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Variation in Gene Expression and Aberrantly Regulated Chromosome Regions in Cloned Mice¹

Department of Epigenetics,³ Medical Research Institute, Tokyo Medical and Dental University, Chiyoda-ku, Tokyo 101-0062, Japan CREST,⁴ Japan Science and Technology Agency (JST), Kawaguchi, Saitama 332-0011, Japan BioResource Center,⁵ RIKEN, Tsukuba, Ibaraki 305-0074, Japan School of Health Sciences,⁶ Tokai University, Bohseidai, Isehara, Kanagawa 259-1193, Japan

ABSTRACT

DNA microarray analysis was used to determine the precise genome-wide gene expression profiles of somatic cloned mice derived from Sertoli and cumulus cells. It demonstrated unexpectedly large epigenetic diversity in neonatal cloned mice, despite their normal appearance and genetic identity. In three neonatal tissues of the cloned mice, the expression of 9–40% of the genes examined was more than two times higher or lower in donor cell-dependent or -independent manners compared with normal controls. Relatively few (0.4–4%) of the genes exhibited up- or downregulation in the same manner in both types of clone. A cluster analysis of the variation in gene expression led to the identification of several chromosome regions in which gene expression was aberrantly controlled in the somatic clones. These results provide a more complete understanding of how somatic clones differ from each other and from normal individuals produced by sexual reproduction and indicate the significant difficulties that face the application of somatic cloning in regenerative medicine.

cumulus cells, developmental biology, gene regulation, Sertoli cells

INTRODUCTION

Animals cloned from somatic cells have attracted a great deal of attention due to their peculiar manner of reproduction. The somatic nuclear cloning technique itself has also had a major impact on human society because nuclear-transferred embryonic stem (ES) cells are expected to be the material best suited for therapeutic cloning. However, one must still ask: What exactly are somatic clones? Does somatic cloning produce animals and tissues identical to those of the nucleus donor animals? Unlike ES cell-derived clones, which have high rates of neonatal abnormalities [1–3], we and others have previously reported that the majority of term pups cloned from somatic cells using nuclear transfer appeared healthy on delivery, gained active movement rapidly, and grew into fertile adults, despite the low success rate due to high embryonal lethality, particularly at early postimplantation stages [4–7]. Abnormal enlarged placentas are commonly observed in both ES and somatic clones and there have been many reports on their abnormal epigenetic status, such as aberrant gene expression [6, 8–11] and DNA methylation [12]. However, it has been reported that a relatively small number of genes exhibit abnormal expression in the livers of neonatal clones derived from cumulus and ES cells compared with the number of genes expressed abnormally in the corresponding placentas of clones [8]. Recently, the long-term health consequences of somatic clones, such as obesity [13], short life span, and immune-system anomalies [14], have been reported. These results suggest that broad effects also occur in neonatal or adult somatic clones, in addition to the commonly observed enlarged placentas. Here, we present detailed gene expression data from somatically cloned mice to further understanding of their biology, a necessary step before these techniques can be applied to regenerative medicine.

MATERIALS AND METHODS

Production of Cloned Mice

Cloning by nuclear transfer was performed according to the method developed by Wakayama et al. [4], with slight modification [5]. Immature Sertoli and cumulus cells were collected from newborn male (0–8 days after birth) and mature female (2–3 mo old) C57BL/6 x DBA/2 F1 (BDF1) mice, respectively, and used as nucleus donor cells. Cloned pups were obtained from the recipients at term (Day 19.5) by cesarean section. All control mice were genetically identical to the somatic cell clones, e.g., BDF1. As the majority (95% and 82% of immature Sertoli clones and cumulus clones, respectively) of term pups appeared healthy on delivery and rapidly gained active movement under our experimental conditions, we did not select individuals but instead used all of the eight pups that we obtained. After confirmation of the start of respiration at the time of cesarean section, we killed the pups and collected tissues and organs. The control neonates were produced by in vitro fertilization and also recovered by cesarean section. All samples from cesarean section were immediately frozen and stored in liquid nitrogen until use. All procedures described here were reviewed and approved by the Animal Experimentation Committee at RIKEN and were performed in accordance with the RIKEN Guiding Principles for the Care and Use of Laboratory Animals.

