Abstract
Cell marking is a very important procedure for identifying donor cells after cell and/or organ transplantation in vivo. Transgenic animals expressing marker proteins such as enhanced green fluorescent protein (EGFP) in their tissues are a powerful tool for research in fields of tissue engineering and regenerative medicine. The purpose of this study was to establish transgenic rabbit lines that ubiquitously express EGFP under the control of the cytomegalovirus immediate early enhancer/beta-actin promoter (CAG) to provide a fluorescent transgenic animal as a bioresource. We microinjected the EGFP expression vector into 945 rabbit eggs and 4 independent transgenic candidate pups were obtained. Two of them died before sexual maturation and one was infertile. One transgenic male candidate founder rabbit was obtained and could be bred by artificial insemination. The rabbit transmitted the transgene in a Mendelian manner. Using fluorescence in situ hybridization analysis, we detected the transgene at 7q11 on chromosome 7 as a large centromeric region in two F1 offspring (one female and one male). Eventually, one transgenic line was established. Ubiquitous EGFP florescence was confirmed in all examined organs. There were no gender-related differences in fluorescence. The established CAG/EGFP transgenic rabbit will be an important bioresource and a useful tool for various studies in tissue engineering and regenerative medicine.
References
Ajiki T, Kimura A, Sato Y, Murakami T, Hakamata Y, Kariya Y, Hoshino Y, Kobayashi E (2005) Composite tissue transplantation in rats: fusion of donor muscle to the recipient site. Transplant Proc 37(1):208–209
Al-Gubory KH, Houdebine LM (2006) In vivo imaging of green fluorescent protein-expressing cells in transgenic animals using fibred confocal fluorescence microscopy. Eur J Cell Biol 85(8):837–845. Epub 2006 June 14
Attaran M, Schneider A, Grote C, Zwiens C, Flemming P, Gratz KF, Jochheim A, Bahr MJ, Manns MP, Ott M (2004) Regional and transient ischemia/reperfusion injury in the liver improves therapeutic efficacy of allogeneic intraportal hepatocyte transplantation in low-density lipoprotein receptor deficient Watanabe rabbits. J Hepatol 41(5):837–844
Chesne P, Adenot PG, Viglietta C, Baratte M, Boulanger L, Renard JP (2002) Cloned rabbits produced by nuclear transfer from adult somatic cells. Nat Biotechnol 20(4):366–369
Chiocchetti A, Tolosano E, Hirsch E, Silengo L, Altruda F (1997) Green fluorescent protein as a reporter of gene expression in transgenic mice. Biochim Biophys Acta 1352(2):193–202
Chrenek P, Vasicek D, Makarevich AV, Jurcik R, Suvegova K, Parkanyi V, Bauer M, Rafay J, Batorova A, Paleyanda RK (2005) Increased transgene integration efficiency upon microinjection of DNA into both pronuclei of rabbit embryos. Transgenic Res 14(4):417–428
Fan J, Watanabe T (2003) Inflammatory reactions in the pathogenesis of atherosclerosis. J Atheroscler Thromb 10(2):63–71
Fan J, Challah M, Watanabe T (1999) Transgenic rabbit models for biomedical research: current status, basic methods and future perspectives. Pathol Int 49(7):583–594
Furukawa H, Oshima K, Tung T, Cui G, Laks H, Sen L (2005) Liposome-mediated combinatorial cytokine gene therapy induces localized synergistic immunosuppression and promotes long-term survival of cardiac allografts. J Immunol 174(11):6983–6992
Hakamata Y, Tahara K, Uchida H, Sakuma Y, Nakamura M, Kume A, Murakami T, Takahashi M, Takahashi R, Hirabayashi M, Ueda M, Miyoshi I, Kasai N, Kobayashi E (2001) Green fluorescent protein-transgenic rat: a tool for organ transplantation research. Biochem Biophys Res Commun 286(4):779–785
Hashimoto S, Kuramochi T, Aoyagi K, Takahashi R, Ueda M, Hirao M, Kamei M, Kitada K, Hirasawa K (2004) Refined porcine follicle stimulating hormone promotes the responsiveness of rabbits to multiple-ovulation treatment. Exp Anim 53(4):395–397
Hayes H, Rogel-Gaillard C, Zijlstra C, De Haan NA, Urien C, Bourgeaux N, Bertaud M, Bosma AA (2002) Establishment of an R-banded rabbit karyotype nomenclature by FISH localization of 23 chromosome-specific genes on both G- and R-banded chromosomes. Cytogenet Genome Res 98(2–3):199–205
