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1 Department of Immunology, Graduate School of Medical Sciences, Kumamoto University, 1-1-1, Honjo, Kumamoto 860-8556, Japan
2 PRESTO, Japan Science and Technology Agency (JST), Saitama, 332-0012 Japan
3 Division of Isotope Science, Center for Resource Analysis (CRA), Kumamoto University, 2-2-1, Honjo, Kumamoto 860-0811, Japan
4 Division of Reproductive Engineering, Center for Animal Resources and Development (CARD), Kumamoto University, 2-2-1, Honjo, Kumamoto 860-0811, Japan
5 Laboratory of Virus Immunology, Center for AIDS Research, Institute for Virus Research, Kyoto University, 53, Shogoin, Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan
6 CREST, Japan Science and Technology Agency (JST), Saitama, 332-0012 Japan
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Abstract |
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Introduction |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
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In Saccharomyces cerevisiae, an inhibitor of actin assembly, Sac3, is critical for regulation of the transcription-coupled DNA recombination process (Bauer & Kölling 1996). Sac3 is associated with Thp1 to form a complex that is involved in RNA export from the nucleus to the cytoplasm (Fischer et al. 2002). Lack of Sac3 causes DNA hyper-recombination in yeast cells (Gallardo et al. 2003). In mammals, however, little is known about the regulation of DNA recombination via Sac3-like molecule(s). A mammalian homologue GANP is expressed in GC B-cells after immunization with T-cell-dependent Ags (Kuwahara et al. 2000). Lack of GANP caused by CD19-Cre conditioned gene targeting impaired the affinity maturation of Ag-driven B-cells with decreased Ig V-region somatic hypermutation (Kuwahara et al. 2004). The mutant B-cells showed altered Ig V-region DSBs during the immune response in GC B-cells (Kawatani et al. 2005). These results indicate that GANP regulates either generation or repair of DNA DSBs in B-cells.
The sequence similarity between Saccharomyces Sac3 and the middle portion (600 amino acids) of mammalian GANP is 23% at amino acid level but not significant in the nucleotide level. This domain is conserved in all species with 20%–30% homology at the amino acid level from yeast to human, suggesting that it may participate in a similar function. However, mammalian GANP carries additional functional domains, named RNA-primase domain in the amino terminal side region and MCM3-binding/acetylating domain in the carboxyl terminal region (Kuwahara et al. 2000, 2001; Takei et al. 2001). There are no similar regions in Saccharomyces Sac3 that correspond to the RNA-primase and MCM3-binding/acetylating domains, which may suggest the unique function of GANP other than that of Saccharomyces Sac3. Taking the molecular function of Saccharomyces Sac3 into consideration, we addressed whether GANP participates in regulation of DNA recombination in mammalian cells. Here, we studied the effect of GANP on DNA repair by using a direct-repeat ß-galactosidase (lacZ) gene vector introduced into mammalian cells.
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Results |
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Here, we investigate whether loss or gain of GANP alters DNA recombination. We created heterozygous ganp gene targeted ES cells by HR to disrupt the 5'-promoter region, exon I and II regions of the ganp gene (Fig. 1A). Southern blotting with Probe A after BamHI digestion of heterozygous knockout (ganp+/d) mouse DNA revealed the targeted allele as a 5.0-kb band distinct from the 6.4-kb band of the wild-type (wt) allele (Fig. 1B). Targeted disruption of the ganp gene was confirmed by PCR analysis of genomic DNAs (data not shown). We concluded that homozygous mutant (ganpd/d) mice were embryonic lethal after examining more than 400 offspring. Therefore, a MEF cell line was generated from hetero-insufficient ganp+/d mice (MEFganp+/d) by SV40 infection. We measured DNA recombination in MEFganp+/d cells after introduction of a recombination substrate reporter construct (Fig. 1C). The vector contains two mutated lacZ genes, lacZ' and lacZ'', with stop codons at different HpaI sites. This precludes expression of productive lacZ, but is capable of generating productive lacZ after homology-mediated DNA recombination in the transfectants. The extrachromosomal plasmid DNA from the MEF transfectant was extracted and the frequency of productive lacZ after transformation in Escherichia coli was measured (Fig. 1D). The formation of a productive lacZ gene either by DNA recombination or by inter-plasmid recombination can be measured by assessing the number of colonies with LacZ activity. MEFganp+/d showed more DNA recombination (5.2-fold at day 2 and 4-fold at day 4) than wt MEFganp+/+. DNA recombination occurred mostly with deletion of the intervening DNA (Fig. 1E). The experiments carried out in the yeast cells used the nutrition-inducible promoter constructs for the reporter assay as stably introduced in the mutant cells for DNA recombination assay (Gallardo et al. 2003). We examined whether the hyper-recombination in MEFganp+/d is transcription-coupled or not by comparing the effect of GANP deficiency with promoterless construct and the construct with the SV40 promoter region. The effect of GANP deficiency was compared between MEFganp+/d and MEFganp+/+ cells by the ratio of DNA recombination frequency. The effect was marked only when measured with a recombination substrate reporter construct with SV40 promoter as 5.2-fold DNA recombination but was minimum using a promoterless construct as 1.2-fold after 2-day culture (Fig. 1F). The results indicated that the effect of GANP on DNA recombination is dependent on the transcription. However, MEFganp+/d cells express similar levels of transcriptions of various genes in comparison with MEFganp+/+ (data not shown), suggesting that the direct effect of GANP on transcriptional regulation is not a major cause of regulating DNA recombination.
