Skip to main content

Advertisement

SpringerLink
Log in

Development and characterization of a new inbred transgenic rat strain expressing DsRed monomeric fluorescent protein

  • Original Paper
  • Published:
Transgenic Research Aims and scope Submit manuscript

Abstract

The inbred rat is a suitable model for studying human disease and because of its larger size is more amenable to complex surgical manipulation than the mouse. While the rodent fulfills many of the criteria for transplantation research, an important requirement is the ability to mark and track donors cells and assess organ viability. However, tracking ability is limited by the availability of transgenic (Tg) rats that express suitable luminescent or fluorescent proteins. Red fluorescent protein cloned from Discosoma coral (DsRed) has several advantages over other fluorescent proteins, including in vivo detection in the whole animal and ex vivo visualization in organs as there is no interference with autofluorescence. We generated and characterized a novel inbred Tg Lewis rat strain expressing DsRed monomeric (DsRed mono) fluorescent protein under the control of a ubiquitously expressed ROSA26 promoter. DsRed mono Tg rats ubiquitously expressed the marker gene as detected by RT-PCR but the protein was expressed at varying levels in different organs. Conventional skin grafting experiments showed acceptance of DsRed monomeric Tg rat skin on wild-type rats for more than 30 days. Cardiac transplantation of DsRed monomeric Tg rat hearts into wild-type recipients further showed graft acceptance and long-term organ viability (>6 months). The DsRed monomeric Tg rat provides marked cells and/or organs that can be followed for long periods without immune rejection and therefore is a suitable model to investigate cell tracking and organ transplantation.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

Abbreviations

BM-MSCs:

Bone marrow-mesenchymal stromal cells

HHT:

Heterotopic heart transplantation

LEW:

Lewis

Mono:

Monomeric

MRI:

Magnetic resonance imaging

RFP:

Red fluorescent protein

Tg:

Transgenic

References

  • Baird GS, Zacharias DA, Tsien RY (2000) Biochemistry, mutagenesis, and oligomerization of DsRed, a red fluorescent protein from coral. Proc Natl Acad Sci U S A 97(22):11984–11989. doi:10.1073/pnas.97.22.11984

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Bevis BJ, Glick BS (2002) Rapidly maturing variants of the Discosoma red fluorescent protein (DsRed). Nat Biotechnol 20(1):83–87. doi:10.1038/nbt0102-83

    Article  CAS  PubMed  Google Scholar 

  • Campbell RE, Tour O, Palmer AE, Steinbach PA, Baird GS, Zacharias DA, Tsien RY (2002) A monomeric red fluorescent protein. Proc Natl Acad Sci U S A 99(12):7877–7882. doi:10.1073/pnas.082243699

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Cerkovnik P, Perhavec A, Zgajnar J, Novakovic S (2007) Optimization of an RNA isolation procedure from plasma samples. Int J Mol Med 20(3):293–300

    CAS  PubMed  Google Scholar 

  • Chang SP, Opsahl ML, Whitelaw CB, Morley SD, West JD (2013) Relative transgene expression frequencies in homozygous versus hemizygous transgenic mice. Transgenic Res. doi:10.1007/s11248-013-9732-5

    Google Scholar 

  • Conradi L, Pahrmann C, Schmidt S, Deuse T, Hansen A, Eder A, Reichenspurner H, Robbins RC, Eschenhagen T, Schrepfer S (2011) Bioluminescence imaging for assessment of immune responses following implantation of engineered heart tissue (EHT). J Vis Exp (JoVE) (52). doi:10.3791/2605

  • Cotlet M, Hofkens J, Habuchi S, Dirix G, Van Guyse M, Michiels J, Vanderleyden J, De Schryver FC (2001) Identification of different emitting species in the red fluorescent protein DsRed by means of ensemble and single-molecule spectroscopy. Proc Natl Acad Sci U S A 98(25):14398–14403. doi:10.1073/pnas.251532698

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Cozzi J, Fraichard A, Thiam K (2008) Use of genetically modified rat models for translational medicine. Drug Discov Today 13(11–12):488–494. doi:10.1016/j.drudis.2008.03.021

    Article  CAS  PubMed  Google Scholar 

  • Davis J, Maillet M, Miano JM, Molkentin JD (2012) Lost in transgenesis: a user’s guide for genetically manipulating the mouse in cardiac research. Circ Res 111(6):761–777. doi:10.1161/CIRCRESAHA.111.262717