Hirabayashi M, Takahashi R, Ito K, Kashiwazaki N, Hirao M, Hirasawa K, Hochi S, Ueda M (2001) A comparative study on the integration of exogenous DNA into mouse, rat, rabbit, and pig genomes. Exp Anim 50(2):125–131
Inamatsu M, Tochio T, Makabe A, Endo T, Oomizu S, Kobayashi E, Yoshizato K (2006) Embryonic dermal condensation and adult dermal papilla induce hair follicles in adult glabrous epidermis through different mechanisms. Dev Growth Differ 48(2):73–86
Inoue S, Tahara K, Kaneko T, Ajiki T, Takeda S, Sato Y, Hakamata Y, Murakami T, Takahashi M, Kaneko M, Kobayashi E (2004) Long-lasting donor passenger leukocytes after hepatic and intestinal transplantation in rats. Transpl Immunol 12:123–131
Inoue H, Ohsawa I, Murakami T, Kimura A, Hakamata Y, Sato Y, Kaneko T, Takahashi M, Okada T, Ozawa K, Francis J, Leone P, Kobayashi E (2005) Development of new inbred transgenic strains of rats with LacZ or GFP. Biochem Biophys Res Commun 329:288–295
Judas F, Figueiredo MH, Cabrita AM, Proenca A (2005) Incorporation of impacted morselized bone allografts in rabbits. Transplant Proc 37(6):2802–2804
Kaneda M, Fujii S, Aoyama H, Teramoto S (1993) Usefulness of a simplified artificial insemination technique in the rabbit for teratology studies. Jikken Dobutsu 42(2):217–220
Kurome M, Ueda H, Tomii R, Naruse K, Nagashima H (2006) Production of transgenic-clone pigs by the combination of ICSI-mediated gene transfer with somatic cell nuclear transfer. Transgenic Res 15(2):229–240
Li Z, Li ZB (2005) Repair of mandible defect with tissue engineering bone in rabbits. ANZ J Surg 75(11):1017–1021
Niwa H, Yamamura K, Miyazaki J (1991) Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene 108(2):193–199
Ohya M, Yamada Y, Ozawa R, Ito K, Takahashi M, Ueda M (2005) Sinus floor elevation applied tissue-engineered bone. Comparative study between mesenchymal stem cells/platelet-rich plasma (PRP) and autogenous bone with PRP complexes in rabbits. Clin Oral Implants Res 16(5):622–629
Okabe M, Ikawa M, Kominami K, Nakanishi T, Nishimune Y (1997) ‘Green mice’ as a source of ubiquitous green cells. FEBS Lett 407(3):313–319
Sato Y, Matsui K, Ajiki T, Igarashi Y, Takahashi M, Murakami T, Hakamata Y, Tabata Y, Kobayashi E (2005) Can a bone marrow cell contribute to organ regeneration? In vivo analysis using transgenic rats with reporter genes. Transplant Proc 37(1):273–275
Tahara K, Murakami T, Fujishiro J, Takahashi M, Inoue S, Hashizume K, Matsuno K, Kobayashi E (2005) Regeneration of the rat neonatal intestine in transplantation. Ann Surg 242:124–132
Wang HJ, Lin AX, Zhang ZC, Chen YF (2001) Expression of porcine growth hormone gene in transgenic rabbits as reported by green fluorescent protein. Anim Biotechnol 12(2):101–110
Watanabe Y, Ebukuro M, Yagami K, Sugiyama F, Ishida J, Murakami K, Nomura T, Katoh H (1996) Chromosomal mapping of human angiotensinogen gene and human renin gene by fluorescence in situ hybridization (FISH) in transgenic mice. Exp Anim 45(3):265–269
Yoshida S, Sekine Y, Saitoh Y, Yasufuku K, Iwata T, Fujisawa T (2005) Surgical technique of experimental lung transplantation in rabbits. Ann Thorac Cardiovasc Surg 11(1):7–11
Zhuo L, Sun B, Zhang CL, Fine A, Chiu SY, Messing A (1997) Live astrocytes visualized by green fluorescent protein in transgenic mice. Dev Biol 187(1):36–42
Acknowledgments
We greatly acknowledge Mr Kazuo Hirasawa, president of Kitayama Labes Co. Ltd, for providing the experimental facilities and animals. We would like to thank Mr Kensaku Kitada, Mr Masao, Hirao Kitayama Labes Co. Ltd, for the skillful technical assistance. We would also like to thank Dr Kazuo Gotoh, The Central Institute for Experimental Animals, for his help in FISH analysis of the transgene, and Mr Akira Shiota, the YS institute Inc, for help in genotype analysis of transgenic rabbit.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Takahashi, Ri., Kuramochi, T., Aoyagi, K. et al. Establishment and characterization of CAG/EGFP transgenic rabbit line. Transgenic Res 16, 115–120 (2007). https://doi.org/10.1007/s11248-006-9043-1
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11248-006-9043-1