无胆固醇小鼠克隆成功
http://www.100md.com
2003年12月20日
编者按：这为缺血性心脏病，高血脂等研究会带来启示
Generation of Viable Cholesterol-Free Mice A. Wechsler,1 A. Brafman,1 M. Shafir,1 M. Heverin,2 H. Gottlieb,1 G. Damari,1 S. Gozlan-Kelner,1 I. Spivak,1 O. Moshkin,1 E. Fridman,1,3 Y. Becker,1 R. Skaliter,1 P. Einat,1 A. Faerman,1 I. Björkhem,2 E. Feinstein1*
, http://www.100md.com
1 Quark Biotech, Inc., 10265 Carnegie Avenue, Cleveland, OH 44106, USA.
2 Department of Medical Laboratory Sciences and Technology, Division of Clinical Chemistry, Karolinska Institute, H3/30 Huddinge University Hospital C1 74 SE/141 86, Hudding, Sweden.
3 Department of Pathology, Sheba Medical Center, Sackler School of Medicine, Tel-HaShomer 52621, Israel.
* To whom correspondence should be addressed. E-mail: elenaf@qbi.co.il
, 百拇医药
Cholesterol accounts for
99% of all sterols in mammals, playing multiple biological roles as a major constituent of membranes, a precursor to numerous signaling molecules, and an inducer of the Hedgehog family of morphogens. Even small changes in cholesterol structure would be expected to be incompatible with life. We report here our surprising observation that mice lacking cholesterol have a relatively mild phenotype.
, 百拇医药
We analyzed mice with a targeted disruption of the Dhcr24 gene, which encodes the cholesterol biosynthetic enzyme desmosterol reductase. Plasma and tissues of Dhcr24–/– mice contained almost no cholesterol (Fig. 1A; fig. S1) (1), and desmosterol was shown to account for 99% of total sterols. There was no detectable accumulation of additional sterols and no gender-dependent differences in sterol composition, suggesting that DHCR24 is responsible for all 24,25-reduction reactions in the cholesterol biosynthesis pathway (fig. S2).
Fig. 1. (A) Plasma and liver sterols in 3-month-old mice. Shown are average sterol concentrations in 10 Dhcr24–/– (KO) and 10 Dhcr24+/+ (WT) mice (5 of each gender). CS, cholesterol; DS, desmosterol; TS, total sterols. Error bars represent standard deviation. (B) Growth curves reflecting the weight gain of 10 Dhcr24+/+ (
), 10 Dhcr24+/– (
), and 10 Dhcr24–/– (
) female mice. (C) Sterol analysis in Dhcr24–/– embryos. Shown are average sterol concentrations measured in 5 Dhcr24–/– and 5 Dhcr24+/+ embryos at gestation days (gd) 11.5 and 17.5. These time points were chosen because steroid synthesis increases in rodent embryos between gd 10 and gd 18 (9). [View Larger Version of this Image (21K GIF file)]
, 百拇医药
Dhcr24–/– pups were born from Dhcr24+/– matings at a frequency of 10 to 17% (versus the expected 25%), indicative of some prenatal death. The Dhcr24–/– newborns were25% smaller in size than Dhcr24+/+ and Dhcr24+/– littermates. When kept isolated from their larger littermates, the Dhcr24–/– mice survived to adulthood, albeit with poorer growth characteristics. Whereas Dhcr24–/– females reached normal weight by 4 to 5 weeks after weaning (Fig. 1B), male Dhcr24–/– mice did not (fig. S3). The accelerated growth rate of Dhcr24–/– mice after weaning suggests that loss of Dhcr24 might impair utilization of dietary fat, which accounts for 13% of mouse milk (1). This suggestion is supported by two additional findings. Our preliminary data indicate that although Dhcr24–/– mice can synthesize the normal profile of bile acids, in comparison with Dhcr24+/+ mice they produce lower amounts of cholic acid, which is needed for optimal absorption of cholesterol and lipids. Second, the livers of weaned Dhcr24–/– mice contain threefold less phytosterols than normal mice.
, http://www.100md.com
A comprehensive pathomorphological analysis of Dhcr24–/– mice at the age of 3 months revealed that all organs were normal in structure with the exception of testes, which were histologically identical to the degenerated testes seen in adult mice treated with a chemical inhibitor of DHCR24 (2). The Dhcr24–/– mice also had smaller stores of subcutaneous and mesenteric fat, and both males and females were infertile (1).