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Day CD, Lee E, Kobayashi J, Holappa LD, Albert H, Ow DW (2000) Transgene integration into the same chromosome location can produce alleles that express at a predictable level, or alleles that are differentially silenced. Genes Dev 14(22):2869–2880

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Doyle A, McGarry MP, Lee NA, Lee JJ (2012) The construction of transgenic and gene knockout/knockin mouse models of human disease. Transgenic Res 21(2):327–349. doi:10.1007/s11248-011-9537-3

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Filipiak WE, Saunders TL (2006) Advances in transgenic rat production. Transgenic Res 15(6):673–686. doi:10.1007/s11248-006-9002-x

    Article  CAS  PubMed  Google Scholar 

  • Gama Sosa MA, De Gasperi R, Elder GA (2010) Animal transgenesis: an overview. Brain Struct Funct 214(2–3):91–109. doi:10.1007/s00429-009-0230-8

    Article  CAS  PubMed  Google Scholar 

  • Gambotto A, Dworacki G, Cicinnati V, Kenniston T, Steitz J, Tuting T, Robbins PD, DeLeo AB (2000) Immunogenicity of enhanced green fluorescent protein (EGFP) in BALB/c mice: identification of an H2-Kd-restricted CTL epitope. Gene Ther 7(23):2036–2040

    Article  CAS  PubMed  Google Scholar 

  • Garcia-Parajo MF, Koopman M, van Dijk EM, Subramaniam V, van Hulst NF (2001) The nature of fluorescence emission in the red fluorescent protein DsRed, revealed by single-molecule detection. Proc Natl Acad Sci U S A 98(25):14392–14397. doi:10.1073/pnas.251525598

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Goulding AM, Rahimi Y, Shrestha S, Deo SK (2008) Dual function labeling of biomolecules based on DsRed-Monomer. Bioconjug Chem 19(11):2113–2119. doi:10.1021/bc800147k

    Article  CAS  PubMed  Google Scholar 

  • Grieshammer U, McGrew MJ, Rosenthal N (1995) Role of methylation in maintenance of positionally restricted transgene expression in developing muscle. Development (Cambridge, England) 121(7):2245–2253

    CAS  Google Scholar 

  • Gross LA, Baird GS, Hoffman RC, Baldridge KK, Tsien RY (2000) The structure of the chromophore within DsRed, a red fluorescent protein from coral. Proc Natl Acad Sci U S A 97(22):11990–11995. doi:10.1073/pnas.97.22.11990

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • 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. doi:10.1006/bbrc.2001.5452

    Article  CAS  PubMed  Google Scholar 

  • Hakamata Y, Murakami T, Kobayashi E (2006) “Firefly rats” as an organ/cellular source for long-term in vivo bioluminescent imaging. Transplantation 81(8):1179–1184. doi:10.1097/01.tp.0000203137.06587.4a

    Article  PubMed  Google Scholar 

  • Hara M, Murakami T, Kobayashi E (2008) In vivo bioimaging using photogenic rats: fate of injected bone marrow-derived mesenchymal stromal cells. J Autoimmun 30(3):163–171. doi:10.1016/j.jaut.2007.12.007

    Article  PubMed  Google Scholar 

  • Heron I (1971) A technique for accessory cervical heart transplantation in rabbits and rats. Acta Pathol Microbiol Scand Sect A Pathol 79(4):366–372

    CAS  Google Scholar 

  • Hofmann A, Kessler B, Ewerling S, Kabermann A, Brem G, Wolf E, Pfeifer A (2006) Epigenetic regulation of lentiviral transgene vectors in a large animal model. Mol Ther J Am Soc Gene Ther 13(1):59–66. doi:10.1016/j.ymthe.2005.07.685

    Article  CAS  Google Scholar 

  • Hung CJ, Yao CL, Cheng FC, Wu ML, Wang TH, Hwang SM (2010) Establishment of immortalized mesenchymal stromal cells with red fluorescence protein expression for in vivo transplantation and tracing in the rat model with traumatic brain injury. Cytotherapy 12(4):455–465. doi:10.3109/14653240903555827

    Article  CAS  PubMed  Google Scholar 

  • 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(1):288–295. doi:10.1016/j.bbrc.2005.01.132