DNA Microarray Experiments

The CodeLink system (Amersham Biosciences) was used to determine and compare the expression levels of 10012 genes (UniSet mouse I) in the neonatal tissues of four Sertoli cell-derived clones, four cumulus cell-derived clones, and four normal controls (two males and two females each). Total RNA was purified using Isogen (Nippon Gene). The cRNA preparation and CodeLink hybridization were conducted according to the manufacturers' protocols. The expression levels of the different genes were assessed using GenePix Pro software. The signals were normalized using the qspline algorithm implemented in the Bioconductor package of the statistics program R [15]. Genes with similar expression profiles were clustered and displayed using Cluster 3.0 and Java TreeView, originally developed by Eisen et al. [16]. The data from the individual microarrays are accessible for download through the National Center for Biotechnology Information's Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/) via series accession number GSE3128.

Quantitative Reverse Transcription-Polymerase Chain Reaction

Genomic DNA and total RNA were prepared from neonatal tissues of the Sertoli cell- and cumulus cell-derived clones and from normal controls using ISOGEN (Nippon Gene), as described previously [17]. All RNA samples were treated with RNase-free DNase I. The cDNA was synthesized from 1 µg total RNA using Superscript II reverse transcriptase (Life Technologies) with an oligo(dT) primer. For reverse transcription-polymerase chain reaction (RT-PCR), 1 ng cDNA in a 100-µl reaction mixture containing 1x ExTaq buffer (TaKaRa), 2.5 mM dNTP mixture, primers, and 2.5 U ExTaq enzyme (TaKaRa) was subjected to 30 PCR cycles at 96°C for 15 sec, 65°C for 30 sec, and 72°C for 30 sec on a Perkin Elmer GeneAmp PCR system 9600. Target cDNA fragments were cloned into plasmids to use as standards in the quantitative analysis of gene expression. The primers used for quantification were Alb1-F, 5'-TCA GAG ACT GCC TTG TGT GG-3'; Alb1-R, 5'-CAG CCT TGC AAC ATG TAT CC-3'; Trf-F, 5'-GTC CTG GAT AAC ACC GAA GG-3'; Trf-R, 5'-ACA GAT TGC ATG TAC TCC GC-3'; Ahsg-F, 5'-ATA GCC ACC ACT GAA GCT GG-3'; Ahsg-R, 5'-TGA AGA TTG GCA AGA GCA CC-3'; Actb-F, 5'-AAG TGT GAC GTT GAC ATC CG-3'; and Actb-R, 5'-GAT CCA CAT CTG CTG GAA GG-3'. The gene expression levels were measured using an ABI PRISM 7700 detection system and SYBR Green PCR Core Reagents (Applied Biosystems). No amplification of RT-PCR products was detected in the reverse transcriptase-minus controls.

RESULTS

Using DNA microarrays, we analyzed the gene expression profiles of the liver, kidney, and brain tissues of four neonatal clones derived from immature Sertoli cells, four neonatal clones derived from cumulus cells, and four neonatal normal control mice produced by in vitro fertilization. Figure 1A illustrates the gene expression profiles of the liver for each individual. The fold change in the expression level of each gene in each animal was calculated relative to the mean expression level of the corresponding gene in the four normal control individuals. The results were plotted in the gene order derived from the cluster analysis of the expressed genes in the microarray. It was apparent that, in the normal controls, the expression of all genes was tightly regulated within a factor of two (as indicated by the horizontal lines in each panel of Fig. 1A) for each individual. Therefore, the overall pattern of gene expression was uniform, with little divergence among the normal control individuals (Fig. 1A, right panels). In contrast, in the clones derived from Sertoli (Fig. 1A, left panels) and cumulus (Fig. 1A, middle panels) cells, many genes were expressed abnormally in each clone, and the gene expression profiles were divergent among the cloned individuals. In all clones, between 4% and 20% (419-2072 of the 10012 genes in the microarray) of the genes exhibited a more than two-fold increase or decrease in signal intensity compared with the mean expression level in the controls (Table 1). The extent of abnormal gene expression in the kidney and brain was similar to that in the liver (2–20%; Table 1 and Fig. 1, B and C).

