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Transgenic animal models: Their role in the acceleration and modification of the drug development process
Poster Session 1B. Transgenic Models
IP18491
ARTIFICIALHETEROZYGOTE ANALYSIS: A NOVEL APPROACH FORTHE EFFICIENT IDENTIFICATION OF POINTMUTATIONS
F. Staedtler *, A. Pospiech, S. Steiner, M. Looser. Novartis Phanna AG, Preclinical Safety-Toxicology-PathologylNovartis Services AG, Scientific Services - BioAnalytics, Basel, Switzerland DNA sequencing is regarded as the gold standard in mutation analysis. However, it is rate limiting in experimental systems if DNA sequence data from large numbers of mutants are required. Therefore, improvements of the current protocols aiming to increase sample throughput and to reduce analysis costs are needed. Here we describe a new, efficient procedure for the identification of point mutations, the artificial heterozygote (AH) analysis. DNA sequencing templates of individual mutants of the target gene are amplified by polymerase chain reaction (PCR). By mixing the PCR fragments from individual mutants in a defined ratio, samples of artificial heterozygote (AH) composition are prepared. These AH samples are then submitted to automated DNA sequencing. A specific base-calling algorithm is used for the automated detection of the heterozygous positions. At these positions the individual intensities of the base signals reflect the amount of each template in the AH sample. Thus the identified point mutations can be assigned to the corresponding mutants. The feasibility of this procedure was shown by identifying point mutations in the laeI mutational target gene recovered from Big BlueR transgenic mice.
I P1850 I USEOF METALLOTHIONEIN-TRANSGENIC AND KNOCK-OUT MOUSEMODELSFORTHE STUDY OF CADMIUM TOXICITY C.D. Klaassen *, J. Liu. Univ Kansas Medical Center, Kansas City, KS, USA The role of metallothionein (MT) in Cd toxicology has been studied using MT-I transgenic (MT-TO) and MT-I and II knock-out (MTnull) mice. We have shown that (I) MT-TO and MT-null mice appear to be normal except for altered tissue MT levels; (2) MT does not appear to affect Cd absorption from gastrointestinal tract, and tissue Cd distribution, but MT plays an important role in the elimination and tissue retention of Cd; (3) MT protects against acute inorganic Cd-induced lethality and hepatotoxicity, and the mechanism of protection appear to be sequestration of Cd in the cytosol, thus reducing amount of Cd in critical organelles; (4) MT modulates Cd-induced expression of protooncogene (c-jun) and tumor suppressor gene (p53) in the mouse liver; (5) MT protects against chronic Cd-induced nephrotoxicity, but does not appear to be a major factor in modulating acute CdMT-induced renal injury and Zn-induced protection; (6) MT protects against chronic Cd-induced hematotoxicity, ameliorates Cd-induced elevation of serum TNF-a and IL-I,B levels, and splenomegaly; (7) MT protects against chronic Cd-induced hepatotoxicity, as evidenced by decreased hepatic inflammation, apoptosis, and granulomas; and (8) MT-null mice are more susceptible than controls to chronic Cd osteotoxicity. MT-TO and MT-null mice are excellent tools in advancing our understanding of the role of MT in Cd toxicology, especially in Cd-induced nephrotoxicity. (Supported by NIH grant ES-0l142).
IP1851 I BACKGROUND DATAOF TRANSGENIC MICE CARRYING THE HUMANPROTOTYPE c-Ha-ras GENE
K. Hioki, H. Ohashi, K. Urano, S. Wakana, S. Yamamoto, T. Usui *, T. Nomura. The Central Institute For Experimental Animals. Kawasaki, Japan Transgenic mice carrying the prototype human c-Ha-ras gene,
49
namely rasH2 mice, was originally established by Saitoh et al. at CIEA. Approximately five to six copies of human c-Ha-ras gene are integrated into the genome of each Tg mouse in a tandem array. Transgenes are expressed in both tumors and normal tissues and the total amount of P21 that is detected by immunoblot analysis is 2 to 3 times higher in Tg mice than in non-Tg mice. No mutations of the transgene has been detected in normal tissues of the Tg mice. CIEA has estblished the maintenance and production of the Tg mice having reproductive difficulties by employing the reproductive engineering techniques such as in vitro fertilization, cryo-preservation of embryos and embryo transfer, and has facilitated a number of background studies in recent years on the Tg mice to support validation of the Tg mouse as an animal model for rapid carcinogenicity testing in which carcinogenic potential of chemical compounds is determined within 26 weeks is now under way in CIEA as well as in volunteered pharmaceutical companies worldwide. The survivability and spontaneous incidences of tumors in the Tg and non-Tg mice during 80 weeks and other biological parameters have been determined to consider in the interpretation of neoplastic phenotypes in carcinogenicity studies.
