Many animals derived from somatic cell nuclear transfer (SCNT) have abnormal phenotypes. Little is known in gene expression of cloned pigs by SCNT. In the present study, the expression profiles of deceased neonatal and one-month old live clones (n=5 for each age group) were compared with those of age-matched controls (n=5 for each age group) using a 13K oligonucleotide microarray. The cloned pigs were generated from fetal fibroblasts. Significantly reduced body weights and weights of the lungs (neonatal group) were found in cloned animals. Samples from the brain, kidney, and lung were chosen for microarray analysis to represent tissues from the endoderm, mesoderm and ectoderm in origin. In deceased neonatal clones, 88, 178, and 121 genes were differentially expressed in brain [fold change (FC)>1.5], kidney (FC>1.5), and lung (FC>2), respectively (p<0.05). Functional annotation and multiplex literature mining revealed disturbances in the following pathways: aberrant myelination and up-regulated MAPK signaling pathways in brain, diabetic nephropathy, down-regulated glycine, serine, and threonine metabolism and up-regulated MAPK signaling pathway in kidney, dysregulated surfactant homeostasis and up-regulated MAPK signaling pathway in lung. In live clones at one month of age, 55, 179, and 154 genes were differentially expressed in brain (FC>1.5), kidney (FC>2), and lung (FC>2), respectively (p<0.05). Abnormalities were found in the following pathways: neural excitotoxicity in brain, diabetic nephropathy and dysregulated adipocytokine signaling pathway in kidney, delayed alveologenesis, and down-regulated adipocytokine and MAPK signaling pathways in lung. Additionally, the maternal lineages of recipient oocytes used in SCNT were traced by sequencing the clones' mitochondrial DNA. Expression of mitochondrial genes in cloned animals, however, were not affected by maternal lineage of recipient oocytes. These findings demonstrate that phenotypic abnormalities found in clones are linked with dysregulated gene expression, and surprisingly, live clones have abnormal gene expression to a more extent than in deceased neonatal clones. Furthermore, epigenetic reprogramming in clones has occurred in a recipient oocyte-independent manner. (poster)