    Article  CAS  PubMed  Google Scholar 

  • Karaoz E, Aksoy A, Ayhan S, Sariboyaci AE, Kaymaz F, Kasap M (2009) Characterization of mesenchymal stem cells from rat bone marrow: ultrastructural properties, differentiation potential and immunophenotypic markers. Histochem Cell Biol 132(5):533–546. doi:10.1007/s00418-009-0629-6

    Article  CAS  PubMed  Google Scholar 

  • Karpen GH (1994) Position-effect variegation and the new biology of heterochromatin. Curr Opin Genet Dev 4(2):281–291

    Article  CAS  PubMed  Google Scholar 

  • Khokha MK, Landini G, Iannaccone PM (1994) Fractal geometry in rat chimeras demonstrates that a repetitive cell division program may generate liver parenchyma. Dev Biol 165(2):545–555. doi:10.1006/dbio.1994.1274

    Article  CAS  PubMed  Google Scholar 

  • Kioussis D, Festenstein R (1997) Locus control regions: overcoming heterochromatin-induced gene inactivation in mammals. Curr Opin Genet Dev 7(5):614–619

    Article  CAS  PubMed  Google Scholar 

  • Kisseberth WC, Brettingen NT, Lohse JK, Sandgren EP (1999) Ubiquitous expression of marker transgenes in mice and rats. Dev Biol 214(1):128–138. doi:10.1006/dbio.1999.9417

    Article  CAS  PubMed  Google Scholar 

  • Kulbatski I, Mothe AJ, Keating A, Hakamata Y, Kobayashi E, Tator CH (2007) Oligodendrocytes and radial glia derived from adult rat spinal cord progenitors: morphological and immunocytochemical characterization. J Histochem Cytochem 55(3):209–222. doi:10.1369/jhc.6A7020.2006

    Article  CAS  PubMed  Google Scholar 

  • Lao Z, Raju GP, Bai CB, Joyner AL (2012) MASTR: a technique for mosaic mutant analysis with spatial and temporal control of recombination using conditional floxed alleles in mice. Cell Rep 2(2):386–396. doi:10.1016/j.celrep.2012.07.004

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Martins PN (2008) Assessment of graft function in rodent models of heart transplantation. Microsurgery 28(7):565–570. doi:10.1002/micr.20544

    Article  PubMed  Google Scholar 

  • Mashimo T, Serikawa T (2009) Rat resources in biomedical research. Curr Pharm Biotechnol 10(2):214–220

    Article  CAS  PubMed  Google Scholar 

  • Menoret S, Remy S, Usal C, Tesson L, Anegon I (2010) Generation of transgenic rats by microinjection of short DNA fragments. Methods Mol Biol (Clifton, NJ) 597:81–92. doi:10.1007/978-1-60327-389-3_6

    Article  CAS  Google Scholar 

  • Moloney TC, Dockery P, Windebank AJ, Barry FP, Howard L, Dowd E (2010) Survival and immunogenicity of mesenchymal stem cells from the green fluorescent protein transgenic rat in the adult rat brain. Neurorehabil Neural Repair 24(7):645–656. doi:10.1177/1545968309357745

    Article  PubMed  Google Scholar 

  • Mothe AJ, Kulbatski I, van Bendegem RL, Lee L, Kobayashi E, Keating A, Tator CH (2005) Analysis of green fluorescent protein expression in transgenic rats for tracking transplanted neural stem/progenitor cells. J Histochem Cytochem 53(10):1215–1226. doi:10.1369/jhc.5A6639.2005

    Article  CAS  PubMed  Google Scholar 

  • Murakami T, Kobayashi E (2005) Color-engineered rats and luminescent LacZ imaging: a new platform to visualize biological processes. J Biomed Opt 10(4):41204. doi:10.1117/1.2007947

    Article  PubMed  Google Scholar 

  • Murakami T, Kobayashi E (2012) GFP-transgenic animals for in vivo imaging: rats, rabbits, and pigs. Methods Mol Biol (Clifton, NJ) 872:177–189. doi:10.1007/978-1-61779-797-2_12

    Article  CAS  Google Scholar 

  • Pan XC, Deng YB, Sugawara Y, Makuuchi M, Okabe M, Ochiya T, Sugiura W, Kitazawa Y, Fuji N, Li XK, Miyamoto M, Kimura H (2009) Immunological behavior of enhanced green fluorescent protein (EGFP) as a minor histocompatibility antigen with a special reference to skin isograft and specific regulation of local graft-versus-host reaction (GvHR). Immunol Lett 123(2):103–113. doi:10.1016/j.imlet.2009.02.004

