Nucleic Acids Research, 1995, Vol. 23, No. 19 3816-3821
© 1995
MOLECULAR BIOLOGY |
Efficient gene activation in mammalian cells by using recombinant adenovirus expressing site-specific Cre recombinase
1Laboaory of Molecular Genetics University of Tokyo 4-6-1 Shirokanedi, Minato-ku, Tokyo 108, Japan 2Deparment of Virology, institute of Medical Science, University of Tokyo 4-6-1 Shirokanedai, Minato-ku, Tokyo 108, Japan 3Discovery Research Laboratories III, Sumitomo Pharmaceuticals Research Center 3-1-98 Kasugade-naka, Konohana-ku, Osaka 554, Japan
*To whom correspondence should be addressed
Received August 8, 1995. Accepted August 23, 1995.
A recombinant adenovirus (Ad) expressing Cre recombinase derived from bacteriophage P1 was constructed. To assay the Cre activity in mammalian colls, another recombinant Ad bearing an on/off-switching reporter unit, where a LacZ-expression unit can be activated by the Cre-mediated exclsionai deletion of an interposed stuffer DNA, was also constructed. Co-infection experiments together with the Cre-expressing and the reporter recombinant Ads showed that the Cre-mediated switching of gene expression was detected in nearly 100% of cultured CV1, HeLa and Jurkat cells. These results suggest that the recombinant Ad efficiently expressed functional Cre and offers a basis for estabilshing a powerful on/off switching strategy of gene expression incultured mammalian cells and presumably in transgenic animals. The method is also applicable to construction of recombinant Ad bearing a gene the expression of which is deleterious to propagation of recombinant Ad.
![]()
CiteULike
Connotea
Del.icio.us What's this?
This article has been cited by other articles:
![]() |
H. E. McMahon, O. Hashimoto, P. L. Mellon, and S. Shimasaki Oocyte-Specific Overexpression of Mouse Bone Morphogenetic Protein-15 Leads to Accelerated Folliculogenesis and an Early Onset of Acyclicity in Transgenic Mice Endocrinology, June 1, 2008; 149(6): 2807 - 2815. [Abstract] [Full Text] [PDF] |
||||
![]() |
T. Sato, Y. Kawamura, R. Asai, T. Amano, Y. Uchijima, D. A. Dettlaff-Swiercz, S. Offermanns, Y. Kurihara, and H. Kurihara Recombinase-mediated cassette exchange reveals the selective use of Gq/G11-dependent and -independent endothelin 1/endothelin type A receptor signaling in pharyngeal arch development Development, February 15, 2008; 135(4): 755 - 765. [Abstract] [Full Text] [PDF] |
||||
![]() |
Y. Yamagishi, T. Sadaoka, H. Yoshii, P. Somboonthum, T. Imazawa, K. Nagaike, K. Ozono, K. Yamanishi, and Y. Mori Varicella-Zoster Virus Glycoprotein M Homolog Is Glycosylated, Is Expressed on the Viral Envelope, and Functions in Virus Cell-to-Cell Spread J. Virol., January 15, 2008; 82(2): 795 - 804. [Abstract] [Full Text] [PDF] |
||||
![]() |
T. Sadaoka, H. Yoshii, T. Imazawa, K. Yamanishi, and Y. Mori Deletion in Open Reading Frame 49 of Varicella-Zoster Virus Reduces Virus Growth in Human Malignant Melanoma Cells but Not in Human Embryonic Fibroblasts J. Virol., November 15, 2007; 81(22): 12654 - 12665. [Abstract] [Full Text] [PDF] |
||||
![]() |
H. Sato, M. Masuda, M. Kanai, K. Tsukiyama-Kohara, M. Yoneda, and C. Kai Measles Virus N Protein Inhibits Host Translation by Binding to eIF3-p40 J. Virol., November 1, 2007; 81(21): 11569 - 11576. [Abstract] [Full Text] [PDF] |
