(CANCER RESEARCH 58. 1338-1343. April 1. 1998] Advances in Brief Thymie Lymphomas in Mice with a Truncating Mutation in Brca2l Lori S. Friedman,2 Fiona C. Thistlethwaite,2 Ketan J. Patel, Veronica P. C. C. Yu, Hyunsook Lee, Ashok R. Venkitaraman, Kenneth J. Abel, Mark B. L. Carlton, Susan M. Hunter, William H. Colledge, Martin J. Evans, and Bruce A. J. Ponder3 Cancer Research Campaign Human Cancer Cenelics Research Group, Cambridge University, Addenbrooke 's Hospital, Cambridge CB2 2QQ, United Kingdom l L S. F.. B. A. J. P.]; Wellcome/Cancer Research Campaign Institute of Cancer and Developmental Biology and Department of Genetics, Cambridge University, Cambridge CB2 1QR, United Kingdom ¡F.C.T.. M.B.L.C., S. M. H., M. J.E.I; Medical Research Council, Laboratory of Molecular Biology. Cambridge CB2 2QH, United Kingdom ¡K.J. P., V. P. C. C. Y., H. L, A. R. V.¡;Sequana Therapeutics, La lotta, California 92037 [K. J. A.¡: and Physiological Laboratory, Cambridge University, Cambridge CB2 3EG. United Kingdom ¡W.H. C.) Abstract Inherited mutations in the BRCA2 gene predispose women to breast and ovarian cancer. We created a mutation in the mouse Brca2 gene that terminates translation in exon 11 at 45% of the normal transcript length. Ninety % of Brca2""ICl"n homozygous mutant mice die prenatally or perinatally. The location of the Brca2""'c<"" mutation differs from those reported previously, and this phenotype suggests a correlation with gen otype analogous to that previously reported in humans. Although hét érozygotemice have remained free of tumors for 10 months, Brca2""'Cam homozygous mutants that survived to adulthood died with thymic lym- phomas between 12 and 14 weeks of age. Introduction Mutations in BRCA2 predispose carriers to female and male breast cancer and to ovarian cancer (1). BRCA2 is predicted to be a tumor suppressor gene, and tumors from mutation carriers consistently show loss of the wild-type alÃ-ele(2). Nearly 90% of germ-line BRCA2 mutations are truncating mutations.4 A genotype-phenotype correla tion suggests that women carrying germ-line mutations within a large region of exon 11 (the OCCR) have an increased risk of ovarian cancer (3). It is not known whether this increased risk is due to the mutations affecting a specific functional domain or to an effect of the mutations on RNA or protein stability. Amino acid motifs found in BRCA2 are the granin motif in exon 27 (4) and eight BRC repeats of unknown significance within exon 11 (5). A transcriptional activation domain was reported at the NH2 terminus of BRCA2 and suggests that BRCA2 may stimulate transcription of unknown genes (6). Brcal was reported to interact with the Rad51 protein and may indicate a role in DNA double-strand break repair (7, 8). Brca2 mutant alÃ-elesin mice result in phenotypes of either early embryonic lethality (8-10) or neonate survival (11). In this study, we report a novel targeted mutation in Brca2 exon 11 that results in a different, intermediate phenotype of embryonic lethality and survival, suggesting a genotype- phenotype correlation. A small proportion of these Brca2 homozy gous mutant mice survive to adulthood, and these survivors develop thymic lymphomas between 12 and 14 weeks of age. Received 12/4/97; accepted 2/13/98. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ' This research was supported by a programme grant from the Cancer Research Campaign [CRC] to B. A. J. P.. The Wellcome Trust, and the Medical Research Council. L. S. F. is a Hitchings-Elion Fellow. B. A. J. P. is a Gibb Fellow of the CRC. 2 These authors contributed equally to this study. 