Hochedlinger and Jaenisch accurately summarized our paper's claims1 and then made three criticisms: (i) that our conclusions are not novel in light of previous work; (ii) that because our differentiated cell population is 99.4% (not 100%) pure, the clones we obtained from this population could be derived from a rare subset of undifferentiated cells and (iii) that our data are insufficient to compare the efficiency of cloning from differentiated versus undifferentiated adult stem cells. We address these points in turn. Our study addresses the efficiency of reprogramming somatic nuclei at different differentiation stages. We are surprised that Hochedlinger and Jaenisch question the novelty of our findings, given that they themselves, in a review published a few months ago2, identified this as an important question: "Whether the genomes of adult stem cells are similar to ES cells in that they are easier to reprogram than the genomes of terminally differentiated cells is an open question." Several excellent studies3, 4, 5, all of which we cited, have reported the derivation of mice by nuclear transfer from terminally differentiated cells. However, they differ from ours in that none of those studies addressed the efficiency of reprogramming of somatic nuclei at different stages of differentiation and none succeeded in making a cloned mouse directly. They had to resort to a two-step process where a donor nucleus from a differentiated cell was used to make a cloned blastocyst from which ES cells were derived and used to make a fetal-placenta chimera via tetraploid complementation procedures3, 4, 5 or for a second round of nuclear transfer4. These studies (with peer commentaries6, 7, 8, 9) differ from ours, as shown in Figure 1. Our studies are therefore the first unambiguous demonstration of nuclear totipotency of terminally differentiated cells by direct nuclear transfer. We found unambiguous, convincing morphological evidence that our population of purified differentiated granulocytes was 99.4% pure after FACS for Gr-1high expression, a different method1 from the one cited by Hochedlinger and Jaenisch. They suggest that the two cloned mice obtained from this population could have come from the 0.6% of cells that are undifferentiated, but we consider this extremely unlikely. Our estimate of 99.4% purity was based on morphological examination of 1,000 cells by two independent observers. The remaining 0.6% of observed cells were myelocytes or metamyelocytes, which are also lineage-committed terminally differentiated cells; we did not observe any hematopoietic stem cells or monocytes (Gr-1low), although monocytes are fully differentiated cells, too. Hochedlinger and Jaenisch argue that morphologically based identification is inherently unreliable. This is not true, as it is the gold standard for recognizing granulocytes in the field of hematology. The chance that the clones were contaminated by primitive HSCs, which constitute only 1 10-5 to 5 10-5 bone marrow nucleated cells even before sorting, is almost zero. We reported that cloning efficiency increases over the differentiation hierarchy in the hematopoietic cell lineage. Hochedlinger and Jaenisch did not agree with our conclusion because of their trivial explanation for our result: namely, that the low efficiency of HSC nuclei might be because (i) the nuclei of HSCs are more easily damaged by nuclear transfer, FACS or the freeze-thaw procedures or (ii) blastocyst development is not an acceptable measure for judging cloning efficiency, and generation of viable animals or ES cell lines is required for comparing cloning efficiency. This is an ad hoc criticism that could be leveled against any result, but we consider it unlikely as an explanation of our findings. We compared cloning efficiency of four types of hematopoietic cells with varying degrees of differentiation along a single lineage: long-term repopulating HSCs, short-term repopulating HSCs, hematopoietic progenitors and differentiated granulocytes, all from the same animal tissue and all isolated, purified, and frozen and thawed using the exact same standard method1. They argue the possibility of some mechanical damage to HSCs during FACS or freezing and thawing, which is very unlikely. Our validations showed that the FACS-isolated HSCs were highly functional in engraftment or culture assays1 and that HSCs either freshly isolated or used after freezing and thawing had similarly poor cloning efficiency in both our studies1, 10, suggesting that the freeze-thaw procedure had no impact. The efficiency of cloning, defined as the percentage of reconstructed embryos that reach morula/blastula stage, shows a consistent trend: efficiency becomes progressively higher as differentiation proceeds, with more than an eightfold difference in efficiency between the least and the most differentiated cells (4% versus 35%). This conclusion is based on over 3,000 nuclear transfer injections. Hochedlinger and Jaenisch argue that the efficiency of development to the blastocyst stage is not an acceptable measure of cloning efficiency. We disagree with their opinion, as this is a commonly used measure in most animal cloning reports, and we note that they3, 4 as well as others that they cited5, 10 have used similar measures in previous studies. While we agree that live births would be the most definitive measurement for totipotency, this is not feasible given the numbers that would be needed. We did transfer some cloned blastocysts derived from HSCs (n = 28, stated as "data not shown"), but none went to term. In our two studies, we failed to generate clones with B6D2F1 HSCs1, 10, although we (K.I.) produced 2 cloned pups from 302 (B6129)F1 HSC–derived embryos10. As many know, the 129 genome helps efficient genomic reprogramming, and it is not reasonable to do a direct comparison between different mouse strains, as shown in their table. We have generated cloned ES cell lines from HSCs (data not shown). The focus of this study was not to generate ES cells or 'clonal' mice via two-step cloning as previously reported3, 4, 5, but to directly compare the developmental potential of differentiated cells versus adult stem cells from the same tissue lineage using the conventional nuclear transfer method that led to the creation from adult somatic cells of cloned animals like Dolly. Our surprising finding provides unequivocal evidence that efficiency of conventional methods of cloning increases with the differentiation hierarchy within a hematopoietic cell lineage, although more studies are required to determine whether this trend is reversed within other cell lineages or under modified conditions.

治疗学的克隆从从体的房间导出的胚胎指胚胎的干细胞(ntESC ) 的推导原子转移(SCNT ) 也作为克隆知道。克隆涉及移植一个区分的房间进把它的原子核(DNA ) 移开的一个卵母细胞。重建的卵母细胞能被激活划分并且发展成一个胚胎。允许这发生的进程被称为原子 reprogramming，并且被定义为通过的机制区分的房间 de 区分或回来到一个 totipotent 状态(能够产生任何房间类型，包括胚胎外) 并且指导胚胎的开发。从胚囊舞台的房间克隆胚胎能被用来产生 ntESC 线。

In the version of this article originally published, Kacie J. Meyer (University of Iowa, Iowa City) was inadvertently omitted from the list of authors, and the names of three authors (Frederieke Koop, Marjolein Langemeijer and Channa Hijmans) were misspelled. Also, the third sentence of the abstract incorrectly stated that 1,168 families were analyzed. The correct number is 1,181 families. Finally, the last paragraph of the Discussion mistakenly identified one of the linkage regions as 11q13-12. This should read 11p13-12. These errors have been corrected in the HTML and PDF versions of the article.