View larger version (34K): [in this window] [in a new window]   FIG. 1. Gene expression profiles of somatic cell clones and control mice. The gene expression profiles of somatic cell clones and control mice in the liver (A), kidney (B), and brain (C). The intensity of each signal from the DNA microarray was normalized by dividing it by the mean signal intensity of the normal controls. If the value exceeded 1, this value was used as the fold increase; if the value was less than 1, the inverse was used as the fold decrease. To calculate the fold change for each normal control, the mean values were calculated from the data for the other three controls to avoid underestimation. Genes with similar expression patterns among the cloned individuals were clustered using Cluster 3.0, and the fold increase or decrease was plotted in the gene order resulting from the cluster analysis. Each graph represents the data from one cloned or normal individual (upper 12 panels; four Sertoli cell-derived clones, c1(s) to c4(s); four cumulus cell-derived clones, c5(c) to c8(c); and four normal controls, n1–n4). The three lower panels show the mean fold changes for the Sertoli cell clones, the cumulus cell clones, and for all cloned individuals. The red dots represent genes of which expression levels exceeded a two-fold change in all of the individual clones. Note that the gene orders among liver (A), kidney (B) and brain (C) differ because the gene clustering was carried out independently

These analyses demonstrated the existence of genes exhibiting both donor cell-dependent and -independent effects on expression in somatic clones, as has been reported previously [8]. Approximately 200 genes were up- or downregulated in the same manner in both types of clone in at least one of the three tissues (Table 2). For example, the expression of Il1b was upregulated in the liver and kidney of both the cumulus cell- and Sertoli cell-derived clones (Fig. 2A).

View larger version (18K): [in this window] [in a new window]   FIG. 2. Genes exhibiting donor cell-independent and -dependent dysregulation effects. The gene expression levels were determined using quantitative RT-PCR in triplicate and normalized using the detected expression level of Actb. The mean level of gene expression in the normal controls was set to 1. The standard errors are shown using error bars. A) The expression levels of interleukin 1 beta (Il1b) in the liver and kidney. Il1b was 1 of 200 genes exhibiting increased expression in all eight clones. B) The expression levels of three genes exhibiting abnormal expression in the kidney that were Sertoli cell clone-specific. Alb1, albumin; Trf, transferrin; Ahsg, alpha-2-HS-glycoprotein

Interestingly, in the class II major histocompatibility complex (MHC) region on chromosome 17, seven genes within a 500-kilobase (kb) region exhibited increased expression levels in the liver, including class II MHC and MHC-related genes (Fig. 3A).

View larger version (43K): [in this window] [in a new window]   FIG. 3. Chromosome regions containing multiple upregulated genes in cloned mice. The physical maps of the genes were drawn using the mouse genome sequence assembly coordinates from NCBI Build 33, version 1. Black and gray arrows represent genes with and without expression profiles, respectively. The lack of expression profiles for some genes was the result of the absence of a probe in the DNA microarray or expression levels below the detection threshold. The gray background in the genome maps indicates regions outside the hypothesized area of coregulation. Scale bars = 100 kb. The gene expression profiles are charted as follows. The fold changes in gene expression were calculated from the signal intensities on the microarrays, normalized to the mean value of the controls. Panels with a white background represent the profiles of coregulated genes, while panels with a gray background represent the profiles of genes adjacent to the coregulated gene clusters. A) Chromosome region containing the class II MHC gene cluster. B) Chromosome regions containing the fibrinogen, apolipoprotein, and serine protease inhibitor gene clusters

