86.1 ± 7.1 (a total of 3 blastocysts). In the third series, one blastocyst was obtained in which 53 cells were counted. The number of cells in blastocysts in the preactivated group seemed unrelated to the series of cloning (Table 3; see also Fig. 2); however, the mean cell number in all preactivated blastocysts was conspicuously lower than in the blastocysts that had developed in vitro from in vivo-produced two-cell embryos (control, Table 3). The latter embryos had a definite developmental advantage over experimental reconstituted embryos, but still, the highest numbers of cells are similar in series 1 and 2 (144 and 141, respectively) and in controls (140).
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Serial Cloning Efficiency
The number of clones obtained from one round of cloning can be calculated when the rate of development (i.e., percent of morulae/blastocysts that developed from fused pairs of ooplasts/blastomeres) is multiplied by the cell number of the donor embryo [6]. Applying this method to the results of the preactivated group in Table 2 elicits the following numbers of clones per one round: 4.5 for the first round (8 x 56%), 3.9 for the second round (8 x 48%), and 3.8 for the third round (8 x 47%). Indeed, when individual embryos were followed, the numbers of morulae+blastocysts obtained after the first round of NT from 11 eight-cell embryos were 0 + 3, 1 + 2, 1 + 2, 0 + 3, 1 + 3, 2 + 2, 1 + 3, 1 + 4, 3 + 3, 2 + 4, and 3 + 5, with a mean value of 4.45. This value is very close to the earlier calculation on the basis of the rate of development (4.5). Unfortunately, it has never been possible to use all blastomere nuclei from the previous round for the next NT series because the supply of recipient oocytes is insufficient. Therefore, 2–5 cloned embryos were used for recloning (the second series) and 3–4 reconstituted embryos were used for the third round. Under these conditions, up to 26–27 morulae/blastocysts could be obtained from single eight-cell embryos in three series of cloning (Fig. 3).
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Postimplantation Development of Embryos from the Third Round of Cloning
Out of 67 eight-cell embryos transferred to recipients, one female and two males were born (Fig. 4). In December 1999, the oldest rabbit was 1 yr 3 mo old, and the youngest one was 8 mo old; they were normal and healthy.
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DISCUSSION
Electric Field Treatment of Rabbit Oocytes and Embryos
Electrofusion was first applied to rabbit embryonic cells to merge blastomeres of two-cell embryos in an attempt to induce tetraploidy [7]. Control embryos, which remained unfused after electric field treatment, developed into normal young in 49%. Tetraploid fused embryos developed to blastocysts in 86%. These results prove that electric field treatment is not deleterious to rabbit embryos. The parameters that were specifically used for nuclear transfer proved unharmful as exhibited by almost 70% of enucleated zygotes fused with karyoplasts carrying two pronuclei having developed into blastocysts in vitro [5].
Activation and fusion efficiency In a pioneer paper on rabbit NT, electric field treatment was applied in order to both activate the ooplast and fuse it with a 1/8 blastomere. The parameters used were one DC pulse of 160 V for 60 µsec. The efficiency of fusion was 84%. When control oocytes were activated with the same current, 52% of parthenogenetic activation was achieved and 16% of embryos developed to the blastocyst stage [8]. Efficiency of fusion was 82.5% after three subsequent pulses of DC, and control oocytes, which had received three pulses, were activated in 64.3% [5]. When three pulses were given in 30-min intervals, the fusion efficiency was 94% and the activation rate was 85%, with 48% developing to blastocysts [9]. Control oocytes, which received six pulses, were activated in 96%, with 72% developing to blastocysts [10]. One hundred percent of those that had been treated with eight pulses of 0.6 kV/cm for 60 µsec each 12 min apart were activated, and 73% developed into diploid blastocysts [11]. Direct comparison of activation rates induced by different field strengths shows that a stronger field (2.4 kV/cm) is more effective (86% activation) than a weaker field (1.8 and 1.2 kV/cm; 58% and 50% activation, respectively), also in promoting development to morulae and blastocysts (28% vs. 13% vs. 7%) [12]. However, results obtained when repetitive DC pulses have been applied (see earlier discussion) suggest that repeating the stimulus is at least as essential for activation as the actual field strength itself. This conclusion agrees with the assumption of the original finding that a repetitive pulse stimulates rabbit oocyte activation [13].