, http://www.100md.com
This relatively mild phenotype contrasts dramatically with the severe abnormalities observed in two registered patients with desmosterolosis who carry mutations in the DHCR24 gene (3, 4). This discrepancy may be due to the fact that maternal cholesterol is not available during human embryogenesis (5, 6), as it is in mice (Fig. 1C), suggesting that cholesterol's role in development is indispensable. The unchanged amount of total sterols and the nature of the accumulating sterol may explain why Dhcr24–/– mice are viable, whereas mice deficient in other cholesterol biosynthetic enzymes are not viable embryonically (5, 7).
, 百拇医药
Previous cell culture studies have shown that desmosterol can replace cholesterol in mouse fibroblasts without deleterious effects (8); the Dhcr24–/– mice document the inter-changeability of these sterols at the level of the whole organism. The availability of Dhcr24–/– mice should help to resolve many scientific questions raised as a result of the promiscuous nature of chemical inhibitors of steroidogenic enzymes, the inability to distinguish between developmental and direct consequences of inborn defects in cholesterol biosynthesis, and the inability to separate defects caused by reduced concentration of total sterols from those caused by cholesterol deficiency.
, http://www.100md.com
References and Notes
1.
Materials and Methods are available as supporting material on Science Online.
2.
S. K. Singh, S. Chakravarty, Reprod. Toxicol. 17, 37 (2003).[CrossRef][ISI][Medline]
3.
D. R. FitzPatrick et al., Am. J. Med. Genet. 75, 145 (1998).3.3.CO;2-1&link_type=DOI">[CrossRef][ISI][Medline]
, http://www.100md.com
4.
H. C. Andersson et al., J. Inherit. Metab. Dis. 23, 200 (2000).
5.
B. U. Fitzky et al., J. Clin. Invest. 108, 905 (2001).[Abstract/Free Full Text]
6.
J. Herz, R. V. Farese, J. Nutr. 129, 473S (1999).[ISI][Medline]
7.
, 百拇医药
C. A. Wassif et al., Hum. Mol. Genet. 10, 555 (2001).[Abstract/Free Full Text]
8.
G. H. Rothblat et al., Science 169, 880(1970).[ISI][Medline]
9.
F. Chevy et al., J. Lipid Res. 43, 1192 (2002).[Abstract/Free Full Text]
10.
We thank Lexicon Genetics Incorporated for the generation of Dhcr24–/– mice and Mitsubishi Tokyo Pharmaceuticals and Swedish Medical Research Council for financial support.
Supporting Online Material
www.sciencemag.org/cgi/content/full/302/5653/2087/DC1
PDF Version of this Article, 百拇医药
Generation of Viable Cholesterol-Free Mice A. Wechsler,1 A. Brafman,1 M. Shafir,1 M. Heverin,2 H. Gottlieb,1 G. Damari,1 S. Gozlan-Kelner,1 I. Spivak,1 O. Moshkin,1 E. Fridman,1,3 Y. Becker,1 R. Skaliter,1 P. Einat,1 A. Faerman,1 I. Björkhem,2 E. Feinstein1*
, http://www.100md.com
1 Quark Biotech, Inc., 10265 Carnegie Avenue, Cleveland, OH 44106, USA.
2 Department of Medical Laboratory Sciences and Technology, Division of Clinical Chemistry, Karolinska Institute, H3/30 Huddinge University Hospital C1 74 SE/141 86, Hudding, Sweden.
3 Department of Pathology, Sheba Medical Center, Sackler School of Medicine, Tel-HaShomer 52621, Israel.
* To whom correspondence should be addressed. E-mail: elenaf@qbi.co.il
, 百拇医药
Cholesterol accounts for
99% of all sterols in mammals, playing multiple biological roles as a major constituent of membranes, a precursor to numerous signaling molecules, and an inducer of the Hedgehog family of morphogens. Even small changes in cholesterol structure would be expected to be incompatible with life. We report here our surprising observation that mice lacking cholesterol have a relatively mild phenotype. , 百拇医药
We analyzed mice with a targeted disruption of the Dhcr24 gene, which encodes the cholesterol biosynthetic enzyme desmosterol reductase. Plasma and tissues of Dhcr24–/– mice contained almost no cholesterol (Fig. 1A; fig. S1) (1), and desmosterol was shown to account for 99% of total sterols. There was no detectable accumulation of additional sterols and no gender-dependent differences in sterol composition, suggesting that DHCR24 is responsible for all 24,25-reduction reactions in the cholesterol biosynthesis pathway (fig. S2).
