Cloning

Cloning is the creation of a clone. A clone is a set of organisms that are genetically identical. Plants in the wild produce clones by asexual reproduction. In nature, animals can produce clones by the division of a post zygotic state into separate organisms. In humans identical twins are a clone. Clone is a functional definition and to a certain extent no truly genetically identical set of organisms exists, since post zygotic changes in the genome, epigenetic changes, and variations in mitochondrial and plastid genomes are certain among any individuals from a single zygote. Sexual reproduction is the antithesis of a clone and is a process in nature to produce genetically variant offspring.

Technique

The cloning of mammals has revolved around a technique known as somatic cell nuclear transfer, which involves the removal of the nucleus from an oocyte (a precursor cell of female gametes that develops into gametes via meiosis). The nucleus of a somatic cell is then implanted in the oocyte, which is then transferred to a surrogate mother. Somatic cell nuclear transfer was first successfully used to clone a sheep. In that study, somatic cells "were obtained from... [the] mammary gland [of]... a 6-year-old Finn Dorset ewe in the last trimester of pregnancy." The cell cycle of the donor cells was stopped in the G0 stage. The "oocytes were recovered from Scottish Blackface ewes between 28 and 33 h after injection of gonadotropin-releasing hormone", after which the nuclei of the oocytes were immediately removed. The donor cells were implanted in the oocyte via "fusion of the donor cell to the enucleated oocyte and activation of the oocyte... induced by... electrical pulses." The resulting embryos were cultured and implanted into surrogate ewes. "Lamb number 6LL3", latter named "Dolly", was born as a result of this process, and was confirmed to be genetically identical to the ewe used as a somatic cell donor through "DNA microsatellite analysis... at four polymorphic loci." The study also involved nuclear transfer with fetal cells, and several lambs were produced using this technique. On February 14, 2003, Dolly was euthanized because she was suffering from ovine pulmonary adenocarcinoma. Dr Harry Griffin, the Acting Director of the Roslin Institute, stated that this was unrelated to the fact that Dolly was cloned. Further cloning has been performed on many other mammals, including dogs.

Safety of Cloning

Some evidence suggests that cloning may be hazardous to the cloned animals. According to the "telomere hypothesis of ageing", ageing is caused by telomeres shortening each time a cell's DNA is replicated. A recent study found telomere length in normal sheep was negatively correlated with age:

The mean size of the terminal telomere fragment obtained by cutting with restriction enzyme (the mean terminal restriction fragment, or TRF) was found to decrease in control animals with increasing age, at a mean rate of 0.59 kilobases (kb) per year. A linear regression analysis of the sheep DNAs yielded a significant result...

Furthermore, Dolly had substantially shorter telomeres than a normal sheep of the same age:

Mean TRF sizes were smaller in all three nuclear-transfer animals than in age matched controls. DNA in 6LL3[Dolly] showed the greatest diminution of mean TRF size for a one-year-old animal (19.14 versus 23.950.18 kb). The size difference is significant (P*0.005) compared with the age matched control animals.

The authors suggest that it is probable that the telomeres in Dolly's cells were shorter because she was cloned from a somatic cell which had itself experienced telomere shortening due to aging. However, they question if telomere shortening will actually cause significantly adverse effects on Dolly's health. It would seem, however, that if repeated cloning of animals that were themselves clones caused telomere lengths to decrease with each successive generation, a severe health hazard might result. The problem of "cloning of clones" presently appears to be unstudied.

Yet if the heath hazards associated with telomere shortening seem conjectural, the increased mortality associated with pregnancies with cloned animals is a certainty. In the study in which Dolly was cloned, "62% of fetuses were lost, a significantly greater proportion than the estimate of 6% after natural mating." This effect may be attributable to the suppression of genes that allow cells to properly differentiate in the developing embryo. A recent study examined this phenomenon in mice and found that 37.5% of embryos created by somatic cell nuclear transfer did not express one or more of the 11 genes selected because of their relationship to successful cell differentiation. However, 100% of normal embryos expressed these genes:

We examined expression of the Oct4 and 10 Oct4-related genes in a total of 48 individual cumulus cloned preimplantation embryos that developed to phenotypically normal blastocysts, after transfer of a donor cumulus cell nucleus into enucleated oocytes. While each of 15 control embryos derived from fertilized zygotes expressed all 11 genes, 18 of 48 cloned blastocysts failed to correctly reactivate expression of at least one of the 11 test genes.

Furthermore, embryos that activated the 11 genes studied had a far greater probability of maturing to normal birth:

Analysis of expression of the Oct4-related genes in somatic and ES cell-derived clones revealed that successful reactivation of the full set correlates with development of cloned embryos to term.

The health hazards of cloning do not end with birth, however. Instead, a recent research review found that clones suffer from a variety of illnesses throughout their lives:

Many cloned animals display abnormalities at or after birth, which have been collectively termed large-calf or cloned-off-spring syndrome and include increased body weight, placentomegaly, pulmonary hypertension and respiratory problems...

These problems may be attributable to "incomplete epigenetic reprogramming of the somatic nucleus" which alters the expression of genes while retaining the same nucleotide sequence. The methylation of genes plays a particularly prominent role in the suppression of the expression of genes needed for embryo development in somatic cells.

Cloning in plants

Natural cloning is a common type of reproduction in the plant world. Examples would be strawberries which put out runners to start new plants that are genetically identical to the parent; or brambles that bend to touch the ground at the tip, creating new roots that form a clone plant.

In addition, artificial cloning has been common for plant propagation since ancient times. Cuttings, either as self rooted new plants or as scions for grafting are taken from the growing tips of plants. Layering, where part of the plant is buried underground to stimulate rooting, or is wrapped in a moist medium and sealed in a vaporproof outer wrap, is another common means of reproduction. Artificial rooting hormone can be applied to speed up rooting. Once roots are established the new cloned plant can be cut from the parent. This is primarily a means of preserving desirable plant cultivars.

More recently cloning through tissue culture has been developed. A primary reason for the newer techniques is that cell reproduction can be stimulated to outrun virus replication, thus creating new stocks of virus-free cultivars.