Preimplantation Development of NT Rabbit Embryos In Vitro
In the first description of preimplantation development after rabbit NT, 16.9% of reconstituted embryos developed into blastocysts, two of which hatched. In this trial, oocytes recovered 17–18 h after hCG treatment were enucleated and activated and fused with 1/8 blastomeres by applying three DC pulses of 2–2.5 kV/cm for 50–70 µsec each [5]. The yield of about 15% of blastocysts remains almost a standard in subsequent trials that applied similar experimental parameters ([14], also the untreated group in the present paper). Applying an alternating current (AC) pulse of 0.1 kV/cm for 5–10 sec followed by two DC pulses of 2.4 kV/cm for 60 µsec with an interval of 30 min. Du et al. [15] obtained 34% of morulae and blastocysts after NT from 1/16 blastomeres and 23% after NT from a cultured cell line. Electrical stimulation of six pulses of 2.2 kV, 60 µsec each, applied every 30 min yielded 36% blastocysts in vitro when fusion between 1/32 blastomere and enucleated oocyte occurred after the first pulse, whereas 22% blastocysts were obtained when it occurred after the sixth pulse [10]. Blastomeres 1/32 synchronized in G1 yielded up to 71% of blastocysts when the six-pulse method was used [16].
Procedures aimed at reducing MPF levels In the work by Stice and Robl [8], activation of oocytes just after their isolation (16–20 h after hCG) was compared with that of oocytes that had been aged in vitro (20–24 h after hCG). Only 4% of young oocytes could be activated as opposed to 52% of old oocytes. This suggested the beneficial effect of aging on recipient oocyte competence for NT. To reduce the level of MPF in the recipient cytoplasm, rabbit oocytes recovered either 19 h postcoitum (hpc) or 14 hpc, were aged in vitro for 5 h, and then activated and fused with 1/32 blastomeres [17]. Oocytes aged 19 hpc yielded 31.5% blastocysts after NT versus 3.7% in the 14-hpc control group.
Preactivation of 15-hpc rabbit oocytes with ionomycin (5 min) and 6-dimethylaminopurine (DMAP) (2 h) before introducing 1/16 blastomeres by cell fusion produced 39.3% blastocysts (versus 15.1% in controls). The second series of cloning by this method supplied 35.7% of blastocysts [14]. Blastocyst formation in the preactivated group shown in this paper suggests that electric field preactivation is even more effective (56% vs. 15%). The postactivation method, when inositol 1,4,5-triphosphate and DMAP were applied 5 h after electrofusion, yielded fewer blastocysts (29.6%) [18], although nuclear donor fibroblasts were presumably synchronized in G0 by serum deprivation.
Synchronization of blastomeres in G1 Positive effects of synchronizing nuclear donor cells in G1 for development of NT rabbit embryos were proved by Collas et al. [16]. Blastomeres 1/16, which were used for NT 1 h after mitosis, yielded 59% blastocysts in vitro, whereas 31% blastocysts developed from blastomeres that had been used 3–4 h after mitosis. A method of synchronizing blastomeres in G1 of the cell cycle was worked out that consisted of applying colcemid followed by aphidicolin. Blastomeres 1/32 synchronized in G1 promoted preimplantation development to an even higher level: 71% of blastocysts developed as opposed to 15% from blastomeres in S phase [16].
Here, 1/8 blastomeres synchronized in G1 by nocodazole yield a 42% rate of development as opposed to 15% in the untreated control group. Regretfully, this effect was lost in the second round of cloning.
Cell numbers of blastocysts It is difficult to directly compare cell numbers of NT blastocysts because the duration of in vitro culture differed between experiments. The highest cell number reported (109 ± 15.77) was accompanied by 123.66 ± 13.77 cells in control blastocysts [5]. The number achieved in the present study (89.2 ± 25.8 vs. 117.7 ± 12.9 in controls) is comparable although lower than that in the report by Stice and Robl [8], which was 91 ± 10.2 vs. 106 ± 5.1 in controls. The comparison of the cell number of blastocysts between three NT series (the present paper) suggests no change in subsequent rounds.
Postimplantation Development of NT Rabbit Embryos
In the first NT in the rabbit, 3.7% of reconstituted embryos (6 out of 164) developed into normal young after embryo transfer [8]. After three DC pulses, Collas and Robl [9] obtained 23 young out of 230 reconstituted embryos (10%), which was 21% of (110) two and four-cell embryos transferred. Yang et al. [12] reported 3% (8 out of 243 cleaving embryos transferred) of the young born after NT from in vitro-cultured 1/32–1/64 blastomeres. Three young born after transfer of 67 eight-cell embryos (4.5%) were obtained here. Although this developmental rate is low, the birth of young that originated from the third round of NT is worth noting.
Preactivation in Other Species
In the sheep, preactivation of oocytes before introducing 1/16 blastomere nuclei improved development to blastocysts (55.4% vs. 21.3% in controls) [19]. In the cow, preactivation of oocytes before fusion with 1/8 blastomeres yielded 16%–17% blastocysts vs. 6%–10% in controls [20]. In this case, minor improvement might be attributed to control oocytes being referred to as "aged" (35–37 h of maturation), which, by itself, markedly increases activation rate and reduces MPF level as shown in rabbit NT embryos [17]. Applying preactivation to serial NT from cow morulae resulted in obtaining 20%–37% blastocysts [21]. Preactivation was also applied to the goat, in which fusing 1/32 blastomeres with enucleated oocytes resulted in 25%–31.6% development to morulae in vitro [22] and in the cow, for recloning first-generation morulae obtained after NT from adult cells, in which 57.5% of morulae and blastocysts developed [23].