In the analysis of gene expression in the kidney, approximately 35 genes exhibited increased expression levels that were specific to the Sertoli cell-derived clones (Table 3), apart from male-specific genes that showed common expression in Sertoli cell-derived clones and male control individuals, and were mapped on the Y chromosome. This group included markers of the acute phase of inflammation, such as Trf, Alb1, Ahsg, Hp, Serpina1a, Serpina1d, and Serpina1e. Semiquantitative RT-PCR confirmed that the expression of Trf, Alb1, and Ahsg was increased 5- to 25-fold in the kidneys of Sertoli cell-derived clones compared with the normal controls (Fig. 2B). The microarray signal intensities for these genes were more than 50 times greater in the liver samples compared with brain and kidney samples (data not shown), indicating that the expression of these genes is highly tissue specific and may be under the control of a common gene regulatory mechanism. We found that the genes showing upregulation in a Sertoli-clone-specific manner also formed clusters in three chromosome regions, including apolipoproteins (Apoc2, Apoc4, Apoc1), fibrinogens (Fgg, Fga, Fgb), and serine protease inhibitors (Serpina1b, Serpina1d, Serpina1a, Serpina1e; Fig. 3B).

The large difference between the total number of aberrantly expressed genes and the number of genes exhibiting aberrant expression in all individual clones indicated that mean expression levels did not represent the real expression profiles of individual somatic cell clones. Therefore, another analytical method is needed to define the genetic character of cloned individuals more precisely. Closer examination of the data revealed that the number of genes showing increased or decreased expression differed among the cloned individuals (Table 1 and Fig. 1), suggesting that the extent of the initial perturbation on the affected chromosome regions and the extent of the abnormalities caused by nuclear transfer differed among cloned individuals.

However, some clones had similar gene expression profiles, including clones 1, 7, and 8 and clones 4, 5, and 6, which exhibited similar general expression patterns (Figs. 1 and 4A). Interestingly, this similarity in the expression profiles did not depend on the donor cell type; both groups mentioned above included Sertoli cell- and cumulus cell-derived clones. When the genes that exhibited Sertoli-specific expression abnormalities in the kidney were excluded, comparable pattern similarities were also observed for the expression profiles in the brain and kidney. The two groups corresponded to the individuals exhibiting fewer gene expression abnormalities (9–12%) and to those exhibiting many abnormalities (34–41%; Table 1 and Fig. 4). These results suggest that the susceptibility of genes to dysregulation caused by somatic cloning varies in a nonrandom manner.

View larger version (52K): [in this window] [in a new window]   FIG. 4. Clustered gene expression profiles and the numbers of genes with altered levels of expression. A) Dendrogram (upper part): cloned and control individuals were clustered according to their gene expression profiles using a hierarchical clustering algorithm and GeneCluster version 3.0. The dendrogram was drawn using Java TreeView. Heat map representation (lower part): genes with similar expression patterns in the clones were clustered using GeneCluster version 3.0 and illustrated using Java TreeView. The mean expression level in the normal controls is indicated in black. The expression profiles are presented as a heat map in which red indicates increased expression levels above the mean and increasing green intensity indicates reduced expression. The intensity of the red or green corresponds to the extent of the change from normal. Sertoli cell clone-specific abnormally expressed genes (red bands in c2, c3, c1, and c4 in kidney) are clearly seen in the middle column as indicated by the parentheses. B) The numbers of genes affected in each individual are indicated on a cumulative bar graph. Red and green bars indicate values for genes with increased and decreased expression, respectively

Next, we clustered the genes into 20 groups and assigned group numbers using k-means clustering with nonparametric analysis of similarities according to the expression pattern among cloned and control individuals in each tissue, as presented in Figure 4. Then, the assigned group numbers were aligned along the mouse genome according to their gene order using assembled mouse genome sequences (NCBI mouse build 33) so that regions with the same assigned numbers could be abstracted automatically. Finally, we checked the gene expression pattern of each gene in these regions manually, in detail, and confirmed that genes with similar patterns of varied expression were clustered. Twenty-one such gene clusters consisting of three or more neighboring genes (Figs. 5 and 6) and more than 70 gene pairs (Supplementary Table, available online at http://www.biolreprod.org) were identified in the genome, in addition to the genes that showed aberrant expression in all clones (described above; Figs. 2 and 3). This analysis revealed that gene expression is regulated abnormally in clusters within many chromosome regions of somatic clones and that the dysregulating effect of somatic cloning sometimes influences genes within one to several hundred kilobases in the genome.

