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Lawrence Roberge

Author, Bioethicist, Biomedical Researcher
Posted October 18, 2004

Cloning: Scientific, Technological, and Ethical Considerations
Part I

ABSTRACT:

This paper will examine the technology of cloning, both non-human and human. The paper will first examine the advantages of cloning non-human animals in such applications as tissue transplantation, pharmaceuticals, and species restoration. Also, the paper will examine the environmental shortcomings of non-human cloning, such as limiting genetic diversity.

The paper will next explore human cloning from the theoretical aspects as well as examining various international and business strategies underway to achieve human cloning. Also, the paper will attempt to distinguish the forces of nature and nurture as it relates to human cloning. Finally, the paper will briefly discuss the effects of mitochondrial DNA on the creation of "pure" human genetic clones. Various technical, ethical, and moral problems from a scientific, social, and Catholic viewpoint will be discussed.

KEY WORDS: BIOETHICS, BIOPHARMING, EUGENICS, CHIMERIA, CLONING, EMBRYO, EMBRYO SPLITTING, HUMAN CLONING, MITOCHONDRIA, NUCLEAR TRANSFER, NUCLEAR TRANSPLANTATION

This paper was presented at the OCTOBER 22-23,1998 Society of Catholic Social Scientists Conference (Franciscan University of Steubenville).

1. INTRODUCTION

Cloning is by definition the replication of an exact genetic copy of an organism via use of a somatic tissue (or cell) from the donor organism. The word, clone, comes from the Greek word (klon), meaning twig. Gardeners and horticulturists for centuries have created a duplicate plant from a sliced off piece of the original plant. Zoologists are familiar with cutting off an arm of a starfish to create a clonal duplicate of the original animal. Recent developments in reproductive technology have brought cloning technology and the issue of human cloning to the forefront (7, 8, 19, 29). This paper explores the scientific, technological, and ethical considerations of cloning technology as well as the questions raised by the technological development of human cloning.

2. METHODS OF CLONING

First, a brief explanation of the two primary types of cloning techniques is required. The two methods are embryo splitting and nuclear transfer (AKA nuclear transplantation). Although each method, at present, requires gestation in the uterus to attain complete development (i.e. live birth), each method is different in the source of the cloning material.

Embryo splitting is a method where an embryo at the 2, 4, or 8 cell stage has the cells (referred to as blastomeres) separated. Each cell at this stage is believed to retain the property of "totipotency". Totipotency is the property where the genetic material has not been programmed to develop into a select tissue yet, as such, the cell may develop into a complete new organism. The cell is considered to be in an undifferentiated stage and thus it can develop into a variety of tissues (and even a complete organism under the proper set of conditions).

Modern techniques begin with the stripping of the embryo of its protective layer, the zona pellucida. After each blastomere has been separated from the embryo mass, the cell is encased in its own protective synthetic zona pellucida layer, usually derived from seaweed (1,2). Each blastomere cell, is now considered a new separate embryo-a clone-from the original donor embryo, and is cultured in vitro and later in vivo in a surrogate mother until birth. This process of embryo splitting is akin to the method by which identical twins (AKA monozygotic twins) are believed to be created (3).

This technique has been successful in cloning sheep in 1979 (2) and was attempted on defective human embryos at George Washington University by Dr. Robert J. Stillman and Dr. Jerry L. Hall in 1993 (4). If this technique was used by In Vitro Fertility (IVF) clinics, it could help increase the available number of embryos for infertile couples (67). But due to the present controversy over human cloning and the uncertainty of the risk of damaging the embryo, most IVF clinics refuse to perform the procedure (67).

The second technique, nuclear transfer, has been met with moderate success with frogs in the 1950's through the 1970's (See 5 for an excellent summary of amphibian cloning). In the 1980's, researchers reported success using the nuclear transfer technique in mice (5, 50) in 1983 and in sheep (6) in 1986. McGrath and Solter (50) developed a nuclear transfer technique in mice, where a donated nuclei was injected between the plasma membrane of an enucleated oocyte and the zona pellucida. Using the micropipette to insert the nuclei next to the plasma membrane, the researchers used inactivated Sendai virus to fuse the donor nuclei into the oocyte cytoplasm.