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We studied the effect of GANP on DNA recombination in NIH3T3 cells carrying the reporter construct by counting LacZ+ cells (Fig. 2A). Introduction of ganp cDNA suppressed spontaneous DNA recombination during cell culture almost completely (> 95%) after 2 days at the lowest concentration measured. This effect was also observed at day 3 (data not shown). The region involved in inhibition of DNA recombination was examined using various truncated ganp cDNAs (Fig. 2B). Full-length ganp, carrying an RNA-primase domain, the first nuclear localization signal (NLS), Sac3-homology region, second NLS and MCM3AP region with acetylation domain suppressed DNA recombination almost completely. A similar effect was observed in the various 5'-deleted mutant cDNAs. The truncation of the MCM3AP region also had suppressive effects if the construct carried the first NLS or the second NLS added artificially. This indicated that the second NLS is also active. The other constructs lacking the Sac3-homology region or the NLSs did not suppress DNA recombination effectively, suggesting that the expression of the Sac3-homology region in the nucleus is important. The construct lacking RNA-primase, first NLS and the Sac3-highly conserved region caused an increase in DNA recombination (> 2.3-fold). The construct expressing only the MCM3AP region (g-mcm3ap) caused high frequency DNA recombination (threefold), which was inhibited by co-introduction of full-length ganp cDNA (Fig. 2B). The construct, containing a Sac3-homology region, the nuclear localization sequence and an RNA-primase domain exerted full inhibitory activity on DNA recombination, which was dependent on the Sac3-homology and -highly conserved region (660–720 aa). This tendency was also observed in the cells stably transfected with the reporter construct (Fig. 2F). To examine whether GANP suppresses the transcription of the substrate DNA in the transfectants, we compared the GFP signal that was transcribed under SV40 promoter of the reporter construct and expressed as the GFP protein in the transfectants. The GFP signal did not show marked change in mock-, ganp- or mcm3ap-cotransfected cells, indicating that the effect of GANP on DNA recombination is not simply due to the regulation of transcription activity in this assay system (Fig. 2C).
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To further confirm the effect of GANP on DNA recombination after DNA damage, the recombination substrate reporter construct was prepared by inserting an I-sceI recognition sequence in the upstream lacZ' gene, and then transfected into NIH3T3 cells with cDNA of yeast I-sceI that generates resected ends. The effect of ganp was examined in comparison with mock and g-mcm3ap transfectants (Fig. 2E). Western blot analysis confirmed the presence of similar levels of I-sceI in all three transfected cells. The DNA recombination generated by I-sceI was high, and was not affected by the expression of flag-g-mcm3ap. However, introduction of ganp effectively suppressed DNA recombination caused by I-sceI in comparison with mock and g-mcm3ap transfections. The frequency of I-sceI-induced DNA recombination was increased (approximately threefold) in comparison to that of spontaneous DNA recombination within 48 h, suggesting that the introduced I-sceI efficiently digested the I-sceI recognition site in the substrate reporter construct. We next asked whether the effect of GANP is only active upon the transcription-coupled DNA DSBs as measured by the extrachromosomal assay or it is also effective on the integrated reporter construct as in Fig. 2F. NIH3T3 cells were stably transfected with the same recombination substrate lacZ gene reporter construct with the I-sceI cleavable site, and the individual clones were transfected with I-sceI cDNA and measured the frequency of DNA recombination. DNA recombination occurred rarely in NIH3T3 cells by measuring the LacZ staining in the absence of I-sceI cDNA transfection estimated as 0.04% of all the cells. Introduction of I-sceI cDNA into the NIH3T3 stable line showed the frequency of LacZ+ cells estimated as 0.84% based on the transfection efficiency, indicating that the DNA recombination observed in this experiment is dependent on the cleavage by I-sceI. We compared the effect of ganp introduction by comparing to the control only with I-sceI cDNA (100%). GANP also suppresses the DNA recombination significantly (average 37.5% of the control) occurring in the integrated DNA damages. The truncated flag-g-mcm3ap caused the marked DNA recombination of the integrated DNA similarly to the results measured with extrachromosomal DNA in Fig. 2E presumably as the dominant negative form. These results confirmed that GANP suppressed DNA recombination at the DNA damages occurring in both extrachromosomal and integrated DNAs.