View larger version (39K): [in this window] [in a new window]   FIG. 5. Chromosome regions containing genes with related functions or structures that were coregulated in the clones. The physical maps of the genes and the gene expression profiles are as shown in Figure 3. Scale bars = 100 kb. As shown in Figure 4B, each clone individual showed increased or decreased expression by more than a factor of two in 2–10% of genes. Assuming that the gene expression of all clones was affected independently, the probability of a nearest neighbor gene showing the same pattern of aberrant gene expression in at least three clone individuals is less than 10–3 to 10–6. Therefore, the gene clusters observed here are significant

View larger version (35K): [in this window] [in a new window]   FIG. 6. Chromosome regions con-taining genes that were coregulated in the clones, including gene(s) with nonrelated function(s). The physical maps ofthe genes and the gene expression profiles are as shown in Figure 3 and Supplementary Figure 5 (available online at http://www.biolreprod.org)

Approximately half of these clusters consisted of genes with similar functions (Fig. 5), while the other half contained genes with no functional or structural similarities (Fig. 6). Coregulation in the former clusters may arise from the abnormal expression of common upstream regulatory genes. In contrast, coregulation of the nonrelated gene clusters implies the presence of functional chromosome domains for regional gene regulation and perturbation of these regulatory systems by somatic cell cloning.

Note that cluster analysis based on variation in gene expression was useful for identifying abnormalities in several metabolic pathways of the clones. We found that genes in some important pathways, including glycolysis, heme synthesis, and steroid synthesis, were aberrantly coregulated in individual cloned mice (Supplementary Figs. 1–3, available online at http://www.biolreprod.org).

DISCUSSION

Our data demonstrated a general disturbance of gene expression in cloned mice from somatic cells that included dysregulation associated with the donor cell type, as well as dysregulation inherent to cloning; whereas a few genes were consistently affected in all clones, many genes showed variation in gene expression among the clones. Unexpectedly, normal-appearing mice cloned from somatic cells had a large number of affected genes compared with normal controls, and the expression profiles differed among the cloned individuals despite their identical genotypes and normal appearance. This was in marked contrast with the observations in the offspring of the control produced by in vitro fertilization, which exhibited homogeneous, strictly regulated gene-expression profiles. Most cloned individuals die before developing to term: the birth rate of mice cloned using somatic cells is only 2–5%. Therefore, the clones we analyzed represented selected individuals, and the number of genes affected was likely the minimum number that met all essential conditions for survival and development, i.e., the changes in gene expression observed here fell within the tolerance levels of mouse development and subsequent growth.

As shown in Figures 2 and 3, the abnormally expressed genes in the neonatal cloned mice included many genes associated with the immune system, including cytokines, specific markers of immune system cells, such as histocompatibility antigens, and markers of inflammation. Although we did not assess the impact of this variation in gene expression on the physiology or life span of the clones [14], these abnormalities may represent a disturbance of the immune system, as reported for other clones [18].

Our data clearly demonstrated major disturbances of gene expression in cloned animals, including effects that were inherent to cloning (donor cell-independent) and donor cell-dependent effects. Note that these common effects accounted for only a small portion of the affected genes; the remaining abnormally expressed genes showed wide variation across the individual somatic clones. One important consequence of this variation is that the calculation and comparison of mean expression levels tend to mask many of the gene expression abnormalities in each individual. Genes that were expressed abnormally in all of the individual Sertoli cell- or cumulus cell-derived clones accounted for only 0.4% or 4% of the total examined genes, respectively (red dots in the lower panels of Fig. 1, A–C, and Table 2). These values were consistent with those in a previous report, which found that less than 0.1% of the genes in the liver of neonatal clones derived from cumulus or embryonic stem cells showed expression levels that differed by more than two-fold [8].