Therapeutic cloning, whereby somatic cell nuclear transfer (SCNT) is used to generate patient-specific embryonic stem cells (ESCs) from blastocysts cloned by nuclear transfer (ntESCs), holds great promise for the treatment of many human diseases. ntESCs have been derived in mice and cattle, but thus far there are no credible reports of human ntESCs. Here we review the recent literature on nuclear reprogramming by SCNT, including studies of gene expression, DNA methylation, chromatin remodeling, genomic imprinting and X chromosome inactivation. Reprogramming of genes expressed in the inner cell mass, from which ntESCs are derived, seems to be highly efficient. Defects in the extraembryonic lineage are probably the major cause of the low success rate of reproductive cloning but are not expected to affect the derivation of ntESCs. We remain optimistic that human therapeutic cloning is achievable and that the derivation of patient-specific ntESC lines will have great potential for regenerative medicine.

In vitro production (IVP) has been shown to affect embryonic gene expression and often result in large offspring syndrome (LOS) in cattle and sheep. To dissect the effects of in vitro maturation, fertilization and culture on bovine embryos, we compared the expression profiles of single blastocysts generated by: (1) in vitro maturation, fertilization and culture (IVF); (2) in vivo maturation, fertilization and in vitro culture (IVD); and (3) in vivo maturation, fertilization and development (AI). To conduct expression profiling, total RNA was isolated from individual embryos, linearly amplified and hybridized to a custom bovine cDNA microarray containing approximately 6,300 unique genes. There were 306, 367, and 200 genes differentially expressed between the AI and IVD, IVF and IVD, and AI and IVF comparisons, respectively. Interestingly, 44 differentially expressed genes were identified between the AI embryos and both the IVF and IVD embryos, making these potential candidates for LOS. There were 60 genes differentially expressed between the IVF embryos and the AI and IVD embryos. The Gene Ontology category “RNA processing” was over‐represented among the genes that were down‐regulated in the IVF embryos, indicating an effect of in vitro oocyte maturation/fertilization on the ability to transcribe maternal RNA stores. A culture effect on the expression of genes involved in translation was also observed by the comparison of AI with IVD embryos. Mol. Reprod. Dev. 76: 38–47, 2009. © 2008 Wiley‐Liss, Inc.

Since the creation of Dolly via somatic cell nuclear transfer (SCNT)1, more than a dozen species of mammals have been cloned using this technology2. One hypothesis for the limited success of cloning via SCNT (1%–5%)3 is that the clones are likely to be derived from adult stem cells4. Support for this hypothesis comes from the findings that the reproductive cloning efficiency for embryonic stem cells is five to ten times higher than that for somatic cells as donors5,6 and that cloned pups cannot be produced directly from cloned embryos derived from differentiated B and T cells or neuronal cells7,8,9,10. The question remains as to whether SCNT-derived animal clones can be derived from truly differentiated somatic cells. We tested this hypothesis with mouse hematopoietic cells at different differentiation stages: hematopoietic stem cells, progenitor cells and granulocytes. We found that cloning efficiency increases over the differentiation hierarchy, and terminally differentiated postmitotic granulocytes yield cloned pups with the greatest cloning efficiency.

The technology is now available for commercial cloning of farm animals for food production, but is the food safe for consumers? Here, we provide data on >100 parameters that compare the composition of meat and milk from beef and dairy cattle derived from cloning to those of genetic- and breed-matched control animals from conventional reproduction. The cloned animals and the comparators were managed under the same conditions and received the same diet. The composition of the meat and milk from the clones were largely not statistically different from those of matched comparators, and all parameters examined were within the normal industry standards or previously reported values. The data generated from our match-controlled experiments provide science-based information desired by regulatory agencies to address public concerns about the safety of meat and milk from somatic animal clones.