||||
![]() |
A. Rikimaru, K. Komori, T. Sakamoto, H. Ichise, N. Yoshida, I. Yana, and M. Seiki Establishment of an MT4-MMP-deficient mouse strain representing an efficient tracking system for MT4-MMP/MMP-17 expression in vivo using {beta}-galactosidase Genes Cells, September 1, 2007; 12(9): 1091 - 1100. [Abstract] [Full Text] [PDF] |
||||
![]() |
H. Konishi, K. Namikawa, K. Shikata, Y. Kobatake, T. Tachibana, and H. Kiyama Identification of Peripherin as a Akt Substrate in Neurons J. Biol. Chem., August 10, 2007; 282(32): 23491 - 23499. [Abstract] [Full Text] [PDF] |
||||
![]() |
K. Kohara, H. Yasuda, Y. Huang, N. Adachi, K. Sohya, and T. Tsumoto A Local Reduction in Cortical GABAergic Synapses after a Loss of Endogenous Brain-Derived Neurotrophic Factor, as Revealed by Single-Cell Gene Knock-Out Method J. Neurosci., July 4, 2007; 27(27): 7234 - 7244. [Abstract] [Full Text] [PDF] |
||||
![]() |
P. Yang, J. J. Peairs, R. Tano, N. Zhang, J. Tyrell, and G. J. Jaffe Caspase-8-Mediated Apoptosis in Human RPE Cells Invest. Ophthalmol. Vis. Sci., July 1, 2007; 48(7): 3341 - 3349. [Abstract] [Full Text] [PDF] |
||||
![]() |
N. Dali-Youcef, C. Mataki, A. Coste, N. Messaddeq, S. Giroud, S. Blanc, C. Koehl, M.-F. Champy, P. Chambon, L. Fajas, et al. Adipose tissue-specific inactivation of the retinoblastoma protein protects against diabesity because of increased energy expenditure PNAS, June 19, 2007; 104(25): 10703 - 10708. [Abstract] [Full Text] [PDF] |
||||
![]() |
S. Unezaki, R. Horai, K. Sudo, Y. Iwakura, and S. Ito Ovol2/Movo, a homologue of Drosophila ovo, is required for angiogenesis, heart formation and placental development in mice Genes Cells, June 1, 2007; 12(6): 773 - 785. [Abstract] [Full Text] [PDF] |
||||
![]() |
M. Wakayama, M. Abei, R. Kawashima, E. Seo, K. Fukuda, H. Ugai, T. Murata, N. Tanaka, I. Hyodo, H. Hamada, et al. E1A, E1B Double-Restricted Adenovirus with RGD-Fiber Modification Exhibits Enhanced Oncolysis for CAR-Deficient Biliary Cancers Clin. Cancer Res., May 15, 2007; 13(10): 3043 - 3050. [Abstract] [Full Text] [PDF] |
||||
![]() |
M. Takehashi, M. Kanatsu-Shinohara, K. Inoue, N. Ogonuki, H. Miki, S. Toyokuni, A. Ogura, and T. Shinohara Adenovirus-mediated gene delivery into mouse spermatogonial stem cells PNAS, February 20, 2007; 104(8): 2596 - 2601. [Abstract] [Full Text] [PDF] |
||||
![]() |
T. Matsuda and C. L. Cepko Controlled expression of transgenes introduced by in vivo electroporation PNAS, January 16, 2007; 104(3): 1027 - 1032. [Abstract] [Full Text] [PDF] |
||||
![]() |
Y. Kitao, Y. Imai, K. Ozawa, A. Kataoka, T. Ikeda, M. Soda, K. Nakimawa, H. Kiyama, D. M. Stern, O. Hori, et al. Pael receptor induces death of dopaminergic neurons in the substantia nigra via endoplasmic reticulum stress and dopamine toxicity, which is enhanced under condition of parkin inactivation Hum. Mol. Genet., January 1, 2007; 16(1): 50 - 60. [Abstract] [Full Text] [PDF] |
||||
![]() |
S. Haupt, F. Edenhofer, M. Peitz, A. Leinhaas, and O. Brustle Stage-Specific Conditional Mutagenesis in Mouse Embryonic Stem Cell-Derived Neural Cells and Postmitotic Neurons by Direct Delivery of Biologically Active Cre Recombinase Stem Cells, January 1, 2007; 25(1): 181 - 188. [Abstract] [Full Text] [PDF] |
