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P XV.18 Development of transgenic mouse mutation test model containing XYLE target gene
S-XV: Transgenic model for studying environmental mutagenesis
POSTERS B
Ir Xv,lsl
Ip Xv,2ol
Development of transgenic mouse mutallon test model contalnlna XYLE ta'1:et gene
Mu-Quan Vin l , Jian Huang", Vao-Fu Chen" , Jian-Quan Chen l , Guo-X iang Cheni, Shao-Fu Xu l , Jerry L. Hsuelr', Xin-Fang Qiu 3. 'Department of Toxicology, Second Military Medical University, Shanghai 100433 . PR China; 21nstitute of Biotechnology. Yangzhou University, Agricultural OJIlege. Yangzhou 150009, PR China; J Institute ofgenetics. Fudan University. Shanghai 200433, PR China In this study, we constructed two transgenic mouse lines containing XYLE gene (0.9 Kb) encoded a catechal 2,3-dioxygenase (CatOlase) as mutat ional target gene in a recoverable pESnX plasmid (12.7 Kb) by microinjection. 549 fertil ized eggs of ICR mice were microinjected with pESnX DNA, 352 survival embryos were then transferred to oviducts of 24 pseudopregment females respect ively which produced 41 offsprings. II of them died in few days after birth . The 30 survival offspnngs were screened for incorporation of the foreign DNA. There arc 17 positive offsprings were identified in PCR (with specific primers for XYLE gene) and Southern blot analyses. Finally, two (Y, and Y31) positive and stout male mice with intact pESnX DNA in their genomes were chosen as founders for establishing transgenic lineages respectively. V, and Y31 mated with Wild-type ICR females respectively and then produced G I offspring. We got 36190 positive G I from V9 and 44/69 positive G I from Y31. G I posinve mice m same litter inbred to produce G2 offspring, we got 20127 pos itive G2 from Y9 and 38/46 positive G2 from Y31. It indicated lite integrated vector can stably transm it to next generat ion. We rescue lite target gene from mouse by digest ing genom ic DNA, circularizing the plasmid and then transforming E. coli for measuring mutation frequency and sequencing.
Toxic and mutagenic effects of %-amlno-l-methyl-6phenyllmldazo-I4,5-bl-pyrldlne (PhIP) In XPA deftclent mIce
Johanna Klein, Edwin Zwart, Coen van Kreijl, Harry van Steeg. RlVM, Laboratory ofHealth Effects Research, Dept. ofCarcinogenesis. Mutagenesis and Genetics , PO Box J, 3720 BA Bilthooen, The Netherlands PhlP is the most predominant heterocyclic aromatic amine formed during cooking of meat and fish. This compound is a potent bacterial mutagen, and has been shown to cause colon and mammary carcinomas in rats and lymphomas in mice. We investigated lite effects of PhlP in XPA deficient mice, which are currently being evaluated as model to be used for shortterm in vivo carcinogenicity test ing. The mice lack a functional XPA protein , which is involved in recognition of DNA damage by the nucleotide excision repair (NER) pathway, and are, therefore, expected to accumulate PhIPinduced DNA lesions . Mice were given varying amounts of PhlP in the diet (ad libitum). XPA-I- animals treated with doses of 0.02% or 0.01 % (w/w) died within 3 and 6 weeks, respectively, while wildtype mice survived a dose of 0.03% for at least two years. Of thirteen animals treated with a dose of 0.004%, only three survived a l3-week exposure period . Intercurrent deaths could always be related to intestinal failure and starvation. Mice treated with lower doses of PhlP (i.e, 0.001% and 0.0025%) did not show any signs of toxicity, nor did they die from developing tumors up to 40 weeks after start ofthe experiment. XPA-I- mice were crossed with lacZ transgenic mice in order to investigate mutation induction by PhiP in various tissues. Elevated lacZ mutat ion frequenc ies were found in a dose dependent way in DNA isolated from spleen, liver, duodenum and ileum of lite XPA-I- mice . In repair proficient mice, however, mutation frequenc ies were only increased in lacZ DNA isolated from the duodenum.