Introduction of ganp does not affect RAG-mediated Ig recombination
We next examined the effect of GANP on Ig gene recombination using reporter constructs of two differently oriented recombination signal sequences (RSSs) (pJH200 and pML110). This approach allows detection of two types of Ig gene recombination of signal joint and coding joint by introduction into NIH3T3 with expression vectors for RAG1 and RAG2. The colonies grown on chloramphenicol plates were counted (Table 1). Introduction of full-length ganp cDNA did not result in significant changes of RAG-mediated RSS recombination in comparison with mock or g-mcm3ap cDNA, either in signal or coding joints, which indicated that GANP does not play a role in Ig gene DNA recombination.
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Discussion |
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The suppressive effect to homology-mediated DNA recombination was marked in the assay with DpnI-treated reporter DNA, suggesting that GANP regulates DNA repair of transcription-coupled DNA injuries. We compared the effect of GANP expression upon the transcription activity by the expression of GFP signal under the same SV40 promoter construct, indicating that the suppressive activity to DNA recombination is undertaken as post-transcriptional process (Fig. 2C). It remains to be determined whether GANP is involved in the RNA exportation mechanism as a mammalian homologue of Sac3. There are at least three genes that potentially encode Sac3-homology members in mammalian cells in the genomic database (Khuda et al. 2004; K. Kuwahara & N. Sakaguchi, unpublished data). GANP is the largest protein (210 kDa) with two additional functional domains as RNA-primase and MCM3-binding/acetylating region as an unique homologue involved in regulation of DNA recombination activity in mammalian cells with much complicated genomic configuration.
This is the first report regarding the direct molecular function of the mammalian Sac3-homologue GANP on DNA repair mechanisms. In addition to the Sac3-homology region, GANP carries an RNA-primase domain on the amino terminal side (Kuwahara et al. 2001) and an MCM3AP region at the carboxyl terminal (Takei et al. 2001), suggesting a unique role for GANP in mammalian cells. Full-length GANP suppresses homology-mediated DNA recombination initiated during culture or after DNA damage caused by AID and the restriction enzyme I-sceI, indicating that GANP acts during the process of DNA repair rather than before or at the same time as DNA damage. GANP does not suppress Ig gene DNA recombination repaired by NHEJR after the cleavage of specific RSS with RAG1 and RAG2. The effects of GANP might be selective for DNA recombination, which is required for DNA repair during cell proliferation. However, the extreme case of DNA recombination could represent a potential risk that causes chromosomal translocation, DNA truncation and genome instability in rapidly proliferating cells. GANP suppresses DNA recombination in a gene dosage-dependent manner, which is presumably necessary to maintain genome integrity and is essential for mammalian early embryonic development.
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Experimental procedures |
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All mice were maintained under specific-pathogen-free conditions in The Center for Animal Resources and Development (CARD) of Kumamoto University. A conventional ganp gene knockout mouse line was established by the standard HR method using TT2 ES cells (Yagi et al. 1993) and a targeting vector carrying the Neo and diphtheria toxin genes, as shown in Fig. 1A. Heterozygous deletion was examined by PCR of genomic DNA with the primers (not shown) and by Southern blot analysis with Probe A. The heterozygous ganp-gene-targeted mice were maintained normally and used for preparation of MEF cells. Establishment of homozygous ganp gene knockout mice was attempted by crossing heterozygous ganp-deficient mice but was unsuccessful due to embryonic death by day 12 of gestation.
