Overview of SCNT Method

The cloning method requires the substitution of a somatic cell genome in place of a normal embryonic genome (see Figure 46.1).

Early efforts to accomplish this using fertilized 1-cell cytoplasts as recipients failed to produce advanced development. It is now clear that the oocyte possesses a myriad of activities that mediate important early events that normally prepare the incoming sperm-derived nucleus for function. One such activity is the ability to disrupt the nuclear envelope (nuclear envelope breakdown, or NEB) (Latham, 1999). Another is the ability to exchange chromatin proteins. Normally, prot-amines must be removed and replaced with histones.

Cloning in Mouse

Remove SCC

Cloning in Mouse

Remove SCC

Lyse somatic cell, Remove cytoplasm, Inject nucleus into ooplasm using piezo

For large nuclei, use electrofusion

Egg Activation Pseudo PN

Lyse somatic cell, Remove cytoplasm, Inject nucleus into ooplasm using piezo

Egg Activation Pseudo PN

Cleavage ^

50-60% Blastocysts

-> Term Development

Figure 46.1 Basic steps of cloning in mice. The MII stage oocyte genetic material is first removed along with the second meiotic spindle. A donor nucleus is lysed with a narrow bore pipet and the nucleus injected into the oocyte using a piezo pipet drill to assist. If donor cells are larger and have larger nuclei, electrofusion can be employed. During the following hour, the nuclear envelope breaks down, chromosomes condense, and a new spindle forms. The oocyte is then activated to initiate cleavage development. As indicated, development to term remains somewhat inefficient.

Additionally, oocyte-specific chromatin proteins typically are assembled onto the incoming sperm DNA (e.g., oocyte-specific histones; Latham (1999); Gao and Latham (2004)). Another activity is the ability to direct nuclear envelope formation and DNA decondensation, leading ultimately to the transcriptional activation of the genome (Latham, 1999). All these activities are devel-opmentally regulated, and essentially lost within a few hours of oocyte activation (Latham, 1999).

Somatic cell nuclei need to recapitulate the same sequence of events as does the incoming sperm nucleus, but when zygote stage recipients are employed, these activities are no longer present, the corresponding processes do not occur, and embryos fail to develop. For this reason, oocytes are most often employed for nuclear transfer, and this typically involves the use of metaphase II (MII) stage oocytes. The first step in the procedure is to remove the oocyte genome, typically by aspirating the spindle-chromosome complex (SCC) into a small glass micropipette. This can be accomplished by a variety of means, each particularly suited to a given species. It should be noted that this procedure eliminates not only the oocyte DNA, but also other factors that are specifically associated with the SCC. After SCC removal, the donor cell nucleus can be introduced. There are three basic kinds of methods employed for nuclear transfer to MII stage oocytes. One involves fusion of the entire donor cell to the oocyte. Fusion is accomplished most often by electrofusion, wherein one or more electrical pulses are delivered in order to induce membrane fluidity changes that lead to fusion. Some alternative fusion methods involve the use of inactivated Sendai virus, or chemicals such as polyethylene glycol coupled with the use of phyto-hemagluttin. Fusion methods have the advantage of being somewhat noninvasive, but the disadvantages are that the entire contents of the donor cell are transferred to the oocyte, and that the efficiency of cell fusion is reduced by smaller donor cell size, which reduces physical contact between donor cell and oocyte. The second method of nuclear transfer involves simple injection of the nucleus into the oocyte. This works with some species, but not with all (e.g., rodents), due to an increased propensity for the oocyte to lyse after injection. The third method, first applied in rodents, is to employ a piezo-assisted pipette driver, which permits microinjection of nuclei, for example, into rodent oocytes, without lysis. With the injection methods, the donor cell can be lysed and repeatedly aspirated and expelled in order to remove the bulk of the cytoplasm before injection.

Once the nuclear transfer has been accomplished, typically one hour or more is permitted for the ooplasm to act upon the nucleus, bringing about NEB and then condensation of chromosomes (CC) and their assembly on a newly formed spindle. Then, the nuclear transfer construct is activated to begin development, whereupon pseudo-pronuclei form and the embryo eventually undergoes cleavage. Activation can be accomplished either as a result of electrical pulses (in some cases delivered at the time of fusion) or the use of chemical treatments, such as the use of ionophores, cycloheximide, 6-dimethylaminopurine (DMAP), or strontium chloride in calcium-free medium. Recent studies in fertilized embryos revealed a potentially important role for sperm-induced calcium oscillations in long-term embryo viability, with particular relevance on the amplitude, number, and frequency of such oscillations (Ducibella et al., 2002). As such oscillations likely are not recapitulated during artificial oocyte activation, cloned embryos may be initially disadvantaged relative to fertilized embryos due to an absence of correct calcium signaling and downstream events.

Once the cloned constructs are activated, they must be cultured in vitro for some period before being returned to the reproductive tract of a suitable foster mother for continued gestation. Recent studies reveal that cloned embryos have radically different culture medium requirements as compared with fertilized embryos (Latham, 2004). This is most likely the result of continued expression of genes from the donor cell nucleus. Reprogramming of gene expression appears to be a rather prolonged process, possibly continuing until just before the time of gastrulation. As a result, when gene transcription commences in the early cloned embryo, many somatic cell-expressed genes will be aberrantly expressed in the embryo. The products of these aberrantly expressed genes likely alter basic processes of osmo-regulation, homeostasis, and metabolism (these processes differ substantially between somatic cells and normal fertilized embryos). Thus, although traditional embryo culture media have been most widely employed for culturing cloned embryos, such media are likely to be grossly suboptimal. In fact, somatic cell formulations have produced superior results in several instances (Latham, 2004). The cloning technique is thus faced with a fundamental problem: in order to achieve continued development, embryos must be returned to the reproductive tract, and yet the environment there is likely optimized for fertilized embryos and thus poorly suited to cloned embryos. If we accept the possibility that nuclear reprogramming continues up through the time of gastrulation, then it is likely that the exposure of the cloned embryos to a suboptimal environment, either in vitro or in vivo, inhibits the reprogramming process and reduces clone viability.

The overall outcome of the cloning procedure is that only about 1 to 5% of all nuclear transfer constructs develop to term. About half of all constructs can complete preimplantation development, and then there are additional waves of loss during the peri-implantation period, and around the time of gastrulation (Yanagimachi, 2002). From the standpoint of contemplating therapeutic uses for cloning, it is fortunate that a substantial number of constructs can form blastocysts and eventually give rise to an inner cell mass population, from which can be derived embryonic stem cells (ESCs).

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