Ip XV,211
Keyword(s): XYLE gene ; Transgenic mouse; Mutagenes is
S 151
The orIgin of spontaneous mutations In the PlIIX174 AM3, CS70 transgenic system
Heinrich V. Mailing, Robert P. Weaver. NIEHS, Research Triangle Parle, NC 27709, USA
Ir
XV,191
Environmental carcinogen (DI\fBA) aels syne'1: 1stlcally In Int-S/aromatase transgenic mice that have Increased mammary estrogen activity l
NagaJakshmi Kesbava", C. Fang2 , Kapil N. BhaJlal , Rajeshwar Rao Tekmal • I Department of Gynecology and Obstetrics of Winship Cancer Center. Atlanta, GA 30322 . USA; 2Department of Medicine of Winship Cancer Center. Emory University School of Medicine, Atlanta, GA 30322. USA Our studies using int-5/aromatase transgenic model have shown that early exposure ofmammasy epithelium to in Situ estrogen as a result ofoverexpression of int-5lasomatase appear to predispose mammary tissue to preneoplastic changes. Here, we hypothesize that the preneoplasric changes induced by mammary estrogen in int-5/aromatase transgenic females may be susceptible to environmental carcinogen like 7,12 dimethyl benz[a]anthracene (DMBA), and may result in acceleration and/or increase in the incidence of breast cancer. In lite present study we show that there was increased preneoplastic/neoplastic changes when lite transgenic mice were treated with DMBA . Tumors appeared in 25% of the mice that were treated with DMBA and all ammaJs had microscopic evidence of tumor formation . Control non-transgenic females did not have significant changes even after treat ing with DMBA. There was also increased neoplastic changes in the involuted transgenic females due to carcinogen exposure indicating that DMBA may be acting synergistically as a tumor promoter. Furthermore, these animals showed increased telomerase acnvity which is an indication of neoplastic transformation. Therefore, our studies show that early estrogen exposure leads to preneoplastic changes in the mammasy epithelial cells which, in tum, increase the risk of developing neoplasia and increased susceptibility to environmental carcinogens. Kcyword(s): Aromatase; Dimethyl Benz[a)anthracene; breast cancer
The target genes in most transgenic systems have a higher spontaneous mutation frequency than comparable intrinsic mammalian target genes . This higher mutat ion frequency in transgenic systems decreases the sensitivity of sueh systems to detect weak mutagenic activity. Spontaneous mutations in transgenes arise predominantly from three sources- in the animals, during rescue of the vector, and under the selection conditions to detect the expressed mutant phenotype. We have used the PhiX-transgenic system in mice to evaluate the extent to which each primary source of mutations contr ibutes to the total frequency. Mutations in this system are detected as revertants of an amber mutation (am3) in the vector. The totaJ number of phages recovered from animals tissues was determ ined by plating on E. coli CQ-2 (permissible, su"). Revertants were detected by plating on E. coli C (non-permissible, su"), E. coli C has been in culture in various laboratories for many yeass, genetic drift may have occurred. and genetic differences accumulated between lite sub-strains. Various sub-strains were tested for their ability to accurately detect revertants by compasing a) the plating efficiency of known revertants and b) the revertant frequency among a standard suspens ion of phages . Significant differences were found among four sub-strains ofE. coli C and with in each sub-strain results differed among single colony isolates. Approaches to est imate the frequency of revertants that are fixed in lite animal will be discussed. Keyword(s): Transgenic; PhiX; Spontaneous Mutations
POSTERS B
Ir Xv,lsl
Ip Xv,2ol
Development of transgenic mouse mutallon test model contalnlna XYLE ta'1:et gene
Mu-Quan Vin l , Jian Huang", Vao-Fu Chen" , Jian-Quan Chen l , Guo-X iang Cheni, Shao-Fu Xu l , Jerry L. Hsuelr', Xin-Fang Qiu 3. 'Department of Toxicology, Second Military Medical University, Shanghai 100433 . PR China; 21nstitute of Biotechnology. Yangzhou University, Agricultural OJIlege. Yangzhou 150009, PR China; J Institute ofgenetics. Fudan University. Shanghai 200433, PR China In this study, we constructed two transgenic mouse lines containing XYLE gene (0.9 Kb) encoded a catechal 2,3-dioxygenase (CatOlase) as mutat ional target gene in a recoverable pESnX plasmid (12.7 Kb) by microinjection. 549 fertil ized eggs of ICR mice were microinjected with pESnX DNA, 352 survival embryos were then transferred to oviducts of 24 pseudopregment females respect ively which produced 41 offsprings. II of them died in few days after birth . The 30 survival offspnngs were screened for incorporation of the foreign DNA. There arc 17 positive offsprings were identified in PCR (with specific primers for XYLE gene) and Southern blot analyses. Finally, two (Y, and Y31) positive and stout male mice with intact pESnX DNA in their genomes were chosen as founders for establishing transgenic lineages respectively. V, and Y31 mated with Wild-type ICR females respectively and then produced G I offspring. We got 36190 positive G I from V9 and 44/69 positive G I from Y31. G I posinve mice m same litter inbred to produce G2 offspring, we got 20127 pos itive G2 from Y9 and 38/46 positive G2 from Y31. It indicated lite integrated vector can stably transm it to next generat ion. We rescue lite target gene from mouse by digest ing genom ic DNA, circularizing the plasmid and then transforming E. coli for measuring mutation frequency and sequencing.
