Search tips
Search criteria 


Logo of molcellbPermissionsJournals.ASM.orgJournalMCB ArticleJournal InfoAuthorsReviewers
Mol Cell Biol. 1996 December; 16(12): 6804–6809.
PMCID: PMC231683

Targeted disruption of the mouse topoisomerase I gene by camptothecin selection.


Topoisomerase I has ubiquitous roles in important cellular functions such as replication, transcription, and recombination. In order to further characterize this enzyme in vivo, we have used gene targeting to inactivate the mouse Top-1 gene. A selection protocol using the topoisomerase I inhibitor camptothecin facilitated isolation of embryonic stem cell clones containing an inactivated allele; isolation of correctly targeted clones was enhanced 75-fold over that achieved by normal selection procedures. The disrupted Top-1 allele is embryonic lethal when homozygous, and development of such embryos fails between the 4- and 16-cell stages. Both sperm and oocytes containing the inactive allele maintain viability through the fertilization point, and thus gene expression of topoisomerase I is not required for gamete viability. These studies demonstrate that topoisomerase I is essential for cell growth and division in vivo. The Top-1 gene was also shown to be linked to the agouti locus.

Full Text

The Full Text of this article is available as a PDF (268K).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Baumgärtner B, Klett C, Hameister H, Richter A, Knippers R. Mouse genes encoding DNA topoisomerase I. Mamm Genome. 1994 Jan;5(1):19–25. [PubMed]
  • Bjornsti MA, Benedetti P, Viglianti GA, Wang JC. Expression of human DNA topoisomerase I in yeast cells lacking yeast DNA topoisomerase I: restoration of sensitivity of the cells to the antitumor drug camptothecin. Cancer Res. 1989 Nov 15;49(22):6318–6323. [PubMed]
  • Burris HA, 3rd, Fields SM. Topoisomerase I inhibitors. An overview of the camptothecin analogs. Hematol Oncol Clin North Am. 1994 Apr;8(2):333–355. [PubMed]
  • D'Arpa P, Machlin PS, Ratrie H, 3rd, Rothfield NF, Cleveland DW, Earnshaw WC. cDNA cloning of human DNA topoisomerase I: catalytic activity of a 67.7-kDa carboxyl-terminal fragment. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2543–2547. [PubMed]
  • Di Mauro E, Camilloni G, Verdone L, Caserta M. DNA topoisomerase I controls the kinetics of promoter activation and DNA topology in Saccharomyces cerevisiae. Mol Cell Biol. 1993 Nov;13(11):6702–6710. [PMC free article] [PubMed]
  • DiNardo S, Voelkel KA, Sternglanz R, Reynolds AE, Wright A. Escherichia coli DNA topoisomerase I mutants have compensatory mutations in DNA gyrase genes. Cell. 1982 Nov;31(1):43–51. [PubMed]
  • Dubrez L, Goldwasser F, Genne P, Pommier Y, Solary E. The role of cell cycle regulation and apoptosis triggering in determining the sensitivity of leukemic cells to topoisomerase I and II inhibitors. Leukemia. 1995 Jun;9(6):1013–1024. [PubMed]
  • Gilmour DS, Pflugfelder G, Wang JC, Lis JT. Topoisomerase I interacts with transcribed regions in Drosophila cells. Cell. 1986 Feb 14;44(3):401–407. [PubMed]
  • Giovanella BC, Stehlin JS, Wall ME, Wani MC, Nicholas AW, Liu LF, Silber R, Potmesil M. DNA topoisomerase I--targeted chemotherapy of human colon cancer in xenografts. Science. 1989 Nov 24;246(4933):1046–1048. [PubMed]