IP18521 TRANSGENIC ANIMAL MODELS: THEIRROLE IN THE ACCELERATION AND MODIFICATION OFTHE DRUG DEVELOPMENT PROCESS C.B. Spainhour *, V.B. Ciofalo. Chrysalis Preclinical Services-North America, 100 Discovery Drive, Olyphant, USA
Our organization has developed and validated numerous transgenic animal models. These models can be used to improve the quality of lead candidates generated from the basic research process, shorten the preclinical safety evaluation process and accelerate the timelines required for the introduction of new drug entities into man. Such relevant human disease models include CETP Mice, Apo B 100 Mice, CETP/Apo B 100 Mice pertinent to the study of human atherosclerosis as well as sPLAz Mice and TNFa Mice relevant to the study of a wide spectrum of human inflammatory diseases. The drug development process is currently undergoing a re-evaluation as a result of numerous technological advancements. However, this change needs to continue and build momentum in order to improve the level of scrutiny present in the safety evaluation process and accommodate the anticipated explosion of lead candidates produced as a result of such technologies as combinatorial chemistry and high throughput screening. To this end, we are providing several theoretical examples which compare and contrast the traditional or current pharmaceutical development process to one which incorporates the strategic use of transgenic animal models. Our examples illustrate the advantages of such an approach: decreased costs of drug development, extension of useful patent life and increases in potential product revenues.
IP18531
STUDIES ONTAMOXIFEN -INDUCED ~UTATIONS IN THE LIVERSAND UTERIOF BIG BLUE TRANSGENIC F344RATS
lA. Styles *, R. Davies, S. Fenwick, J. Walker, E. Martin, I.N.H. White, L.L. Smith. MRC Toxicology Unit, Hodgkin Building, University ofLeicester, PO Box 138, Lancaster Road, Leicester, LEI 9HN,UK The rat liver carcinogen tamoxifen (20, 10 or 5 mg/kgjwas administered by gavage daily for 6 weeks to female Big Blue transgenic F344 rats (6-8 weeks old) homozygous for the laeI gene. Control animals were given the solvent tricaprylin. The chemical was withdrawn for a period of 2 weeks before the animals were sacrificed. Liver and uterine DNA were extracted and mutations at the laeI gene
IP18491
ARTIFICIALHETEROZYGOTE ANALYSIS: A NOVEL APPROACH FORTHE EFFICIENT IDENTIFICATION OF POINTMUTATIONS
F. Staedtler *, A. Pospiech, S. Steiner, M. Looser. Novartis Phanna AG, Preclinical Safety-Toxicology-PathologylNovartis Services AG, Scientific Services - BioAnalytics, Basel, Switzerland DNA sequencing is regarded as the gold standard in mutation analysis. However, it is rate limiting in experimental systems if DNA sequence data from large numbers of mutants are required. Therefore, improvements of the current protocols aiming to increase sample throughput and to reduce analysis costs are needed. Here we describe a new, efficient procedure for the identification of point mutations, the artificial heterozygote (AH) analysis. DNA sequencing templates of individual mutants of the target gene are amplified by polymerase chain reaction (PCR). By mixing the PCR fragments from individual mutants in a defined ratio, samples of artificial heterozygote (AH) composition are prepared. These AH samples are then submitted to automated DNA sequencing. A specific base-calling algorithm is used for the automated detection of the heterozygous positions. At these positions the individual intensities of the base signals reflect the amount of each template in the AH sample. Thus the identified point mutations can be assigned to the corresponding mutants. The feasibility of this procedure was shown by identifying point mutations in the laeI mutational target gene recovered from Big BlueR transgenic mice.