||||
![]() |
H. Fukumitsu, M. Ohtsuka, R. Murai, H. Nakamura, K. Itoh, and S. Furukawa Brain-Derived Neurotrophic Factor Participates in Determination of Neuronal Laminar Fate in the Developing Mouse Cerebral Cortex J. Neurosci., December 20, 2006; 26(51): 13218 - 13230. [Abstract] [Full Text] [PDF] |
||||
![]() |
S. Ueda, K. Fukamachi, Y. Matsuoka, N. Takasuka, F. Takeshita, A. Naito, M. Iigo, D. B. Alexander, M. A. Moore, I. Saito, et al. Ductal origin of pancreatic adenocarcinomas induced by conditional activation of a human Ha-ras oncogene in rat pancreas Carcinogenesis, December 1, 2006; 27(12): 2497 - 2510. [Abstract] [Full Text] [PDF] |
||||
![]() |
T. Tanaka, J. Huang, S. Hirai, M. Kuroki, M. Kuroki, N. Watanabe, K. Tomihara, K. Kato, and H. Hamada Carcinoembryonic Antigen-Targeted Selective Gene Therapy for Gastric Cancer through FZ33 Fiber-Modified Adenovirus Vectors. Clin. Cancer Res., June 15, 2006; 12(12): 3803 - 3813. [Abstract] [Full Text] [PDF] |
||||
![]() |
Y.-i. Fujimura, K.-i. Isono, M. Vidal, M. Endoh, H. Kajita, Y. Mizutani-Koseki, Y. Takihara, M. van Lohuizen, A. Otte, T. Jenuwein, et al. Distinct roles of Polycomb group gene products in transcriptionally repressed and active domains of Hoxb8 Development, June 15, 2006; 133(12): 2371 - 2381. [Abstract] [Full Text] [PDF] |
||||
![]() |
T. Yamashita, M. Ninomiya, P. Hernandez Acosta, J. M. Garcia-Verdugo, T. Sunabori, M. Sakaguchi, K. Adachi, T. Kojima, Y. Hirota, T. Kawase, et al. Subventricular zone-derived neuroblasts migrate and differentiate into mature neurons in the post-stroke adult striatum. J. Neurosci., June 14, 2006; 26(24): 6627 - 6636. [Abstract] [Full Text] [PDF] |
||||
![]() |
A. Izawa, K. Sano, M. Takehara, M. Inobe, J.-i. Suzuki, H. Imamura, M. Takahashi, U. Ikeda, M. Isobe, and T. Uede Cre/loxP-mediated CTLA4IgG gene transfer induces clinically relevant immunosuppression via on-off gene recombination in vivo Cardiovasc Res, January 1, 2006; 69(1): 289 - 297. [Abstract] [Full Text] [PDF] |
||||
![]() |
J. Sun, B. Tumurbaatar, J. Jia, H. Diao, F. Bodola, S. M. Lemon, W. Tang, D. G. Bowen, G. W. McCaughan, P. Bertolino, et al. Parenchymal Expression of CD86/B7.2 Contributes to Hepatitis C Virus-Related Liver Injury J. Virol., August 15, 2005; 79(16): 10730 - 10739. [Abstract] [Full Text] [PDF] |
||||
![]() |
J. Fujishiro, S.-i. Takeda, Y. Takeno, K. Takeuchi, Y. Ogata, M. Takahashi, Y. Hakamata, T. Kaneko, T. Murakami, T. Okada, et al. Gene transfer to the rat kidney in vivo and ex vivo using an adenovirus vector: factors influencing transgene expression Nephrol. Dial. Transplant., July 1, 2005; 20(7): 1385 - 1391. [Abstract] [Full Text] [PDF] |
||||
![]() |
T. Miyamoto, T. Kaneko, M. Yamashita, Y. Tenda, M. Inami, A. Suzuki, S. Ishii, M. Kimura, K. Hashimoto, H. Shimada, et al. Prolonged skin allograft survival by IL-10 gene-introduced CD4 T cell administration Int. Immunol., June 1, 2005; 17(6): 759 - 768. [Abstract] [Full Text] [PDF] |
||||
![]() |