Efficiency of Serial Cloning
Some theoretical problems related to NT, such as oocyte aging and applying serial NT, have already been addressed in the paper by Stice and Robl [8]. These authors suggested that serial transfer would be beneficial for developing advanced (donor) nuclei and for multiplying the original (eight-cell) embryo. For the former purpose (i.e., enhancement of remodeling of donor nucleus in recipient cytoplasm), serial cloning was successfully used in the mouse [24, 25] and in the cow, in which adult cells served as nuclear donors [23].
To multiply the original embryo, four rounds of cloning were achieved in the cow, with 17%–36% of morulae in subsequent series and a decreasing rate of calving (10%, 2%, 3%, 0%) [6]. Ectors et al. [26] reported 12.9% and 14.9% of blastocysts and 20% newborn in two rounds of NT from morulae. Takano et al. [21] obtained 20%–37% of blastocysts in three NT series and 25% offspring (two calves) in the third series. In the goat, the rate of development to morulae was 25%–31.6% in six series of cloning from 1/32 blastomeres. However, an unusually high percentage (30%–33%) of postimplantation development was claimed in five subsequent rounds of cloning [22], which requires confirmation from other laboratories. In rabbits, this paper is the first to report successful preimplantation development (47.2%–56.3%, Table 2, preactivated group) of three series of cloned embryos, and full-term development of the embryos from the third series. The calculated number of clones per one round of cloning was 4.5–3.8 (see Results). If this parameter is calculated for the results of Stice and Keefer [6] on cow embryos developed after NT from 28- to 41-cell embryos, the number of clones is 5.9–3.0. For goat embryos [22] it is as high as 10–8; however, if, on the other hand, the rate of development obtained in the present paper (0.56–0.47 for the preactivated group, see Results) was achieved using 30-cell embryos as donors of nuclei for NT, which is comparable with those used by the authors discussed earlier [6, 22], then the number of clones per one round could reach 17–14. Speculative as it is, this comparison shows that donor nuclei for embryo cloning should possibly originate from the most advanced stages of preimplantation embryos.
The number of rabbit morulae/blastocysts obtained from individual eight-cell embryos in three rounds of NT (up to 27) is obviously lower than what was reached in the cow, when morulae were used as individual nuclear donors. After two rounds of NT, 21 morulae and blastocysts were obtained [26], and after three rounds, 54 morulae from two single embryos [6] and 43 blastocysts from a single embryo [21] were produced. However, the yield of successfully developed embryos in serial NT experiments is still limited by the availability of recipient oocytes and by the number of scientists simultaneously performing NT rather than by biological efficiency of the system employed.
ACKNOWLEDGMENTS
The authors are grateful for the technical assistance offered by Mr. Bogus
aw Wa
, who injected the rabbits with hormones. K.P. thanks Dr. Andrzej Guszkiewicz for introducing her to micromanipulation.
FOOTNOTES
First decision: 2 February 2000.
1 Financed by grant 5 PO6D 01910 from the Committee for Scientific Research. 
2 Correspondence. FAX: 4822 756 1699; panighz{at}atos.warman.com.pl
Accepted: April 3, 2000.
Received: December 9, 1999.
REFERENCES
ski JA, Smorg Z. Preimplantation development of rabbit embryos after transfer of embryonic nuclei into different cytoplasmic environment. Mol Reprod Dev 1991; 28:361–372.[CrossRef][Medline]ski JA. Effects of electric field on fusion rate and survival of 2-cell rabbit embryo. J Embryol Exp Morphol 1986; 96:211–228.[Medline]This article has been cited by other articles:
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  E. Popova, M. Bader, and A. Krivokharchenko Full-Term Development of Rat after Transfer of Nuclei from Two-Cell Stage Embryos Biol Reprod, October 1, 2006; 75(4): 524 - 530. [Abstract] [Full Text] [PDF]
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S. Novak, F. Paradis, C. Savard, K. Tremblay, and M.-A. Sirard Identification of Porcine Oocyte Proteins That Are Associated with Somatic Cell Nuclei after Co-Incubation Biol Reprod, October 1, 2004; 71(4): 1279 - 1289. [Abstract] [Full Text] [PDF]
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S. M. Mitalipov, R. R. Yeoman, K. D. Nusser, and D. P. Wolf Rhesus Monkey Embryos Produced by Nuclear Transfer from Embryonic Blastomeres or Somatic Cells Biol Reprod, May 1, 2002; 66(5): 1367 - 1373. [Abstract] [Full Text]
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