I P1850 I USEOF METALLOTHIONEIN-TRANSGENIC AND KNOCK-OUT MOUSEMODELSFORTHE STUDY OF CADMIUM TOXICITY C.D. Klaassen *, J. Liu. Univ Kansas Medical Center, Kansas City, KS, USA The role of metallothionein (MT) in Cd toxicology has been studied using MT-I transgenic (MT-TO) and MT-I and II knock-out (MTnull) mice. We have shown that (I) MT-TO and MT-null mice appear to be normal except for altered tissue MT levels; (2) MT does not appear to affect Cd absorption from gastrointestinal tract, and tissue Cd distribution, but MT plays an important role in the elimination and tissue retention of Cd; (3) MT protects against acute inorganic Cd-induced lethality and hepatotoxicity, and the mechanism of protection appear to be sequestration of Cd in the cytosol, thus reducing amount of Cd in critical organelles; (4) MT modulates Cd-induced expression of protooncogene (c-jun) and tumor suppressor gene (p53) in the mouse liver; (5) MT protects against chronic Cd-induced nephrotoxicity, but does not appear to be a major factor in modulating acute CdMT-induced renal injury and Zn-induced protection; (6) MT protects against chronic Cd-induced hematotoxicity, ameliorates Cd-induced elevation of serum TNF-a and IL-I,B levels, and splenomegaly; (7) MT protects against chronic Cd-induced hepatotoxicity, as evidenced by decreased hepatic inflammation, apoptosis, and granulomas; and (8) MT-null mice are more susceptible than controls to chronic Cd osteotoxicity. MT-TO and MT-null mice are excellent tools in advancing our understanding of the role of MT in Cd toxicology, especially in Cd-induced nephrotoxicity. (Supported by NIH grant ES-0l142).
IP1851 I BACKGROUND DATAOF TRANSGENIC MICE CARRYING THE HUMANPROTOTYPE c-Ha-ras GENE
K. Hioki, H. Ohashi, K. Urano, S. Wakana, S. Yamamoto, T. Usui *, T. Nomura. The Central Institute For Experimental Animals. Kawasaki, Japan Transgenic mice carrying the prototype human c-Ha-ras gene,
49
namely rasH2 mice, was originally established by Saitoh et al. at CIEA. Approximately five to six copies of human c-Ha-ras gene are integrated into the genome of each Tg mouse in a tandem array. Transgenes are expressed in both tumors and normal tissues and the total amount of P21 that is detected by immunoblot analysis is 2 to 3 times higher in Tg mice than in non-Tg mice. No mutations of the transgene has been detected in normal tissues of the Tg mice. CIEA has estblished the maintenance and production of the Tg mice having reproductive difficulties by employing the reproductive engineering techniques such as in vitro fertilization, cryo-preservation of embryos and embryo transfer, and has facilitated a number of background studies in recent years on the Tg mice to support validation of the Tg mouse as an animal model for rapid carcinogenicity testing in which carcinogenic potential of chemical compounds is determined within 26 weeks is now under way in CIEA as well as in volunteered pharmaceutical companies worldwide. The survivability and spontaneous incidences of tumors in the Tg and non-Tg mice during 80 weeks and other biological parameters have been determined to consider in the interpretation of neoplastic phenotypes in carcinogenicity studies.
IP18521 TRANSGENIC ANIMAL MODELS: THEIRROLE IN THE ACCELERATION AND MODIFICATION OFTHE DRUG DEVELOPMENT PROCESS C.B. Spainhour *, V.B. Ciofalo. Chrysalis Preclinical Services-North America, 100 Discovery Drive, Olyphant, USA
Our organization has developed and validated numerous transgenic animal models. These models can be used to improve the quality of lead candidates generated from the basic research process, shorten the preclinical safety evaluation process and accelerate the timelines required for the introduction of new drug entities into man. Such relevant human disease models include CETP Mice, Apo B 100 Mice, CETP/Apo B 100 Mice pertinent to the study of human atherosclerosis as well as sPLAz Mice and TNFa Mice relevant to the study of a wide spectrum of human inflammatory diseases. The drug development process is currently undergoing a re-evaluation as a result of numerous technological advancements. However, this change needs to continue and build momentum in order to improve the level of scrutiny present in the safety evaluation process and accommodate the anticipated explosion of lead candidates produced as a result of such technologies as combinatorial chemistry and high throughput screening. To this end, we are providing several theoretical examples which compare and contrast the traditional or current pharmaceutical development process to one which incorporates the strategic use of transgenic animal models. Our examples illustrate the advantages of such an approach: decreased costs of drug development, extension of useful patent life and increases in potential product revenues.
IP18531
STUDIES ONTAMOXIFEN -INDUCED ~UTATIONS IN THE LIVERSAND UTERIOF BIG BLUE TRANSGENIC F344RATS
lA. Styles *, R. Davies, S. Fenwick, J. Walker, E. Martin, I.N.H. White, L.L. Smith. MRC Toxicology Unit, Hodgkin Building, University ofLeicester, PO Box 138, Lancaster Road, Leicester, LEI 9HN,UK The rat liver carcinogen tamoxifen (20, 10 or 5 mg/kgjwas administered by gavage daily for 6 weeks to female Big Blue transgenic F344 rats (6-8 weeks old) homozygous for the laeI gene. Control animals were given the solvent tricaprylin. The chemical was withdrawn for a period of 2 weeks before the animals were sacrificed. Liver and uterine DNA were extracted and mutations at the laeI gene