The large variation in gene expression among individuals may be attributable to initial errors in the reprogramming process after nuclear transfer or may simply represent an abnormal epigenetic state that arises in the donor cells despite avoiding in vitro culture steps in their preparation. In either case, the first event affects target genes at random and may induce many secondary effects and ultimately lead to genome-wide, multilayered, mosaic abnormalities in somatically derived clones. In this respect, applications of nuclear transfer to human or veterinary medicine, such as therapeutic cloning, have serious limitations at present.

Recently, extensive evidence has accumulated for the clustering of coexpressed genes in mammalian genomes. The importance of the hierarchical architecture of epigenetic regulation that partitions the mammalian genome functionally, including very large domains such as isochors, as well as relatively small chromosomal domains of several hundred kilobase pairs, has now been recognized [19]. However, detailed, genome-wide surveys of gene regulation in these chromosome regions have not yet been reported for mammalian systems. The abnormal expression patterns observed in some gene clusters in this study may represent disturbances of a regionally regulated architecture of the chromosome. The gene expression variation-based cluster analysis used in this study may be generally applicable to the classification of genes that are coordinately regulated in the mammalian genome by permitting systematic analysis of apparently random biological phenomena that have previously been difficult to study.

FOOTNOTES

¹ Supported by grants from CREST, the research program of the Japan Science and Technology Agency (JST), the Uehara Memorial Science Foundation, the Ministries of Health, Labour, and Welfare for Child Health and Development (14-C) and Education, Culture, Sports, Science and Technology of Japan.

² Correspondence: Fumitoshi Ishino, Department of Epigenetics, Medical Research Institute, Tokyo Medical and Dental University, 2–3–10 Kanda-surugadai, Chiyoda-ku, Tokyo 101-0062, Japan. FAX: 81 3 5280 8073; fishino.epgn{at}mri.tmd.ac.jp

Received: 27 June 2005.

First decision: 4 August 2005.

Accepted: 22 August 2005.