Establishment of MEF cell line
MEF cells were prepared by the standard method from the day-12 embryos of heterozygous ganp+/d or wt mice and cultured for 7 days in Dulbecco's modified minimum essential medium supplemented with heat-inactivated fetal calf serum (10%), L-glutamine (2 mM) and ß-mercaptoethanol (50 µM) and then transformed with SV40 as described (Fresa et al. 1987).
Expression vectors
The expression vectors for the ganp gene were constructed with various forms of ganp cDNAs tagged with Xpress or FLAG in the pCXN2 vector. The AID expression vectors pEF-Xpress-mAID and Xpress-mAID carboxyl terminal deletion mutant (188–198 aa) were modified from pCMV5amAID-HA-FLAG (Ta et al. 2003). The I-sceI endonuclease expression vector (pcBAsce; Rouet et al. 1994) was modified by inserting an HA sequence downstream of the NLS (pCAGGS-HA-I-sceI).
DNA recombination substrate vector of the lacZ-based direct-repeat sequence
As the substrate of DNA recombination with the direct-repeat, pSVlacZ 1351 + 757neo was generated by modification of the construct reported previously (Herzing & Meyn 1993). Two HpaI sites of lacZ gene were mutated individually. Addition of two nucleotides converted the HpaI site to the XhoI site (CTCGAGCC) of the lacZ gene, which induced a frame shift and inactivated LacZ activity. The vector of recombination substrate was constructed by tandem alignment of two differently mutated lacZ' and lacZ'' genes as shown in Fig. 1B. The 5'-side lacZ' has the mutation at the first HpaI site (255 nt) and the 3'-side lacZ'' at the second HpaI site (289 nt). The other vector was also modified by inserting the I-sceI recognition sequence at the XhoI site created after mutation of the second HpaI (*) site, as indicated in Fig. 2D. A Lac promoter sequence derived from pJH200 plasmid was inserted downstream of the SV40 promoter and upstream of the lacZ' gene. The promoterless recombination substrate vector was prepared similarly by using inverse PCR method (Kuwahara et al. 2001).
DNA recombination assay by the extrachromosomal lacZ with direct-repeat sequence vector
Cells were grown to 60% confluence in 6-cm dishes. DNA transfection was conducted with the plasmids (1 µg of each) of the recombination substrate vector with or without the I-sceI or ganp cDNA expression vectors in the presence of 100 µL of serum-free Dulbecco's modified eagle medium and 20 µL of Polyfect (Qiagen, Valencia, CA). In some experiments, cells were transfected by Polyfect with the recombination substrate and aid cDNA expression vectors with or without ganp cDNA. Expression of LacZ was examined by fixation in a solution of 0.1 M sodium phosphate (pH 7.0), 1 mM MgCl2 and 0.25% glutaraldehyde, washed twice in phosphate-buffered saline (PBS) and stained with 2 µg/mL X-gal, 1 mM MgCl2, 150 mM NaCl, 3.3 mM K4Fe(CN)63H2O, 3.3 mM K3Fe(CN)6, 60 mM Na2HPO4 and 40 mM NaH2PO4. Cells were incubated for 12 h at 37 °C and the LacZ-positive cells were counted under the microscope. The restriction enzyme I-sceI was introduced in NIH3T3 by pCAGGS-HA-I-sceI and the expression of I-sceI protein was measured by immunoblotting using anti-HA Ab (Santa Cruz, Santa Cruz, CA). MEF (3.0 x 106) cells were transfected with 20-µg plasmid by electroporation using a gene pulser (Bio-Rad, Hercules, CA) (950 µF, 200V), harvested after 2–4 days, and plasmid DNA prepared by the alkali lysis method for transformation, followed by counting LacZ E. coli colonies. To distinguish the recombination event between deletion and non-deletion types, the plasmid DNA was purified from the individual LacZ colony and digested with HpaI to examine the 600-bp band as the evidence of DNA recombination, and then digested by SalI. The plasmid DNA with gene conversion-type recombination should show the 6-kb band, whereas the one with deletion-type recombination should show the 4-kb band. For the measurement of the GANP effect on the integrated reporter gene, NIH3T3 was stably transfected with the same reporter, and the individual clones were used for DNA recombination assay by the transient DNA transfection with pCAGGS-HA-I-sceI.
FACS analysis
NIH3T3 cells were grown to 60% confluence in 6-cm dishes, and transfected by pSVEGFP pA in the presence of mock, ganp, or mcm3ap expression vector by Polyfect. After 24 or 48 h, cells were harvested and washed twice with PBS containing 3% FCS and 0.05% NaN3. The GFP signal was analyzed by FACSCalibur (BD, Franklin Lakes, NJ) after gating of living cells.
