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A mouse model of familial amyloidotic polyneuropathy type i homozygous for the mutant transthyretin gene
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A MOUSE MODEL OF FAMILIAL AMYLOIDOT1CPOLYNEUROPATHYTYPE I HOMOZYGOUS FOR THE MUTANT TRANSTHYRETIN GENE K. Yokoi 1, S. Ito1, T. Mabuchi 1, K. Miyakawa2, J. A. Palha3, H. lijima4, S. Tsukahara4, W. S. Blaner5, M. J. M. Saraiva3, M. E. Gottesman6, K. Takahashi 2, K. Yamamura 7, K. Shimada8, S. Maeda1 1Department of Biochemistry, Yamanashi Medical University, Japan, 2Department of Pathology, Kumamoto University School of Medicine, Japan, 3Centro de Estudos de Paramiloidose, Portugal, 4Department of Ophthalmology, Yamanashi Medical University, Japan, 5Institute of Human Nutrition, Columbia University, USA, 6Institute of Cancer Research, Columbia University, USA, 7Institute of Molecular Embryology and Genetics, Kumamoto University School of Medicine, Japan, 8Department of Medical Genetics, Research Institute for Microbial Diseases, Osaka University, Japan To generate a closer model of familial amyloidotic polyneuropathy (FAP) type I and to elucidate the function of the human variant -I-I-R, we introduced the human mutant transthyretin (ttr) gene, that is responsible for FAP type I, into the "l-rR-deficient mice generated through gene targeting. The introduced human mutant gene (6.0-hMet30) contained 6 kb of its own upstream region. The serum levels of human "I-I-R Met 30 in these transgenic mice varied from 20 to 60 mg/dl. In these transgenic mice, amyloid composed of human -I-I'R began to deposit at the age 11 months. The deposition of amyloid was independent of the serum levels of human "FrR. There appeared to be no significant difference in the onset, progression, and tissue distribution of amyloid deposition between the "l-FR-deficient and control wild type transgenic mice carrying 6.0-hMet30. The above observation is consistent with the observation that the FAP patients homozygous for the mutant ttr gene do not show more severe symptoms or earlier onset than those heterozygous for the mutant gene. The "l-I-R-deficient mouse carrying 6.0-hMet30 should be useful as a closer model of FAP homozygous for the mutant ttr gene. However, these mice have not yet developed peripheral neuropathy. The introduction of 6.0-hMet30 into the TTR-deficient mice increased the serum level of retin,ol-binding protein (RBP) from 3% to 84% of the wild type mice. The introduction also increased the serum level of thyroid hormone (T4) from 40% to 60% of the wild type mice. These results indicate that the variant human "I-I'R binds T4 and RBP.
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Further characterization of transgenic mice, carrying the human TTR Met 30 gene under the control of mouse metalothionein promoter gene, lacking amyloid deposition Maria Jolo Boniflicio ~, Yoshiuki Sakaki ~, Maria Jo~o Saraiva 1 ICentro de Estudos de Paramiloidos¢, Hospital de Sto Ant6nlo 4000 PORTO and Instituto de Ci~ncias Biom6dicas Abel Salazar, Unlversldad¢ do Porto, 4000 PORTO, PORTUGAL. 2Institute of Medical Science, University of Tokyo, Japan
Transgenic mice, carrying the human Transthyretin fI'TR) Met 30 structural gene directed by the mouse metalothionein promoter have been, described to have amyloid deposits starting at about 6 month of age (Yi et al 1991). Other transgenic mice, also carrying the human TTR Met 30 structural gene under the control of the mouse metalothionein gene promoter, were also described (Sasaki et al 1986), but in these animals no amyloid deposits could be detected. We set out therefore to try to understand the reasons preventing amyloid deposition. These animals had been described to express the human protein in testis/ovary and intestine, however we found out expression also in the retina and skin, as determined by reverse transcription after total RNA isolation. As expected therefore, primary cultures of fibroblasts from these animals were shown to express and synthesize the human protein as demonstrated by ELISA, immunoprecipitation and immunohistochemistry. Serum levels of the human protein were determined by ELISA and it was established that: a) only the animals receiving ZnS04 had the protein in the circulation, b) protein levels varied between animals and were usually below 2mg human TTR Met 30/dl. The human protein was in the tetrameric form as determined by gel filtration HPLC. Functionality was investigated by thyroxine binding studies. A long term study was carried out in which animals, on ZnSO4 for at least 12 months, were sacrificed and processed for histochemistry to assess the presence of amyloid; although some "aggregates" could be identified extracellularly by immunohistochemistry with a monoclonal antibody for human TrR, staining with Congo Red was negative. Experiments are in progress to characterize these aggregates. In summary, although the human protein appears to be functional and expressed in several tissues, the amounts produced are either too low to lead to amyloid deposition or the genetic background of these animals, in some unknown way, prevent amyloid deposition. Yi S., Takahashi K., Wakasugi S., Maeda S., Shimada K., Yamamura, K., Araki, S. (1991) Am. J. Pathol. 138: 403-412 Sasaki H., Tone S,, Nakasato M., Yoshioka K., Matsuo H., Kato Y., Sakaki Y. (1986) Biochem. Biophys. Res. Commun. 139: 794-799.