N. Yamashita, J.-M. Kim, O. Koiwai, K.-i. Arai, and H. Masai Functional analyses of mouse ASK, an activation subunit for Cdc7 kinase, using conditional ASK knockout ES cells Genes Cells, June 1, 2005; 10(6): 551 - 563. [Abstract] [Full Text] [PDF] |
||||
![]() |
Y. Arima, M. Nitta, S. Kuninaka, D. Zhang, T. Fujiwara, Y. Taya, M. Nakao, and H. Saya Transcriptional Blockade Induces p53-dependent Apoptosis Associated with Translocation of p53 to Mitochondria J. Biol. Chem., May 13, 2005; 280(19): 19166 - 19176. [Abstract] [Full Text] [PDF] |
||||
![]() |
M. Nakano, K. Odaka, Y. Takahashi, M. Ishimura, I. Saito, and Y. Kanegae Production of viral vectors using recombinase-mediated cassette exchange Nucleic Acids Res., May 5, 2005; 33(8): e76 - e76. [Abstract] [Full Text] [PDF] |
||||
![]() |
B. Qiao, S. R. Padilla, and P. D. Benya Transforming Growth Factor (TGF)-{beta}-activated Kinase 1 Mimics and Mediates TGF-{beta}-induced Stimulation of Type II Collagen Synthesis in Chondrocytes Independent of Col2a1 Transcription and Smad3 Signaling J. Biol. Chem., April 29, 2005; 280(17): 17562 - 17571. [Abstract] [Full Text] [PDF] |
||||
![]() |
K.-i. Mizutani and T. Saito Progenitors resume generating neurons after temporary inhibition of neurogenesis by Notch activation in the mammalian cerebral cortex Development, March 15, 2005; 132(6): 1295 - 1304. [Abstract] [Full Text] [PDF] |
||||
![]() |
Z. Gao, T. Sasaoka, T. Fujimori, T. Oya, Y. Ishii, H. Sabit, M. Kawaguchi, Y. Kurotaki, M. Naito, T. Wada, et al. Deletion of the PDGFR-{beta} Gene Affects Key Fibroblast Functions Important for Wound Healing J. Biol. Chem., March 11, 2005; 280(10): 9375 - 9389. [Abstract] [Full Text] [PDF] |
||||
![]() |
Y. Ishigaki, H. Katagiri, T. Yamada, T. Ogihara, J. Imai, K. Uno, Y. Hasegawa, J. Gao, H. Ishihara, T. Shimosegawa, et al. Dissipating Excess Energy Stored in the Liver Is a Potential Treatment Strategy for Diabetes Associated With Obesity Diabetes, February 1, 2005; 54(2): 322 - 332. [Abstract] [Full Text] [PDF] |
||||
![]() |
E. Seo, M. Abei, M. Wakayama, K. Fukuda, H. Ugai, T. Murata, T. Todoroki, Y. Matsuzaki, N. Tanaka, H. Hamada, et al. Effective Gene Therapy of Biliary Tract Cancers by a Conditionally Replicative Adenovirus Expressing Uracil Phosphoribosyltransferase: Significance of Timing of 5-Fluorouracil Administration Cancer Res., January 15, 2005; 65(2): 546 - 552. [Abstract] [Full Text] [PDF] |
||||
![]() |
J. Schindehutte, H. Fukumitsu, P. Collombat, G. Griesel, C. Brink, P. C. Baier, M. R. Capecchi, and A. Mansouri In Vivo and In Vitro Tissue-Specific Expression of Green Fluorescent Protein Using the Cre-Lox System in Mouse Embryonic Stem Cells Stem Cells, January 1, 2005; 23(1): 10 - 15. [Abstract] [Full Text] [PDF] |
||||
![]() |
K. Matsuura, H. Wada, T. Nagai, Y. Iijima, T. Minamino, M. Sano, H. Akazawa, J. D. Molkentin, H. Kasanuki, and I. Komuro Cardiomyocytes fuse with surrounding noncardiomyocytes and reenter the cell cycle J. Cell Biol., October 25, 2004; 167(2): 351 - 363. [Abstract] [Full Text] [PDF] |
||||
![]() |
T. Numakawa, T. Ishimoto, S. Suzuki, Y. Numakawa, N. Adachi, T. Matsumoto, D. Yokomaku, H. Koshimizu, K. E. Fujimori, R. Hashimoto, et al. Neuronal Roles of the Integrin-associated Protein (IAP/CD47) in Developing Cortical Neurons J. Biol. Chem., October 8, 2004; 279(41): 43245 - 43253. [Abstract] [Full Text] [PDF] |