  • Hsiang YH, Hertzberg R, Hecht S, Liu LF. Camptothecin induces protein-linked DNA breaks via mammalian DNA topoisomerase I. J Biol Chem. 1985 Nov 25;260(27):14873–14878. [PubMed]
  • Ishimi Y, Sugasawa K, Hanaoka F, Kikuchi A. Replication of the simian virus 40 chromosome with purified proteins. J Biol Chem. 1991 Aug 25;266(24):16141–16148. [PubMed]
  • Kijima T, Kubota N, Nishio K. Establishment of a CPT-11-resistant human ovarian cancer cell line. Anticancer Res. 1994 May-Jun;14(3A):799–803. [PubMed]
  • Kim HS, Smithies O. Recombinant fragment assay for gene targetting based on the polymerase chain reaction. Nucleic Acids Res. 1988 Sep 26;16(18):8887–8903. [PMC free article] [PubMed]
  • Kim RA, Wang JC. Function of DNA topoisomerases as replication swivels in Saccharomyces cerevisiae. J Mol Biol. 1989 Jul 20;208(2):257–267. [PubMed]
  • Koiwai O, Yasui Y, Sakai Y, Watanabe T, Ishii K, Yanagihara S, Andoh T. Cloning of the mouse cDNA encoding DNA topoisomerase I and chromosomal location of the gene. Gene. 1993 Mar 30;125(2):211–216. [PubMed]
  • Kondo K, Tsuneizumi K, Watanabe T, Oishi M. Induction of in vitro differentiation of mouse embryonal carcinoma (F9) cells by inhibitors of topoisomerases. Cancer Res. 1991 Oct 1;51(19):5398–5404. [PubMed]
  • Kretzschmar M, Meisterernst M, Roeder RG. Identification of human DNA topoisomerase I as a cofactor for activator-dependent transcription by RNA polymerase II. Proc Natl Acad Sci U S A. 1993 Dec 15;90(24):11508–11512. [PubMed]
  • Kubota N, Kanzawa F, Nishio K, Takeda Y, Ohmori T, Fujiwara Y, Terashima Y, Saijo N. Detection of topoisomerase I gene point mutation in CPT-11 resistant lung cancer cell line. Biochem Biophys Res Commun. 1992 Oct 30;188(2):571–577. [PubMed]
  • Kunze N, Yang GC, Dölberg M, Sundarp R, Knippers R, Richter A. Structure of the human type I DNA topoisomerase gene. J Biol Chem. 1991 May 25;266(15):9610–9616. [PubMed]
  • Lee MP, Brown SD, Chen A, Hsieh TS. DNA topoisomerase I is essential in Drosophila melanogaster. Proc Natl Acad Sci U S A. 1993 Jul 15;90(14):6656–6660. [PubMed]
  • Loenen WA, Blattner FR. Lambda Charon vectors (Ch32, 33, 34 and 35) adapted for DNA cloning in recombination-deficient hosts. Gene. 1983 Dec;26(2-3):171–179. [PubMed]
  • Mansour SL, Thomas KR, Capecchi MR. Disruption of the proto-oncogene int-2 in mouse embryo-derived stem cells: a general strategy for targeting mutations to non-selectable genes. Nature. 1988 Nov 24;336(6197):348–352. [PubMed]
  • Morham SG, Shuman S. Covalent and noncovalent DNA binding by mutants of vaccinia DNA topoisomerase I. J Biol Chem. 1992 Aug 5;267(22):15984–15992. [PubMed]
  • Mortensen RM, Zubiaur M, Neer EJ, Seidman JG. Embryonic stem cells lacking a functional inhibitory G-protein subunit (alpha i2) produced by gene targeting of both alleles. Proc Natl Acad Sci U S A. 1991 Aug 15;88(16):7036–7040. [PubMed]
  • Porter SE, Champoux JJ. The basis for camptothecin enhancement of DNA breakage by eukaryotic topoisomerase I. Nucleic Acids Res. 1989 Nov 11;17(21):8521–8532. [PMC free article] [PubMed]
  • Potmesil M, Hsiang YH, Liu LF, Bank B, Grossberg H, Kirschenbaum S, Forlenza TJ, Penziner A, Kanganis D, Forlenzar TJ, et al. Resistance of human leukemic and normal lymphocytes to drug-induced DNA cleavage and low levels of DNA topoisomerase II. Cancer Res. 1988 Jun 15;48(12):3537–3543. [PubMed]