Extrachromosomal site-specific Ig gene-type recombination
The pJH200, a signal joint substrate, and pML110, a signal joint and coding joint substrate, were used for site-specific NHEJR assays (Lieber et al. 1988). We transfected the recombination substrates with expression vectors for GST-RAG1 and GST-RAG2 into NIH3T3 cells (Gallo et al. 1994). After 3 days, cells were washed twice with PBS, and the plasmid DNA extracted by the alkali lysis method was then used for counting the recombinant colonies after transformation into DH5
on LB plates with either chloramphenicol or ampicillin selection.
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Acknowledgements |
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Footnotes |
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* Correspondence: E-mail: nobusaka{at}kumamoto-u.ac.jp
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References |
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Bauer, A. & Kölling, R. (1996) The SAC3 gene encodes a nuclear protein required for normal progression of mitosis. J. Cell Sci. 109, 1575–1583.[Abstract]
Casellas, R., Nussenzweig, A., Wuerffel, R., Pelanda, R., Reichlin, A., Suh, H., Qin, X.F., Besmer, E., Kenter, A., Rajewsky, K. & Nussenzweig, M.C. (1998) Ku80 is required for immunoglobulin isotype switching. EMBO J. 17, 2404–2411.[CrossRef][Medline]
Fischer, T., Sträßer, K., Rácz, A., Rodriguez-Navarro, S., Oppizzi, M., Ihrig, P., Lechner, J. & Hurt, E. (2002) The mRNA export machinery requires the novel Sac3p–Thp1p complex to dock at the nucleoplasmic entrance of the nuclear pores. EMBO J. 21, 5843–5852.[CrossRef][Medline]
Fresa, K.L., Karjalainen, H.E. & Tevethia, S.S. (1987) Sensitivity of simian virus 40-transformed C57BL/6 mouse embryo fibroblasts to lysis by murine natural killer cells. J. Immunol. 138, 1215–1220.
Gallardo, M., Luna, R., Erdjument-Bromage, H., Tempst, P. & Aguilera, A. (2003) Nab2p and the Thp1p–Sac3p complex functionally interact at the interface between transcription and mRNA metabolism. J. Biol. Chem. 278, 24225–24232.
Gallo, M.L., Pergola, F., Daniels, G.A. & Lieber, M.R. (1994) Distinct roles for RAG-1 in the initiation of V(D)J recombination and in the resolution of coding ends. J. Biol. Chem. 269, 22188–22192.
Herzing, L.B.K. & Meyn, M.S. (1993) Novel lacZ-based recombination vectors for mammalian cells. Gene 137, 163–169.[CrossRef][Medline]
Honjo, T., Kinoshita, K. & Muramatsu, M. (2002) Molecular mechanism of class switch recombination: linkage with somatic hypermutation. Annu. Rev. Immunol. 20, 165–196.[CrossRef][Medline]
Kawatani, Y., Igarashi, H., Matsui, T., Kuwahara, K., Fujimura, S., Okamoto, N., Takagi, K. & Sakaguchi, N. (2005) Double-stranded DNA breaks in the immunoglobulin V-region gene were detected at lower frequency in affinity-maturation impeded GANP–/– mice. J. Immunol. 175, 5615–5618.
Khuda, S.E., Yoshida, M., Xing, Y., Shimasaki, T., Takeya, M., Kuwahara, K. & Sakaguchi, N. (2004) The Sac3 homologue shd1 is involved in mitotic progression in mammalian cells. J. Biol. Chem. 279, 46182–46190.
Kuwahara, K., Fujimura, S., Takahashi, Y., Nakagata, N., Takemori, T., Aizawa, S. & Sakaguchi, N. (2004) Germinal center-associated nuclear protein contributes to affinity maturation of B cell antigen receptor in T cell-dependent responses. Proc. Natl. Acad. Sci. USA 101, 1010–1015.
Kuwahara, K., Tomiyasu, S., Fujimura, S., Nomura, K., Xing, Y., Nishiyama, N., Ogawa, M., Imajoh-Ohmi, S., Izuta, S. & Sakaguchi, N. (2001) Germinal center-associated nuclear protein (GANP) has a phosphorylation-dependent DNA-primase activity that is up-regulated in germinal center regions. Proc. Natl. Acad. Sci. USA 98, 10279–10283.