Although Willadsen referred to his studies as nuclear transplantation (as did McGrath and Solter), he in fact transplanted a blastomere cell into an enucleated sheep oocyte. Subsequently, he induced donor nuclei and oocyte fusion using the inactivated Sendai virus technique. More recent techniques use a donor nucleus (from adult, embryo, or cultured cell lines) stripped away from the cell and then insert the nucleus into an enucleated oocyte (7, 8, 11). When the newly formed embryo is activated with the proper stimulation, which may range from chemical (10) to electrical stimulation (7), the nucleus begins the process of division and development into a morula.

These successes in the 1980's led the way for further success with sheep in 1996 and 1997 (7,8), primates in 1997 (9), cattle in 1997 (10), and mice in 1998 (11). But more on this later.

The concept of nuclear transfer is based on cell biology concept that the animals' genetic makeup (AKA genome) is located in the cell nucleus. The only exception to this rule is a small loop of DNA about 16,000 bases in length residing in the mitochondria, which code for some mitochondrial proteins (12).

As each cell becomes a differentiated tissue (skin, bone, liver cell, etc.), the genes for the cell are programmed to selective gene expression for that particular tissue. Therefore, to use the nucleus from a differentiated cell requires techniques to re-program the DNA to return to an undifferentiated state. The challenge to the researchers was to devise ways to induce re-programming to the nucleus.

Also, the nuclear DNA must have the characteristic of ploidy stability. That is, the chromosome number of the donor cell must be 2N (diploid state) and thus the chromosomes must not exhibit duplication efforts or breakage as the clone is being created. Previous studies of frog cloning (5) failed in part due to nucleus of the cell underwent DNA replication. If the cell enters the S stage (DNA replication) of the cell cycle, the subsequent cloning attempts will yield chromosome breakage or aneuploidy (excess or shortage of chromosomes for the diploid state). It is ideal to obtain a nucleus of a cell in the cell cycle stage prior to the S stage, know as the G1 stage. Also, many cells undergo quiescence or suspend the cell cycle reproduction stage. This is referred to as a G0 stage. Cells obtained in the G1 or G0 stage are prime targets for nuclear transplantation efforts (11,12). It is believed that the methods of cell culture using media starvation by the Wilmut group (7, 8, 12) and the method of a time delay between nuclear transfer and embryo activation by the Yamagimachi group (12) may allow the cells to achieve genomic re-programming and inducement of G0 status.

There are a host of other factors that extend beyond the scope of this paper (see 12 for Campbell review) that may play a role in nuclear transplantation techniques. Even the work by Wakayama et al (12), suggests that other post-activation steps may play a role in the survival of the cloned embryo and may help determine the success of a live birth. These points will be important to keep in mind later in the paper.

3. ANIMAL CLONING SUCCESS

After the frog cloning studies (5), researchers pursued more complex animals. By 1979, Willadsen has successfully created cloned twin sheep using embryo-splitting techniques. In the early 1980's, nuclear transplantation techniques had yielded cloned mice (50). In 1986, Willadsen's version of nuclear transplantation yielded the birth of three cloned lambs. These lambs came from "reconstituted" embryos in which a blastomere cell from an 8-cell embryo was combined with an enucleated half of an unfertilized egg.

The decade of the 1990's has produced a wealth of cloning milestones. In 1996, two lambs, Megan and Morag were born. Dr. Ian Wilmut's team from the Roslin Institute (United Kingdom) has created the lambs using nuclear transplantation from the cultured embryo-derived epithelial cell line, TNT4 (7). In early 1997, Dr. Wilmut's team announced the birth of Dolly, the first cloned lamb originating from an adult mammary gland cell (8). The technique again relied on nuclear transfer and both studies used techniques of cell culture media starvation to induce the donor cells into a quiescent (G0) state. Although in early 1998, a letter by Sgaramela and Zinder questioned the true clone status of Dolly (13). The Wilmut team responded to the letter (14) and later the Wilmut team and an independent lab using several methods of genetic analysis verified the clone status of Dolly (15,16,17).