||||
![]() |
B. D. Anderson, T. Nakamura, S. J. Russell, and K.-W. Peng High CD46 Receptor Density Determines Preferential Killing of Tumor Cells by Oncolytic Measles Virus Cancer Res., July 15, 2004; 64(14): 4919 - 4926. [Abstract] [Full Text] [PDF] |
||||
![]() |
A. Kageyama, M. Oka, T. Okada, S.-i. Nakamura, T. Ueyama, N. Saito, V. J. Hearing, M. Ichihashi, and C. Nishigori Down-regulation of Melanogenesis by Phospholipase D2 through Ubiquitin Proteasome-mediated Degradation of Tyrosinase J. Biol. Chem., June 25, 2004; 279(26): 27774 - 27780. [Abstract] [Full Text] [PDF] |
||||
![]() |
X. Lin, A. H. Fischer, K.-y. Ryu, J.-y. Cho, T. J. Sferra, R. T. Kloos, E. L. Mazzaferri, and S. M. Jhiang Application of the Cre/loxP System to Enhance Thyroid-Targeted Expression of Sodium/Iodide Symporter J. Clin. Endocrinol. Metab., May 1, 2004; 89(5): 2344 - 2350. [Abstract] [Full Text] [PDF] |
||||
![]() |
K. Tsukiyama-Kohara, S. Tone, I. Maruyama, K. Inoue, A. Katsume, H. Nuriya, H. Ohmori, J. Ohkawa, K. Taira, Y. Hoshikawa, et al. Activation of the CKI-CDK-Rb-E2F Pathway in Full Genome Hepatitis C Virus-expressing Cells J. Biol. Chem., April 9, 2004; 279(15): 14531 - 14541. [Abstract] [Full Text] [PDF] |
||||
![]() |
J. Kawai, K. Ando, A. Tojo, T. Shimosawa, K. Takahashi, M. L. Onozato, M. Yamasaki, T. Ogita, T. Nakaoka, and T. Fujita Endogenous Adrenomedullin Protects Against Vascular Response to Injury in Mice Circulation, March 9, 2004; 109(9): 1147 - 1153. [Abstract] [Full Text] [PDF] |
||||
![]() |
H. Ma, C. M. Sumbilla, I. K. G. Farrance, M. G. Klein, and G. Inesi Cell-specific expression of SERCA, the exogenous Ca2+ transport ATPase, in cardiac myocytes Am J Physiol Cell Physiol, March 1, 2004; 286(3): C556 - C564. [Abstract] [Full Text] |
||||
![]() |
Y. Arima, T. Hirota, C. Bronner, M. Mousli, T. Fujiwara, S.-i. Niwa, H. Ishikawa, and H. Saya Down-regulation of nuclear protein ICBP90 by p53/p21Cip1/WAF1-dependent DNA-damage checkpoint signals contributes to cell cycle arrest at G1/S transition Genes Cells, February 1, 2004; 9(2): 131 - 142. [Abstract] [Full Text] [PDF] |
||||
![]() |
S.-i. Yoshimura, A. Yamamoto, Y. Misumi, M. Sohda, F. A. Barr, G. Fujii, A. Shakoori, H. Ohno, K. Mihara, and N. Nakamura Dynamics of Golgi Matrix Proteins after the Blockage of ER to Golgi Transport J. Biochem., February 1, 2004; 135(2): 201 - 216. [Abstract] [Full Text] [PDF] |
||||
![]() |
G. Nishitai, N. Shimizu, T. Negishi, H. Kishimoto, K. Nakagawa, D. Kitagawa, T. Watanabe, H. Momose, S. Ohata, S. Tanemura, et al. Stress Induces Mitochondria-mediated Apoptosis Independent of SAPK/JNK Activation in Embryonic Stem Cells J. Biol. Chem., January 16, 2004; 279(3): 1621 - 1626. [Abstract] [Full Text] [PDF] |
||||
![]() |
T. Iwano, M. Tachibana, M. Reth, and Y. Shinkai Importance of TRF1 for Functional Telomere Structure J. Biol. Chem., January 9, 2004; 279(2): 1442 - 1448. [Abstract] [Full Text] [PDF] |
||||
![]() |
M. Hashimoto and K. Mikoshiba Neuronal Birthdate-Specific Gene Transfer with Adenoviral Vectors J. Neurosci., January 7, 2004; 24(1): 286 - 296. [Abstract] [Full Text] [PDF] |