1 To whom requests for reprints should be addressed, at CRC Human Cancer Genetics Research Group. Cambridge University. Addenbrooke's Hospital. Box 238. Hills Road. Cambridge. CB2 2QQ. United Kingdom. Phone: +44-1223-336900; Fax: +44-1223- 336902; E-mail: bajp@mole.bio.cam.ac.uk. 4 This information is available at http://www.nhgri.nih.gov/intramuraLresearch/ Iab_transfer/Bic/. Materials and Methods Cloning and Mapping of the Mouse Brca2 Gene. Mouse Brca2 clones were identified by both PCR and hybridization of cDNA libraries. Genomic DNA from mouse strain 129Sv/Ev was amplified using human BKCA2 exon 11 PCR primers. In the second cloning strategy, an 8.5-day C57BL/6 mouse embryo cDNA library (12) was probed with human exon 11. The mouse sequence was homologous to human BRCA2 and was subsequently shown to be in agreement with reported mouse Brcal exon 11 sequences (13, 14). One clone, mila, was found to be variant between C57BL/6 and Mus spretus mice with a TaqI digest. Scoring this variant on a BSS backcross panel (The Jackson Laboratories) revealed that this cDNA mapped to distal chro mosome 5 in the mouse and was closely linked to markers with human homologues in chromosome 13ql2-ql3, as expected for a BKCA2 homologue. Creating the Targeting Construct. Two nonoverlapping regions of Brca2 exon 11 were amplified from mouse strain 129Sv/Ev genomic DNA. These PCR products were ligated into a ß-geoplasrnid containing stop codons in three reading frames, an internal ribosomal entry sequence, lacZ-Neo fusion, and a polyadenylation signal (Fig. M). The targeting construct was linearized with Noti and electroporated into 129Sv/Ev ES cells that were subjected to G418 selection. Homologous recombination was identified in 2 of the 36 clones that grew in selective medium, using a 3' external Southern blot probe detecting the 3.1-kb EcoRI fragment of the wild-type alÃ-eleand the 3.9-kb EcoRl fragment of the mutant alÃ-ele. Two PCR products confirmed the homologous recombination, with cycling conditions of 94°Cfor 1 min, 56°C for 1 min, and 72°Cfor 2 min. Primers (Fig. \A, arrowheads) for the 5' amplification were: 5'-AGA CCG CGA AGA GTT TGT CCT C-3' and 5'-GAC TCA GAT TCA TTA GCT GTG GTC C-3'. Primers for the 3' amplification were: 5'-GGA TCC CCT TCT TGA CGA GTT CTT CTG A-3' and 5'-GGG GCA TGT AAA CAG TGA GCA-3'. Three germ-line transmit ting chimeric male mice were generated from one targeted ES cell line. Chimeric mice were crossed with females from the MF1 (outbred) or 129Sv/Ev (inbred) strains to obtain heterozygous offspring. Genotyping and Analysis of Brca2 Expression. Brca2 genotyping was done by Southern blot (as above) or by PCR, using DNA extracted from the tail tips of adult mice. Genotyping by PCR produces a wild-type product of 310 bp using the primers 5'-ACT GTC ACT CAA TTA CCA GCT C-3' (WT forward) and 5'-TTG CTG AGO TAG CAT TTC ACT C-3' (WT reverse) or a 350-bp product from the mutant alÃ-eleby using the primers 5'-CCT TCT TGA CGA GTT CTT CTG-3' (ß-geoforward) and 5'-TTG CTG AGG TAG CAT TTC ACT C-3' (WT reverse). To obtain RNA for RT-PCR5 analysis, embryos were immediately frozen on dry ice, and RNA was extracted with Triazol (Sigma Chemical Co.) according to the manufacturer's instructions. RNA was reverse transcribed using oligodeoxythymidylic acid primer with avian myeloblastosis virus reverse transcriptase (Promega) according to the manufacturer's instructions. RT-PCR was successfully performed using 12 different primer pairs: five primer combinations amplified regions 5' of the ß-geoinsertion, three primer combinations surrounded the ß-geoinsertion, two primer combinations surrounded exon 11, and two primer combinations amplified regions 3' of the ß-geoinsertion. PCR was performed for 35 cycles ~ The abbreviations used are: RT-PCR, reverse transcription-PCR; ES, embryonic stem; TCR, T-cell receptor; OCCR, ovarian cancer cluster region. 1338 on May 1, 2021. © 1998 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from