| First Author | Schweer T | Year | 1998 |
| Journal | J Biomol Struct Dyn | Volume | 16 |
| Pages | 180-1 (Abstr.) | Mgi Jnum | J:82357 |
| Mgi Id | MGI:2652423 | Citation | Schweer T, et al. (1998) Conditional liver-specific "knock-out" of the murine genes encoding 06-Alkylguanine-DNA Alkyltransferase (AGT) and AP-Endonuclease (APE). J Biomol Struct Dyn 16:180-1 (Abstr.) |
| abstractText | Full text of Abstract: Abstracts. From the Journal of Biomolecular Structure and Dynamics 16, 149-187 (1998): Abstracts: 25th Anniversary Conference, National Foundation for Cancer Research, September 14-16, 1998. Torsten Schweer and Manfred F. Rajewsky*. Institute of Cell Biology (Cancer Research), University of Essen Medical School and West German Cancer Center Essen, Hufeland-Strasse 55, D-45122 Essen, Germany. *Author to whom correspondence should be addressed. Phone: 49-201-723-2802; Fax: +49-201-723-5905: E-mail: rajewsky@uni-essen.de. ConditionaI Liver-Specific "Knock-out" of the Murine Genes Encoding 06-Alkylguanine-DNA Alkyl- transferase (AGT) and AP-Endonuclease (APE). Defects in DNA repair may destabilize the integrity of genomic DNA and result in mutator phenotypes, thereby increasing the cellular risk of malignant conversion. Well-defined and reproducible animal models are required for assessment of the relative importance of specific DNA repair functions and pathways in counteracting oncogenesis, as induced by mutagenic carcinogens with known DNA reaction products. To be able to delete a defined DNA repair function in vivo well and freely timed, and to restrict this inactivation to a controllable number of a particular type of target cells in vivo, we have used the conditional gene "knock-out'' approach in mice. The actual situation in carcinogenesis can thereby be simulated in a more true-to-life manner than would be the case using conventional germlike gene inactivation. Mice were generated in which the last exon of the O6-alkylguanine-DNA alkyltransferase (AGT) DNA repair gene is flanked ("floxed") by two loxP sites, and can thus be inactivated in vivo using the cre-loxP methodology (1). The last exon of AGT encodes the active center of the AGT protein. Embryonic stem cells (ES-cells) homozygous for the deleted allele lacked detectable AGT activity, while cells heterozygous for the "floxed" allele exhibited AGT levels comparable to wild-type cells (400 fmol/mg protein). The elimination of O6-methylguanine (O6-MeGua) and O6-ethylguanine (O6-EtGua) from cellular DNA was strongly reduced in AGT-deleted cells. T50-values, i.e. the time required for elimination from DNA of 50% of the input O6-MeGua and O6-EtGua residues, increased from 8 h to 24 h and from 2.5 h to 30 h, respectively. The cre recombinase coding sequence was cloned under the control of the human c-reactive protein promoter (hCRP-cre) to permit inducible hepatocyte-specific expression of the transgene. One out of five hCRP-cre transgenic lines showed inducible liver-specific expression of cre, as judged by RT-PCR and in situ hybridization. To examine cre recombinase activity at the single-cell level, AGT "floxed" mice were crossed with "reporter mice" carrying a 1acZ expression construct with a "floxed" stop cassete between promoter and coding sequence (2). Staining of Beta-galactosidase with a flourescent antibody revealed that a subpopulation of cre RNA-positive cells had spliced out the stop cassette. Mice with the genotype AGTflox/flox, hCRP-cre-heterozygous are used to evaluate the suppressive effect of O6-alkylguanine repair by AGT on the frequency of carcinogen (e.g., dimethyl- or diethylnitrosamine)-induced transformation in mouse livers containing different proportions of AGT repair-deficient versus repair-proficient hepatocytes. AP-endonuclease (APE) is a bifunctional enzyme which stimulates the binding of transcription factors (e.g,, AP-1, p53) to DNA by a redox reaction and initiates the processing of basic sites by its endonuclease activity. Conventional germlike "knock-out" of the APE gene results in embryonic lethality (3). Focusing on the DNA repair function of the APE protein, we have designed a target vector in which only the fourth exon of APE- which encodes the endonuclease domain- is "floxed", leaving the redox function intact. The corresponding ES-cells are under study. Research supported by the National Foundation for Cancer Research (NFCR) through KREBSFORSCHUNG INTERNATIONAL e.V. (Germany). References and Footnotes 1. H. Gu et al., Science 265, 103-106, 1993. 2. Y. Zhang, et al., Nucleic Acids Res., 24, 543-548, 1996. 3. S. Xanthoudakis, et al., PNAS 93, 8919-8923, 1996. |