Later in July 1997, the Wilmut team announced the successful birth of cloned transgenic lamb (named Polly) that came from a transgenic fetal fibroblast cell line (18, 51). In December of that year, the Wilmut team published their results in the journal, SCIENCE (18). The cell line had inserted into its genome a gene for the coagulation blood protein, Factor IX. The scientists plan to apply this technique to cows, which produce more milk than sheep (51). Using this technique, this blood clotting protein can now be expressed in the milk of these clones and thereby expand the biopharmaceutical production of this valuable drug and accelerate it into the marketplace.

In March 1997, Dr. Don Wolf and other researchers at the Oregon Regional Primate Research Center announced the cloning of rhesus monkeys (8, 19). Using nuclear transplantation techniques, the researchers extracted the nucleus from an 8-cell stage embryo and transferred it into an enucleated oocyte.

In August 1997, ABS Global of De Forest, WI, announced the birth of Gene, a cloned calf. The ABS Global method used a method similar to Willmsen (6). A fetal donor calf cell is fused to an enucleated oocyte, cultured to 16-cell stage, then the blastomere cells are separated and each cell is then fused to new enucleated oocyte. Each fusing procedure used a jolt of electricity (electrofusion) to activate the embryo. Finally, the embryos were implanted into surrogate cows.

In January 1998, a team from the University of Massachusetts at Amherst and from Advanced Cell Therapeutics (Worcester, MA) announced the birth of three cloned calves (49, 52, 53). The researchers created 276 cloned embryos, from which 3 healthy calves were born. The team used fetal fibroblasts as the donor nuclei source. The reasoning for using this cell line was that this cell has a long G1 phase and would therefore be a good candidate for donor nuclei.

In January 1998, Dr. Neal First from the University of Wisconsin at Madison announced the cloning to the pre-implantation embryo stage of clones created from cow oocytes and nuclei from other species, including: sheep, pigs, rats, cattle, and primates (44, 45). The clones are interspecies since their oocyte come from a species other than the cow. This research suggests that the molecular machinery responsible for reprogramming the genome by the cytoplasm may be similar or identical in all mammals.

In 1998, Dr. Ryotu Yamagimachi and his team from University of Hawaii achieved the cloning of mice using nuclear transfer techniques with cumulus cell nuclei (10). The cumulus cells taken from an adult mouse were used as the source of donor nuclei because cumulus cells do not normally divide in adult mice, but remain at the G0/G1 state in the cell cycle. The cloning technique used a piezo-impact pipette drive to reduce the time lag during donor nucleus insertion into the oocyte. Also, the study allowed for a time delay between nuclear transfer and oocyte activation. These techniques may have increased the success rate of blastocyst development (from 39.9% to 6.9%), but the live birth rate remained low (2.8%). The Yamagimachi team suspects several other developmental steps may play a role in successful development from post-implantation embryos to live births (10).

One important detail must also be noted at this point. Despite the successful achievements in the 1990's, all studies have found that the return rate (cloned embryo to live birth) was exceedingly low. For example, the birth of Dolly came after 276 cloning attempts (8). Also, the Yamagimachi team's low rate (2.8%) was achieved after 603 oocytes were injected with donor nuclei. For this technology to become readily available, the techniques must be improved to increase the cloning success rate. This may also require further studies to explore, as the Yamagimachi team hinted (10), if other post-activation steps may have to be mastered to achieve a high cloning return rate (i.e. live births).

© Lawrence Roberge 2004 Reprinted with Permission

LAWRENCE F. ROBERGE M.S.is a biotechnology consultant, college instructor, bioethicist, and biomedical researcher. He is the author of the new book, THE COST OF ABORTION (Four Winds Publications, LaGrange, GA, 1995). He has consulted for pharmaceutical, medical, and biotechnology corporations across the United States, Canada, and Europe. He has published research on neuroscience, biomedicine, abortion vaccine technology, and the adverse effects of abortion on women.