Somatic Cell Cloning Efforts in the Monkey

As noted previously, nuclear reprogramming is thought to be the limiting parameter in our monkey somatic cell cloning protocol. Obviously, the reprogramming required after SCNT is more extensive than the process that occurs during embryonic cell NT. For normal full term development of cloned embryos, genes normally expressed during embryogenesis, but silent in the somatic donor nucleus, must be reactivated in an appropriate temporal and spatial manner. Despite the fact that even terminally differentiated donor cell nuclei can be reprogrammed in egg cytoplasm acquiring the capacity to support full-term development, only a few NT embryos develop to term and, of those, many die shortly after birth in all species where cloning has been successful. The most likely explanation for such outcome is the inability of the cytoplast to reprogram the epigenetic profile of the somatic donor nucleus to that of the fertilized zygote. Our current research on somatic cell cloning in the monkey is focused on the effect of various cell types (younger fetal fibroblasts, cumulus, oviductal epithelial, embryonic stem cells) on NT development in efforts to screen for a more "reprogrammable" source of donor nuclei. Apparently, the primate egg cytoplasm lacks or contains insufficient levels of factors responsible for somatic chromatin remodeling; therefore, another logical approach is to induce nuclear remodeling in cultured cells before nuclear transfer.

The feasibility of such an approach is supported by the recent demonstration that denatured somatic cells subjected to heat treatment can be reactivated after NT with development to blastocysts in vitro and to viable offspring in vivo (11). Such treatments destabilize high-order nucleoprotein complexes and, presumably, denatured chromatin is more readily accessible to the oocyte's reprogramming machinery. Often, fusion of such cells with recipient cytoplasts is not possible because pre-treatment of donor cells results in irreversible membrane damage. We have explored alternative methods of introducing donor chromatin involving direct injection without using piezo manipulators with results comparable to those achieved for cell fusion (see Note 14).

4. Notes

1. The percentage of "nonresponders" varies by season showing an increase during the summer months, reaching more than 35% in June and July. During summer, despite housing in controlled, constant environments, many female monkeys also become anovulatory, and it is impractical to attempt COS (12). Females can be recycled for COSs; however, the response to recombinant human gonadotropins is decreased gradually with increasing numbers of stimulations, apparently the result of an immune reaction. Practically, as many as three stimulations on average can be performed per female monkey, with the recovery of a reasonable number of high quality oocytes. The availability of monkey recombinant gonadotropins would allow the more efficient and extended use of females.

2. The time between aspiration and oocyte recovery should be minimized to avoid the detrimental effects of blood exposure, which usually contaminates the aspirates. The conventional approach of diluting aspirates with medium and searching for oocytes under a dissecting microscope is labor intensive often requiring two to three technicians. The recovery time can be minimized by sifting the aspirates through cell strainers.

3. Before assignment to the project, each animal receives a physical examination from the veterinary staff and is fitted with a special neck chain (using pole and collar method) to facilitate restraint. Only animals with sperm concentrations greater that 60 million per milliliter and with more that 70% motile cells with normal morphology are acceptable

4. Priming is used on those animals that show little or no movement. If an animal is a mover in the chair, stimulation can begin immediately, moving the Current Adjustment dial without priming.

5. It usually takes approx 15 s for priming but when the Current Adjustment dial is moved up, the total time from post prime to the end of trial should be no more that 15-20 s. All samples will be collected within this time frame. If data are available from previous stimulations, the Current Adjustment dial is slowly adjusted to a level that had been previously successful for that particular animal. Total stimulus current time is left at this level for a maximum of 15-20 s during which ejaculation almost always occurs. If no sample is obtained, the stimulation is repeated within 1-1.5 min. No more that five consecutive stimuli attempts are made per animal on each trial. Each animal is given an abstinence period at least 48 h between sample collections.

6. Piercing the plasma membrane during the ICSI procedure is critical and often difficult to assess because the needle can grossly invaginate the membrane without breaking through. For that reason, the injection pipet should be inserted through the zona pellucida and "into" the oocyte across approx one-third of the egg's diameter. Cytoplasm is slowly aspirated into the ICSI pipet (as far back as the needle junction with the zona pellucida) until the plasma membrane brakes. A "pop" or sudden movement of cytoplasm into the pipet indicates the release of membrane tension. Once the membrane is penetrated, the sperm is expelled into the cytoplasm, visualized as a "dive" of the sperm. Frozen-thawed sperm can also be used for ICSI. We have previously reported that fertilization rates by ICSI with cryopre-served sperm injected shortly after thaw are significantly lower than with fresh sperm (13). However, preincubation times of more than 3 h after thaw were associated with ICSI fertilizing capacity similar to that seen with fresh sperm (14,15).