A MOUSE MODEL OF FAMILIAL AMYLOIDOT1CPOLYNEUROPATHYTYPE I HOMOZYGOUS FOR THE MUTANT TRANSTHYRETIN GENE K. Yokoi 1, S. Ito1, T. Mabuchi 1, K. Miyakawa2, J. A. Palha3, H. lijima4, S. Tsukahara4, W. S. Blaner5, M. J. M. Saraiva3, M. E. Gottesman6, K. Takahashi 2, K. Yamamura 7, K. Shimada8, S. Maeda1 1Department of Biochemistry, Yamanashi Medical University, Japan, 2Department of Pathology, Kumamoto University School of Medicine, Japan, 3Centro de Estudos de Paramiloidose, Portugal, 4Department of Ophthalmology, Yamanashi Medical University, Japan, 5Institute of Human Nutrition, Columbia University, USA, 6Institute of Cancer Research, Columbia University, USA, 7Institute of Molecular Embryology and Genetics, Kumamoto University School of Medicine, Japan, 8Department of Medical Genetics, Research Institute for Microbial Diseases, Osaka University, Japan To generate a closer model of familial amyloidotic polyneuropathy (FAP) type I and to elucidate the function of the human variant -I-I-R, we introduced the human mutant transthyretin (ttr) gene, that is responsible for FAP type I, into the "l-rR-deficient mice generated through gene targeting. The introduced human mutant gene (6.0-hMet30) contained 6 kb of its own upstream region. The serum levels of human "I-I-R Met 30 in these transgenic mice varied from 20 to 60 mg/dl. In these transgenic mice, amyloid composed of human -I-I'R began to deposit at the age 11 months. The deposition of amyloid was independent of the serum levels of human "FrR. There appeared to be no significant difference in the onset, progression, and tissue distribution of amyloid deposition between the "l-FR-deficient and control wild type transgenic mice carrying 6.0-hMet30. The above observation is consistent with the observation that the FAP patients homozygous for the mutant ttr gene do not show more severe symptoms or earlier onset than those heterozygous for the mutant gene. The "l-I-R-deficient mouse carrying 6.0-hMet30 should be useful as a closer model of FAP homozygous for the mutant ttr gene. However, these mice have not yet developed peripheral neuropathy. The introduction of 6.0-hMet30 into the TTR-deficient mice increased the serum level of retin,ol-binding protein (RBP) from 3% to 84% of the wild type mice. The introduction also increased the serum level of thyroid hormone (T4) from 40% to 60% of the wild type mice. These results indicate that the variant human "I-I'R binds T4 and RBP.
52
Further characterization of transgenic mice, carrying the human TTR Met 30 gene under the control of mouse metalothionein promoter gene, lacking amyloid deposition Maria Jolo Boniflicio ~, Yoshiuki Sakaki ~, Maria Jo~o Saraiva 1 ICentro de Estudos de Paramiloidos¢, Hospital de Sto Ant6nlo 4000 PORTO and Instituto de Ci~ncias Biom6dicas Abel Salazar, Unlversldad¢ do Porto, 4000 PORTO, PORTUGAL. 2Institute of Medical Science, University of Tokyo, Japan
Transgenic mice, carrying the human Transthyretin fI'TR) Met 30 structural gene directed by the mouse metalothionein promoter have been, described to have amyloid deposits starting at about 6 month of age (Yi et al 1991). Other transgenic mice, also carrying the human TTR Met 30 structural gene under the control of the mouse metalothionein gene promoter, were also described (Sasaki et al 1986), but in these animals no amyloid deposits could be detected. We set out therefore to try to understand the reasons preventing amyloid deposition. These animals had been described to express the human protein in testis/ovary and intestine, however we found out expression also in the retina and skin, as determined by reverse transcription after total RNA isolation. As expected therefore, primary cultures of fibroblasts from these animals were shown to express and synthesize the human protein as demonstrated by ELISA, immunoprecipitation and immunohistochemistry. Serum levels of the human protein were determined by ELISA and it was established that: a) only the animals receiving ZnS04 had the protein in the circulation, b) protein levels varied between animals and were usually below 2mg human TTR Met 30/dl. The human protein was in the tetrameric form as determined by gel filtration HPLC. Functionality was investigated by thyroxine binding studies. A long term study was carried out in which animals, on ZnSO4 for at least 12 months, were sacrificed and processed for histochemistry to assess the presence of amyloid; although some "aggregates" could be identified extracellularly by immunohistochemistry with a monoclonal antibody for human TrR, staining with Congo Red was negative. Experiments are in progress to characterize these aggregates. In summary, although the human protein appears to be functional and expressed in several tissues, the amounts produced are either too low to lead to amyloid deposition or the genetic background of these animals, in some unknown way, prevent amyloid deposition. Yi S., Takahashi K., Wakasugi S., Maeda S., Shimada K., Yamamura, K., Araki, S. (1991) Am. J. Pathol. 138: 403-412 Sasaki H., Tone S,, Nakasato M., Yoshioka K., Matsuo H., Kato Y., Sakaki Y. (1986) Biochem. Biophys. Res. Commun. 139: 794-799.