Fig. 1. (A) Plasma and liver sterols in 3-month-old mice. Shown are average sterol concentrations in 10 Dhcr24–/– (KO) and 10 Dhcr24+/+ (WT) mice (5 of each gender). CS, cholesterol; DS, desmosterol; TS, total sterols. Error bars represent standard deviation. (B) Growth curves reflecting the weight gain of 10 Dhcr24+/+ (), 10 Dhcr24+/– (), and 10 Dhcr24–/– () female mice. (C) Sterol analysis in Dhcr24–/– embryos. Shown are average sterol concentrations measured in 5 Dhcr24–/– and 5 Dhcr24+/+ embryos at gestation days (gd) 11.5 and 17.5. These time points were chosen because steroid synthesis increases in rodent embryos between gd 10 and gd 18 (9). [View Larger Version of this Image (21K GIF file)], 百拇医药
Dhcr24–/– pups were born from Dhcr24+/– matings at a frequency of 10 to 17% (versus the expected 25%), indicative of some prenatal death. The Dhcr24–/– newborns were
25% smaller in size than Dhcr24+/+ and Dhcr24+/– littermates. When kept isolated from their larger littermates, the Dhcr24–/– mice survived to adulthood, albeit with poorer growth characteristics. Whereas Dhcr24–/– females reached normal weight by 4 to 5 weeks after weaning (Fig. 1B), male Dhcr24–/– mice did not (fig. S3). The accelerated growth rate of Dhcr24–/– mice after weaning suggests that loss of Dhcr24 might impair utilization of dietary fat, which accounts for 13% of mouse milk (1). This suggestion is supported by two additional findings. Our preliminary data indicate that although Dhcr24–/– mice can synthesize the normal profile of bile acids, in comparison with Dhcr24+/+ mice they produce lower amounts of cholic acid, which is needed for optimal absorption of cholesterol and lipids. Second, the livers of weaned Dhcr24–/– mice contain threefold less phytosterols than normal mice. , http://www.100md.com
A comprehensive pathomorphological analysis of Dhcr24–/– mice at the age of 3 months revealed that all organs were normal in structure with the exception of testes, which were histologically identical to the degenerated testes seen in adult mice treated with a chemical inhibitor of DHCR24 (2). The Dhcr24–/– mice also had smaller stores of subcutaneous and mesenteric fat, and both males and females were infertile (1).
, http://www.100md.com
This relatively mild phenotype contrasts dramatically with the severe abnormalities observed in two registered patients with desmosterolosis who carry mutations in the DHCR24 gene (3, 4). This discrepancy may be due to the fact that maternal cholesterol is not available during human embryogenesis (5, 6), as it is in mice (Fig. 1C), suggesting that cholesterol's role in development is indispensable. The unchanged amount of total sterols and the nature of the accumulating sterol may explain why Dhcr24–/– mice are viable, whereas mice deficient in other cholesterol biosynthetic enzymes are not viable embryonically (5, 7).
, 百拇医药
Previous cell culture studies have shown that desmosterol can replace cholesterol in mouse fibroblasts without deleterious effects (8); the Dhcr24–/– mice document the inter-changeability of these sterols at the level of the whole organism. The availability of Dhcr24–/– mice should help to resolve many scientific questions raised as a result of the promiscuous nature of chemical inhibitors of steroidogenic enzymes, the inability to distinguish between developmental and direct consequences of inborn defects in cholesterol biosynthesis, and the inability to separate defects caused by reduced concentration of total sterols from those caused by cholesterol deficiency.
, http://www.100md.com
References and Notes
1.
Materials and Methods are available as supporting material on Science Online.
2.
S. K. Singh, S. Chakravarty, Reprod. Toxicol. 17, 37 (2003).[CrossRef][ISI][Medline]
3.
D. R. FitzPatrick et al., Am. J. Med. Genet. 75, 145 (1998).3.3.CO;2-1&link_type=DOI">[CrossRef][ISI][Medline]
, http://www.100md.com
4.
H. C. Andersson et al., J. Inherit. Metab. Dis. 23, 200 (2000).
5.
B. U. Fitzky et al., J. Clin. Invest. 108, 905 (2001).[Abstract/Free Full Text]
6.
J. Herz, R. V. Farese, J. Nutr. 129, 473S (1999).[ISI][Medline]
7.
, 百拇医药
C. A. Wassif et al., Hum. Mol. Genet. 10, 555 (2001).[Abstract/Free Full Text]
8.
G. H. Rothblat et al., Science 169, 880(1970).[ISI][Medline]
9.
F. Chevy et al., J. Lipid Res. 43, 1192 (2002).[Abstract/Free Full Text]
10.
We thank Lexicon Genetics Incorporated for the generation of Dhcr24–/– mice and Mitsubishi Tokyo Pharmaceuticals and Swedish Medical Research Council for financial support.
Supporting Online Material
www.sciencemag.org/cgi/content/full/302/5653/2087/DC1
PDF Version of this Article, 百拇医药