Toxic and mutagenic effects of %-amlno-l-methyl-6phenyllmldazo-I4,5-bl-pyrldlne (PhIP) In XPA deftclent mIce
Johanna Klein, Edwin Zwart, Coen van Kreijl, Harry van Steeg. RlVM, Laboratory ofHealth Effects Research, Dept. ofCarcinogenesis. Mutagenesis and Genetics , PO Box J, 3720 BA Bilthooen, The Netherlands PhlP is the most predominant heterocyclic aromatic amine formed during cooking of meat and fish. This compound is a potent bacterial mutagen, and has been shown to cause colon and mammary carcinomas in rats and lymphomas in mice. We investigated lite effects of PhlP in XPA deficient mice, which are currently being evaluated as model to be used for shortterm in vivo carcinogenicity test ing. The mice lack a functional XPA protein , which is involved in recognition of DNA damage by the nucleotide excision repair (NER) pathway, and are, therefore, expected to accumulate PhIPinduced DNA lesions . Mice were given varying amounts of PhlP in the diet (ad libitum). XPA-I- animals treated with doses of 0.02% or 0.01 % (w/w) died within 3 and 6 weeks, respectively, while wildtype mice survived a dose of 0.03% for at least two years. Of thirteen animals treated with a dose of 0.004%, only three survived a l3-week exposure period . Intercurrent deaths could always be related to intestinal failure and starvation. Mice treated with lower doses of PhlP (i.e, 0.001% and 0.0025%) did not show any signs of toxicity, nor did they die from developing tumors up to 40 weeks after start ofthe experiment. XPA-I- mice were crossed with lacZ transgenic mice in order to investigate mutation induction by PhiP in various tissues. Elevated lacZ mutat ion frequenc ies were found in a dose dependent way in DNA isolated from spleen, liver, duodenum and ileum of lite XPA-I- mice . In repair proficient mice, however, mutation frequenc ies were only increased in lacZ DNA isolated from the duodenum.
Ip XV,211
Keyword(s): XYLE gene ; Transgenic mouse; Mutagenes is
S 151
The orIgin of spontaneous mutations In the PlIIX174 AM3, CS70 transgenic system
Heinrich V. Mailing, Robert P. Weaver. NIEHS, Research Triangle Parle, NC 27709, USA
Ir
XV,191
Environmental carcinogen (DI\fBA) aels syne'1: 1stlcally In Int-S/aromatase transgenic mice that have Increased mammary estrogen activity l
NagaJakshmi Kesbava", C. Fang2 , Kapil N. BhaJlal , Rajeshwar Rao Tekmal • I Department of Gynecology and Obstetrics of Winship Cancer Center. Atlanta, GA 30322 . USA; 2Department of Medicine of Winship Cancer Center. Emory University School of Medicine, Atlanta, GA 30322. USA Our studies using int-5/aromatase transgenic model have shown that early exposure ofmammasy epithelium to in Situ estrogen as a result ofoverexpression of int-5lasomatase appear to predispose mammary tissue to preneoplastic changes. Here, we hypothesize that the preneoplasric changes induced by mammary estrogen in int-5/aromatase transgenic females may be susceptible to environmental carcinogen like 7,12 dimethyl benz[a]anthracene (DMBA), and may result in acceleration and/or increase in the incidence of breast cancer. In lite present study we show that there was increased preneoplastic/neoplastic changes when lite transgenic mice were treated with DMBA . Tumors appeared in 25% of the mice that were treated with DMBA and all ammaJs had microscopic evidence of tumor formation . Control non-transgenic females did not have significant changes even after treat ing with DMBA. There was also increased neoplastic changes in the involuted transgenic females due to carcinogen exposure indicating that DMBA may be acting synergistically as a tumor promoter. Furthermore, these animals showed increased telomerase acnvity which is an indication of neoplastic transformation. Therefore, our studies show that early estrogen exposure leads to preneoplastic changes in the mammasy epithelial cells which, in tum, increase the risk of developing neoplasia and increased susceptibility to environmental carcinogens. Kcyword(s): Aromatase; Dimethyl Benz[a)anthracene; breast cancer
The target genes in most transgenic systems have a higher spontaneous mutation frequency than comparable intrinsic mammalian target genes . This higher mutat ion frequency in transgenic systems decreases the sensitivity of sueh systems to detect weak mutagenic activity. Spontaneous mutations in transgenes arise predominantly from three sources- in the animals, during rescue of the vector, and under the selection conditions to detect the expressed mutant phenotype. We have used the PhiX-transgenic system in mice to evaluate the extent to which each primary source of mutations contr ibutes to the total frequency. Mutations in this system are detected as revertants of an amber mutation (am3) in the vector. The totaJ number of phages recovered from animals tissues was determ ined by plating on E. coli CQ-2 (permissible, su"). Revertants were detected by plating on E. coli C (non-permissible, su"), E. coli C has been in culture in various laboratories for many yeass, genetic drift may have occurred. and genetic differences accumulated between lite sub-strains. Various sub-strains were tested for their ability to accurately detect revertants by compasing a) the plating efficiency of known revertants and b) the revertant frequency among a standard suspens ion of phages . Significant differences were found among four sub-strains ofE. coli C and with in each sub-strain results differed among single colony isolates. Approaches to est imate the frequency of revertants that are fixed in lite animal will be discussed. Keyword(s): Transgenic; PhiX; Spontaneous Mutations