Nature Genetics
30, 253 - 254 (2002)
Published online: 11 February 2002; | doi:10.1038/ng841
Early death of mice cloned from somatic cellsNarumi Ogonuki1, 5, Kimiko Inoue1, 5, Yoshie Yamamoto1, 5, Yoko Noguchi1, Kentaro Tanemura1, 4, Osamu Suzuki1, Hiroyuki Nakayama2, Kunio Doi2, Yukiko Ohtomo3, Michiko Satoh3, Akira Nishida3
& Atsuo Ogura11 Department of Veterinary Science, National Institute of Infectious Diseases, Shinjuku, Tokyo, Japan. 2 Department of Veterinary Pathology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan. 3 Graduate School of Agricultural Science, Faculty of Agriculture, Tohoku University, Sendai, Japan. 4 Present address: Laboratory for Alzheimer's Disease, Brain Science Institute, RIKEN, Saitama, Japan. 5 These authors contributed equally to this work.
Correspondence should be addressed to Atsuo Ogura aogura@nih.go.jpHere we report that the lifespan of mice cloned from somatic cells is significantly shorter than that of genotype- and sex-matched controls, most likely due to severe pneumonia and hepatic failure. This finding demonstrates the possibility of long-term deleterious effects of somatic-cell cloning, even after normal birth.
Animals cloned from somatic cells havebeen examined in many ways. Their true lifespan is controversial, however, and has not yet been determined. From March to September 1999 (ref. 1), we cloned 12 male mice from immature Sertoli cells and started long-term observations, examining their weight gain and serum biochemical values. Each parameter was compared with those of male mice with the same genetic background (hybrid F1 between C57BL/6  DBA/2; B6D2F1) derived from natural mating (n=7) or spermatid injection (n=6)2. All animals, including controls, were maintained under specific pathogen-free conditions.
The weight gain of cloned mice did not differ from that of natural-mating controls (53.5  9.7 g versus 51.6 5.7 g at one year after birth, P>0.05). Serum biochemical parameters were measured at 3 and 14 months of age. Of the 16 serum biochemical values examined, lactate dehydrogenase (LDH) and ammonia (NH3) were found to be significantly higher in the cloned mice than in the controls (P<0.05) at both time points (Table 1).
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The cloned mice started to die 311 days after birth, and 10 of the 12 cloned mice died before 800 days (Fig. 1a). Only one control mouse from natural mating and two from spermatid injection died within the same time span. The survival rate of the clones was significantly different from those of the two control groups (P<0.05, Kaplan-Meier analysis). We carried out histological examination of necropsy samples for six cloned mice and observed no post-mortem degeneration. We found severe pneumonia (6/6 cases, Fig. 1b), extensive necrosis in the liver (4/6, Fig. 1c) and tumors (leukemia and lung cancer, 1/6 each). An analysis of LDH isozyme types indicated that the major source of serum LDH in the cloned mice was the liver (data not shown), indicating the presence of some hepatic damage. A high level of ammonium in the serum is also a reliable index of hepatic failure. We have never encountered similar pathological alterations, except for tumors found in the natural-mating controls. It is thus likely that the morbidity and mortality of the cloned mice was due to a dysfunction of the liver and lungs that occurred specifically in clones.
| | Figure 1. Early death of cloned mice. | | |  | a, The survival rate of cloned mice (pink circles, n=12) differed significantly from those of genotype-matched control mice from natural mating (blue circles, n=7) or spermatid injection (orange circles, n=6; Kaplan-Meier analysis, P<0.05). b, Left: an inflammatory lesion in the lung of a cloned mouse that died at 531 d. The alveolar structure is completely destroyed by thick intraluminal exudates and heavy accumulations of inflammatory cells. Right: lung from a control mouse without inflammatory cells. c, Left: a broad necrotic lesion (arrow) in the liver of a cloned mouse that died at 523 d. Right: liver from a control mouse without necrotic cells. d, Left: significant reduction of the antibody productivity of cloned mice (asterisk indicates P<0.01). At 2 wk after hypodermic inoculation with live Mycobacterium bovis (BCG), the total amounts of plasma anti-BCG IgG, IgM, and IgA were measured by enzyme-linked immunosorbent assay. Right: phagocytic activity of cloned mice, as measured by carbon clearance in vivo11, was also lower than the control level, although the difference was not statistically significant (P>0.05).
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Recent clinical examinations of cloned bovines and goats have implied malfunction in the immune system3,
4. We therefore examined immune responses in another group of Sertoli cell−cloned mice. At as early as 4−5 months of age, the antibody productivity of the clones (n=20) was significantly reduced, compared with that of age- and genotype-matched controls derived from natural mating (n=9, P<0.01; Fig. 1d). Phagocytic activity was also lower than in controls, although the difference was not significant (Fig. 1d). As immunologic function generally decreases with advanced age5, it is probable that the immune incompetency in cloned mice might increase the risk of age-related autogenous infection, which most commonly causes pneumonia in humans and animals6,
7. The cause of liver necrosis in the clones, however, has yet to be determined.
Telomere shortening is known to be associated with cellular aging. It has recently been reported that, after cloning, telomere length can be restored to its original length by nuclear transfer8,
9, implying that the lifespan of clones might not be shortened. In this study, however, we demonstrate that unexpected clone-associated phenotypes, such as functional deficiency of some organs, might compromise the systemic condition of clones and shorten their lifespan. The cloned sheep 'Dolly' is still alive, but she is the sole reported clone under investigation for longevity. Two of our twelve clones are still alive and might have normal lifespans. Moreover, clone-associated phenotypes may vary according to the genotype and cell-type of donors, as reported for obesity in cloned mice10. Much controversy has been generated over the ethics of human cloning. The possible negative long-term effects of cloning, as well as the high incidence of spontaneous abortion and abnormal birth of cloned animals, give cause for concern about attempts to clone humans for reproductive purposes.
Received 9 December 2001; Accepted 15 January 2002; Published online: 11 February 2002.
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Acknowledgments
This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology; the Ministry of Health, Labor and Welfare; and the Human Science Foundation of Japan.
Competing interests statement:
The authors declare that they have no competing financial interests. |