Kuwahara, K., Yoshida, M., Kondo, E., Sakata, A., Watanabe, Y., Abe, E., Kouno, Y., Tomiyasu, S., Fujimura, S., Tokuhisa, T., Kimura, H., Ezaki, T. & Sakaguchi, N. (2000) A novel nuclear phosphoprotein, GANP, is up-regulated in centrocytes of the germinal center and associated with MCM3, a protein essential for DNA replication. Blood 95, 2131–2138.
Lieber, M.R., Hesse, J.E., Lewis, S., Bosma, G.C., Rosenberg, N., Mizuuchi, K., Bosma, M.J. & Gellert, M. (1988) The defect in murine severe combined immune deficiency: joining of signal sequences but not coding segments in V(D)J recombination. Cell 55, 7–16.[CrossRef][Medline]
Manis, J.P., Gu, Y., Lansford, R., Sonoda, E., Ferrini, R., Davidson, L., Rajewsky, K. & Alt, F.W. (1998) Ku70 is required for late B cell development and immunoglobulin heavy chain class switching. J. Exp. Med. 187, 2081–2089.
Meek, K., Gupta, S., Ramsden, D.A. & Lees-Miller, S.P. (2004) The DNA-dependent protein kinase: the director at the end. Immunol. Rev. 200, 132–141.[CrossRef][Medline]
Rooney, S., Chaudhuri, J. & Alt, F.W. (2004) The role of the non-homologous end-joining pathway in lymphocyte development. Immunol. Rev. 200, 115–131.[CrossRef][Medline]
Rouet, P., Smih, F. & Jasin, M. (1994) Expression of a site-specific endonuclease stimulates homologous recombination in mammalian cells. Proc. Natl. Acad. Sci. USA 91, 6064–6068.
Sakaguchi, N., Kimura, T., Matsushita, S., Fujimura, S., Shibata, J., Araki, M., Sakamoto, T., Minoda, C., Kuwahara, K. (2005) Generation of high-affinity antibody against T cell-dependent antigen in ganp gene-transgenic mouse. J. Immunol. 174, 4485–4494.
Ta, V.T., Nagaoka, H., Catalan, N., Durandy, A., Fischer, A., Imai, K., Nonoyama, S., Tashiro, J., Ikegawa, M., Ito, S., Kinoshita, K., Muramatsu, M. & Honjo, T. (2003) AID mutant analyses indicate requirement for class-switch-specific cofactors. Nat. Immunol. 4, 843–848.[CrossRef][Medline]
Takei, Y., Swietlik, M., Tanoue, A., Tsujimoto, G., Kouzarides, T. & Laskey, R. (2001) MCM3AP, a novel acetyltransferase that acetylates replication protein MCM3. EMBO Rep. 2, 119–123.[CrossRef][Medline]
West, S.C. (2003) Molecular views of recombination proteins and their control. Nat. Rev. Mol. Cell Biol. 4, 435–445.[CrossRef][Medline]
Yagi, T., Tokunaga, T., Furuta, Y., Nada, S., Yoshida, M., Tsukada, T., Saga, Y., Takeda, N., Ikawa, Y. & Aizawa, S. (1993) A novel ES cell line, TT2, with high germline-differentiating potency. Anal. Biochem. 214, 70–76.[CrossRef][Medline]
Received: 7 March 2007
Accepted: 18 July 2007
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | ADVANCED SEARCH | TABLE OF CONTENTS |
EMBL3 genomic DNA library of C57BL/6 mice were used for the targeting vector with insertions of the Neo gene cassette and the diphtheria toxin fragment as described previously to disrupt the exon I (Ex I) and part of exon II (Ex II) of ganp gene. ES cells transfected with the vector were selected with G418 and screened by genomic PCR analysis. (B) Southern blot analysis with the Probe A displayed the 5.0-kb targeted allele in addition to the 6.4-kb wt allele in the tail DNA of heterozygous ganp+/d mice after digestion with BamHI. (C) The recombination substrate reporter construct with lacZ direct-repeat DNA sequences. The two lacZ mutants (lacZ' and lacZ'') generate productive lacZ after DNA recombination. Asterisks indicate the mutation of HpaI sites, which generate XhoI sites and result in non-productive lacZ mutants as described in Experimental procedures. (D) MEF cells were obtained from day-12 embryos of heterozygous ganp+/d or wt mice and then transformed by SV40 as indicated. The SV40-transformed MEF cell line was transfected with the lacZ direct-repeat DNA recombination substrate. The extrachromosomal plasmid DNA was purified after the periods indicated and transformed into DH5