REFERENCES

1. Humpherys D, Eggan K, Akutsu H, Hochedlinger K, Rideout WM, III, Biniszkiewicz D, Yanagimachi R, Jaenisch R, Epigenetic instability in ES cells and cloned mice. Science 2001 293:95-97[Abstract/Free Full Text]
2. Rideout WM, Eggan K, Jaenisch R, Nuclear cloning and epigenetic reprogramming of the genome. Science 2001 293:1093-1098[Abstract/Free Full Text]
3. Ohta H, Wakayama T, Generation of normal progeny by intracytoplasmic sperm injection following grafting of testicular tissue from cloned mice that died postnatally. Biol Reprod 2005 73:390-395[Abstract/Free Full Text]
4. Wakayama T, Perry AC, Zuccotti M, Johnson KR, Yanagimachi R, Full-term development of mice from enucleated oocytes injected with cumulus cell nuclei. Nature 1998 394:369-374[CrossRef][Medline]
5. Ogura A, Inoue K, Ogonuki N, Noguchi A, Takano K, Nagano R, Suzuki O, Lee J, Ishino F, Matsuda J, Production of male cloned mice from fresh, cultured, and cryopreserved immature Sertoli cells. Biol Reprod 2000 62:1579-1584[Abstract/Free Full Text]
6. Inoue K, Kohda T, Lee J, Ogonuki N, Mochida K, Noguchi Y, Tanemura K, Kaneko-Ishino T, Ishino F, Ogura A, Faithful expression of imprinted genes in cloned mice. Science 2002 295:297[Free Full Text]
7. Ogura A, Inoue K, Ogonuki N, Lee J, Kohda T, Ishino F, Phenotypic effects of somatic cell cloning in the mouse. Cloning Stem Cells 2002 4:397-405[CrossRef][Medline]
8. Humpherys D, Eggan K, Akutsu H, Friedman A, Hochedlinger K, Yanagimachi R, Lander ES, Golub TR, Jaenisch R, Abnormal gene expression in cloned mice derived from embryonic stem cell and cumulus cell nuclei. Proc Natl Acad Sci U S A 2002 99:12889-12894[Abstract/Free Full Text]
9. Bortvin A, Eggan K, Skaletsky H, Akutsu H, Berry DL, Yanagimachi R, Page DC, Jaenisch R, Incomplete reactivation of Oct4-related genes in mouse embryos cloned from somatic nuclei. Development 2003 130:1673-1680[Abstract/Free Full Text]
10. Suemizu H, Aiba K, Yoshikawa T, Sharov AA, Shimozawa N, Tamaoki N, Ko MS, Expression profiling of placentomegaly associated with nuclear transplantation of mouse ES cells. Dev Biol 2003 253:36-53[CrossRef][Medline]
11. Singh U, Fohn L, Wakayama T, Ohgane J, Steinhoff C, Lipkowitz B, Schulz R, Orth A, Ropers H, Behringer R, Tanaka S, Shiota K, et al Different molecular mechanisms underlie placental overgrowth phenotypes caused by interspecies hybridization, cloning, and Esx1 mutation. Dev Dyn 2004 230:149-164[CrossRef][Medline]
12. Ohgane J, Wakayama T, Kogo Y, Senda S, Hattori N, Tanaka S, Yanagimachi R, Shiota K, DNA methylation variation in cloned mice. Genesis 2001 30:45-50[CrossRef][Medline]
13. Tamashiro K, Wakayama T, Akutsu H, Yamazaki Y, Lachey J, Wortman M, Seeley R, D'Alessio D, Woods S, Yanagimachi R, Sakai R, Cloned mice have an obese phenotype not transmitted to their offspring. Nat Med 2002 8:262-267[CrossRef][Medline]
14. Ogonuki N, Inoue K, Yamamoto Y, Noguchi Y, Tanemura K, Suzuki O, Nakayama H, Doi K, Ohtomo Y, Satoh M, Nishida A, Ogura A, Early death of mice cloned from somatic cells. Nat Genet 2002 30:253-254[CrossRef][Medline]
15. Gentleman R, Carey V, Bates D, Bolstad B, Dettling M, Dudoit S, Ellis B, Gautier L, Ge Y, Gentry J, Hornik K, Hothorn T, et al Bioconductor: open software development for computational biology and bioinformatics. Genome Biol 2004 5:R80[CrossRef][Medline]
16. Eisen M, Spellman P, Brown P, Botstein D, Cluster analysis and display of genome-wide expression patterns. Proc Natl Acad Sci U S A 1998 95:14863-14868[Abstract/Free Full Text]
17. Kaneko-Ishino T, Kuroiwa Y, Miyoshi N, Kohda T, Suzuki R, Yokoyama M, Viville S, Barton SC, Ishino F, Surani MA, Peg1/Mest imprinted gene on chromosome 6 identified by cDNA subtraction hybridization. Nat Genet 1995 11:52-59[CrossRef][Medline]
18. Renard JP, Chastant S, Chesne P, Richard C, Marchal J, Cordonnier N, Chavatte P, Vignon X, Lymphoid hypoplasia and somatic cloning. Lancet 1999 353:1489-1491[CrossRef][Medline]
19. Hurst L, Pál C, Lercher M, The evolutionary dynamics of eukaryotic gene order. Nat Rev Genet 2004 5:299-310[CrossRef][Medline]

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This Article Abstract Full Text (PDF) All Versions of this Article: 73/6/1302    most recent biolreprod.105.044958v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kohda, T. Articles by Ishino, F. Search for Related Content PubMed PubMed Citation Articles by Kohda, T. Articles by Ishino, F. Agricola Articles by Kohda, T. Articles by Ishino, F.