  • Pruss GJ, Manes SH, Drlica K. Escherichia coli DNA topoisomerase I mutants: increased supercoiling is corrected by mutations near gyrase genes. Cell. 1982 Nov;31(1):35–42. [PubMed]
  • Raji A, Zabel DJ, Laufer CS, Depew RE. Genetic analysis of mutations that compensate for loss of Escherichia coli DNA topoisomerase I. J Bacteriol. 1985 Jun;162(3):1173–1179. [PMC free article] [PubMed]
  • Ryan AJ, Squires S, Strutt HL, Johnson RT. Camptothecin cytotoxicity in mammalian cells is associated with the induction of persistent double strand breaks in replicating DNA. Nucleic Acids Res. 1991 Jun 25;19(12):3295–3300. [PMC free article] [PubMed]
  • Sorensen M, Sehested M, Jensen PB. Characterisation of a human small-cell lung cancer cell line resistant to the DNA topoisomerase I-directed drug topotecan. Br J Cancer. 1995 Aug;72(2):399–404. [PMC free article] [PubMed]
  • Stewart AF, Herrera RE, Nordheim A. Rapid induction of c-fos transcription reveals quantitative linkage of RNA polymerase II and DNA topoisomerase I enzyme activities. Cell. 1990 Jan 12;60(1):141–149. [PubMed]
  • Stewart AF, Schütz G. Camptothecin-induced in vivo topoisomerase I cleavages in the transcriptionally active tyrosine aminotransferase gene. Cell. 1987 Sep 25;50(7):1109–1117. [PubMed]
  • Tanizawa A, Beitrand R, Kohlhagen G, Tabuchi A, Jenkins J, Pommier Y. Cloning of Chinese hamster DNA topoisomerase I cDNA and identification of a single point mutation responsible for camptothecin resistance. J Biol Chem. 1993 Dec 5;268(34):25463–25468. [PubMed]
  • Tanizawa A, Pommier Y. Topoisomerase I alteration in a camptothecin-resistant cell line derived from Chinese hamster DC3F cells in culture. Cancer Res. 1992 Apr 1;52(7):1848–1854. [PubMed]
  • Thrash C, Bankier AT, Barrell BG, Sternglanz R. Cloning, characterization, and sequence of the yeast DNA topoisomerase I gene. Proc Natl Acad Sci U S A. 1985 Jul;82(13):4374–4378. [PubMed]
  • Uemura T, Yanagida M. Isolation of type I and II DNA topoisomerase mutants from fission yeast: single and double mutants show different phenotypes in cell growth and chromatin organization. EMBO J. 1984 Aug;3(8):1737–1744. [PubMed]
  • Vosberg HP. DNA topoisomerases: enzymes that control DNA conformation. Curr Top Microbiol Immunol. 1985;114:19–102. [PubMed]
  • Wang JC. Recent studies of DNA topoisomerases. Biochim Biophys Acta. 1987 Jun 6;909(1):1–9. [PubMed]
  • Wobbe CR, Dean FB, Murakami Y, Borowiec JA, Bullock P, Hurwitz J. In vitro replication of DNA containing either the SV40 or the polyoma origin. Philos Trans R Soc Lond B Biol Sci. 1987 Dec 15;317(1187):439–453. [PubMed]
  • Wold MS, Weinberg DH, Virshup DM, Li JJ, Kelly TJ. Identification of cellular proteins required for simian virus 40 DNA replication. J Biol Chem. 1989 Feb 15;264(5):2801–2809. [PubMed]
  • Woychik RP, Generoso WM, Russell LB, Cain KT, Cacheiro NL, Bultman SJ, Selby PB, Dickinson ME, Hogan BL, Rutledge JC. Molecular and genetic characterization of a radiation-induced structural rearrangement in mouse chromosome 2 causing mutations at the limb deformity and agouti loci. Proc Natl Acad Sci U S A. 1990 Apr;87(7):2588–2592. [PubMed]

Articles from Molecular and Cellular Biology are provided here courtesy of American Society for Microbiology (ASM)