The study suggests that even apparently normal cloned animals may have subtle abnormalities in gene expression, say the Science authors, led by Rudolf Jaenisch at the Whitehead Institute for Biomedical Research. Many of the cloned mice in the study survived to adulthood, however, meaning that mammals may be more tolerant of this type of widespread gene dysregulation during development than previously suspected.
As experiments with several animal species have shown, the process used to create clones, called nuclear transfer, is an inefficient one. Most cloned embryos, derived from the insertion of a donor nucleus into an emptied egg, die before birth. The clones that do survive frequently have respiratory and circulatory problems, and abnormally high birth weights.
Some researchers have suggested that abnormal expression of certain genes, called imprinted genes, may be the culprit behind these large offspring, since imprinted gene regulation has been linked to fetal growth. Imprinting is a unique "epigenetic" phenomenon--a variation in gene expression that occurs without variation in the actual DNA sequence of the genes themselves. The expression of imprinted genes is dependent on which parent transmitted the genes in question. In certain cases, a gene passed on by your mother would not be expressed, for instance, while the identical gene from your father would.
The Science researchers decided to test this link between abnormal imprinted gene expression and the peculiar biology of clones. Jaenisch and colleagues examined gene expression for several imprinted genes in the placenta and kidney, heart, and liver of mice cloned from embryonic stem cells. Unlike normal mice and mice created by in vitro fertilization, the expression of these genes varied widely between placentas and tissues of the clones, the researchers found.
They also discovered that the abnormal expression of one particular gene, called H19, was linked to a chemical alteration in part of the gene called methylation, another epigenetic phenomenon. Methylation, the addition of methyl groups to particular DNA nucleotides, makes the gene harder to transcribe and therefore lowers its expression levels. Degrees of methylation of H19 varied between the clones.
Although most of the mice showed increased birth and placental weights, along with the changes in gene expression, the researchers didn't find any significant correlation between any of the specific variations in gene expression and increased weight. The cumulative action of many abnormally expressed genes may be necessary to produce the overgrowth in clones, say the researchers in their report.
After documenting the changes in gene expression for the clones themselves, the investigators turned their attention to a second question: were these abnormalities produced by the cloning process itself, or did they already exist in the embryonic stem cells used to create the clones?
The scientists found that variation in imprinted gene expression was rampant among the stem cells as well. Wide variation in expression was observed between different embryonic stem cell lines, between individual colonies of cells, or subclones, of each line, and even between individual cells within each colony, surprising the researchers.
This research suggests that extreme variability in gene expression may be the hallmark of embryonic stem cells, noted Jaenisch.
The researchers are interested in finding out whether the epigenetic state of human embryonic stem cells cultured for clinical applications will be as unstable as that of mouse embryonic stem cells. Jaenisch emphasized, however, that the unstable nature of the mouse cells doesn't interfere with their in vitro differentiation into different cell types, and doesn't impair the creation of normal mice generated by transplanting these embryonic stem cells into very early stage embryos called blastocysts.
The other members of the research team include D. Humpherys, K. Eggan, K. Hochedlinger, W. Rideout III, D. Biniszkiewicz, and R. Jaenisch at Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology in Cambridge, MA; H. Akutsu and R. Yanagimachi at U. of Hawaii in Honolulu, HI. This research was supported in part by the Victoria and Bradley Geist Foundation, the Kosasa Family Foundation, the Harold Castle Foundation, and NIH.