7. Preimplantation development of the primate embryo as in other mammals includes explicit developmental stages from the formation of the zygote after fertilization to cleavage, morula formation, compaction and finally cavitation with the formation of the blastocyst (Fig. 1). In HECM-9aa + FBS, blastocysts containing 100 or so cells can be seen by day 6 or 7 in vitro. Admittedly, in vivo rates are probably somewhat faster than in vitro, which may reflect suboptimal culture conditions.

8. Monkey oocytes are more fragile than those of the cow and easily lyse during enu-cleation if the cytoskeleton organization has not been "softened" by cytochalasin

Embryo Chimpanzee

Fig. 1. Rhesus monkey preimplantation embryo development in vitro. (A) Pronuclear stage zygote with male and female pronuclei, 12 h after fertilization by intracytoplasmic sperm injection. (B) Day 1, two-cell stage embryo (day of fertilization = day zero). (C) Day 2, four-cell stage embryo. (D) Day 3, eight-cell stage embryo. (E) Day 4, morula stage embryo. (F) Day 5, compact morula. Note that individual blas-tomeres have maximized their intracellular contacts and compacted into a tight cell mass. (G) Day 6 or 7, expanded blastocyst with a single flat layer of trophectoderm surrounding a fluid filled blastocoel and the inner cell mass.

Fig. 1. Rhesus monkey preimplantation embryo development in vitro. (A) Pronuclear stage zygote with male and female pronuclei, 12 h after fertilization by intracytoplasmic sperm injection. (B) Day 1, two-cell stage embryo (day of fertilization = day zero). (C) Day 2, four-cell stage embryo. (D) Day 3, eight-cell stage embryo. (E) Day 4, morula stage embryo. (F) Day 5, compact morula. Note that individual blas-tomeres have maximized their intracellular contacts and compacted into a tight cell mass. (G) Day 6 or 7, expanded blastocyst with a single flat layer of trophectoderm surrounding a fluid filled blastocoel and the inner cell mass.

B exposure. However, prolonged incubation in cytochlasin B during enucleation may cause excessive softening and swelling of the cytoplasm.

9. Penetration through the zona during enucleation is more difficult than in the ICSI procedure because of the larger size of the enucleation pipet, and in some cases, may require extending the pipet all the way to the other side of the egg.

10. As the pipet is pulled away from the egg, the plasma membrane will stretch and form a thin bridge between the egg and the material being removed. A rapid movement of the enucleation pipet toward 6 o'clock will break this cytoplast bridge.

11. Various artificial activation treatments have been developed that mimic sperm-triggered events and induce development of NT embryos. For example, ethanol,

Ionomycin Calcium
Fig. 1. (continued)

electroporation in calcium containing medium, ionomycin, or inositol 1,4,5-trisphosphate. However, M-phase-promoting factor activity at least in the young bovine and rabbit oocyte is quickly restored with recondensation of chromosomes and reentry of activated oocytes into a new M-phase arrest, known as MIII (16). This phase can be circumvented by additional treatments with protein synthesis (cycloheximide), protein phosphorylation (DMAP) or specific M-phase-promoting factor inhibitors (roscovitine). Thus, sequential approaches have evolved with ionomycin/DMAP, ionomycin/cycloheximide, or ionomycin /roscovitin that result in high activation rates in monkey NT embryos (17).

12. Stepwise transfer to fusion medium minimizes osmotic shock and reduces electrolyte contamination in the fusion chamber.

13. For information on optimal timing of embryo transfer, see ref. 15.

14. A blunt transfer pipet (5-7 |im outer diameter) is used to mechanically disrupt a single donor cell by aspiration in a 10% PVP drop. The nucleus is subsequently transferred into a conventionally prepared cytoplast via pipet passage through the hole in the zona and injection into the cytoplasm as described for the ISCI procedure. The efficiency of direct injection is comparable to fusion with 80-90% survival rate and 60-70% pronuclear formation rate.

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