||||
![]() |
N. Jullien, F. Sampieri, A. Enjalbert, and J.-P. Herman Regulation of Cre recombinase by ligand-induced complementation of inactive fragments Nucleic Acids Res., November 1, 2003; 31(21): e131 - e131. [Abstract] [Full Text] [PDF] |
||||
![]() |
T. Numakawa, H. Nakayama, S. Suzuki, T. Kubo, F. Nara, Y. Numakawa, D. Yokomaku, T. Araki, T. Ishimoto, A. Ogura, et al. Nerve Growth Factor-induced Glutamate Release Is via p75 Receptor, Ceramide, and Ca2+ from Ryanodine Receptor in Developing Cerebellar Neurons J. Biol. Chem., October 17, 2003; 278(42): 41259 - 41269. [Abstract] [Full Text] [PDF] |
||||
![]() |
H. Yoshida, L. Beltran-Parrazal, P. Butler, M. Conti, A. C. Charles, and R. I. Weiner Lowering Cyclic Adenosine-3',5'-Monophosphate (cAMP) Levels by Expression of a cAMP-Specific Phosphodiesterase Decreases Intrinsic Pulsatile Gonadotropin-Releasing Hormone Secretion from GT1 Cells Mol. Endocrinol., October 1, 2003; 17(10): 1982 - 1990. [Abstract] [Full Text] [PDF] |
||||
![]() |
K. Shirahama-Noda, A. Yamamoto, K. Sugihara, N. Hashimoto, M. Asano, M. Nishimura, and I. Hara-Nishimura Biosynthetic Processing of Cathepsins and Lysosomal Degradation Are Abolished in Asparaginyl Endopeptidase-deficient Mice J. Biol. Chem., August 29, 2003; 278(35): 33194 - 33199. [Abstract] [Full Text] [PDF] |
||||
![]() |
S. Kondo, A. Okuda, H. Sato, N. Tachikawa, M. Terashima, Y. Kanegae, and I. Saito Simultaneous on/off regulation of transgenes located on a mammalian chromosome with Cre-expressing adenovirus and a mutant loxP Nucleic Acids Res., July 15, 2003; 31(14): e76 - e76. [Abstract] [Full Text] [PDF] |
||||
![]() |
M. Senoo, L. Wang, D. Suzuki, N. Takeda, Y. Shinkai, and S. Habu Increase of TCR V{beta} Accessibility within E{beta} Regulatory Region Influences its Recombination Frequency But Not Allelic Exclusion J. Immunol., July 15, 2003; 171(2): 829 - 835. [Abstract] [Full Text] [PDF] |
||||
![]() |
Y. Iwai, S. Terawaki, M. Ikegawa, T. Okazaki, and T. Honjo PD-1 Inhibits Antiviral Immunity at the Effector Phase in the Liver J. Exp. Med., July 7, 2003; 198(1): 39 - 50. [Abstract] [Full Text] [PDF] |
||||
![]() |
S. Nakagomi, Y. Suzuki, K. Namikawa, S. Kiryu-Seo, and H. Kiyama Expression of the Activating Transcription Factor 3 Prevents c-Jun N-Terminal Kinase-Induced Neuronal Death by Promoting Heat Shock Protein 27 Expression and Akt Activation J. Neurosci., June 15, 2003; 23(12): 5187 - 5196. [Abstract] [Full Text] [PDF] |
||||
![]() |
M. Inoue, Y. Tokusumi, H. Ban, T. Kanaya, M. Shirakura, T. Tokusumi, T. Hirata, Y. Nagai, A. Iida, and M. Hasegawa A New Sendai Virus Vector Deficient in the Matrix Gene Does Not Form Virus Particles and Shows Extensive Cell-to-Cell Spreading J. Virol., June 1, 2003; 77(11): 6419 - 6429. [Abstract] [Full Text] [PDF] |
||||
![]() |
T. Kudo, S. Kure, K. Ikeda, A.-P. Xia, Y. Katori, M. Suzuki, K. Kojima, A. Ichinohe, Y. Suzuki, Y. Aoki, et al. Transgenic expression of a dominant-negative connexin26 causes degeneration of the organ of Corti and non-syndromic deafness Hum. Mol. Genet., May 1, 2003; 12(9): 995 - 1004. [Abstract] [Full Text] [PDF] |
||||

