    Article  CAS  PubMed  Google Scholar 

  • Remy S, Tesson L, Usal C, Menoret S, Bonnamain V, Nerriere-Daguin V, Rossignol J, Boyer C, Nguyen TH, Naveilhan P, Lescaudron L, Anegon I (2010) New lines of GFP transgenic rats relevant for regenerative medicine and gene therapy. Transgenic Res 19(5):745–763. doi:10.1007/s11248-009-9352-2

    Article  CAS  PubMed  Google Scholar 

  • Sacchetti A, Subramaniam V, Jovin TM, Alberti S (2002) Oligomerization of DsRed is required for the generation of a functional red fluorescent chromophore. FEBS Lett 525(1–3):13–19

    Article  CAS  PubMed  Google Scholar 

  • Sato Y, Igarashi Y, Hakamata Y, Murakami T, Kaneko T, Takahashi M, Seo N, Kobayashi E (2003) Establishment of Alb-DsRed2 transgenic rat for liver regeneration research. Biochem Biophys Res Commun 311(2):478–481

    Article  CAS  PubMed  Google Scholar 

  • Sato Y, Endo H, Ajiki T, Hakamata Y, Okada T, Murakami T, Kobayashi E (2004) Establishment of Cre/LoxP recombination system in transgenic rats. Biochem Biophys Res Commun 319(4):1197–1202. doi:10.1016/j.bbrc.2004.04.204

    Article  CAS  PubMed  Google Scholar 

  • Shaner NC, Steinbach PA, Tsien RY (2005) A guide to choosing fluorescent proteins. Nat Methods 2(12):905–909. doi:10.1038/nmeth819

    Article  CAS  PubMed  Google Scholar 

  • Strack RL, Strongin DE, Bhattacharyya D, Tao W, Berman A, Broxmeyer HE, Keenan RJ, Glick BS (2008) A noncytotoxic DsRed variant for whole-cell labeling. Nat Methods 5(11):955–957. doi:10.1038/nmeth.1264

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Strack RL, Hein B, Bhattacharyya D, Hell SW, Keenan RJ, Glick BS (2009) A rapidly maturing far-red derivative of DsRed-Express2 for whole-cell labeling. Biochemistry 48(35):8279–8281. doi:10.1021/bi900870u

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Stripecke R, Carmen Villacres M, Skelton D, Satake N, Halene S, Kohn D (1999) Immune response to green fluorescent protein: implications for gene therapy. Gene Ther 6(7):1305–1312

    Article  CAS  PubMed  Google Scholar 

  • Tang W, Luo XY, Sanmuels V (2001) Gene silencing: double-stranded RNA mediated mRNA degradation and gene inactivation. Cell Res 11(3):181–186. doi:10.1038/sj.cr.7290084

    Article  CAS  PubMed  Google Scholar 

  • Verkhusha VV, Akovbian NA, Efremenko EN, Varfolomeyev SD, Vrzheshch PV (2001) Kinetic analysis of maturation and denaturation of DsRed, a coral-derived red fluorescent protein. Biochemistry (Biokhimiia) 66(12):1342–1351

    Article  CAS  Google Scholar 

  • Wall MA, Socolich M, Ranganathan R (2000) The structural basis for red fluorescence in the tetrameric GFP homolog DsRed. Nat Struct Biol 7(12):1133–1138. doi:10.1038/81992

    Article  CAS  PubMed  Google Scholar 

  • Youssef AR, Otley C, Mathieson PW, Smith RM (2002) Effector mechanisms in murine allograft rejection: comparison of skin and heart grafts in fully allogeneic and minor histocompatibility antigen-mismatched strain combinations. Transpl Int 15(6):302–309. doi:10.1007/s00147-002-0407-z

    Article  CAS  PubMed  Google Scholar 

  • Zambrowicz BP, Imamoto A, Fiering S, Herzenberg LA, Kerr WG, Soriano P (1997) Disruption of overlapping transcripts in the ROSA beta geo 26 gene trap strain leads to widespread expression of beta-galactosidase in mouse embryos and hematopoietic cells. Proc Natl Acad Sci U S A 94(8):3789–3794

    Article  CAS  PubMed Central  PubMed  Google Scholar 

Download references

Acknowledgments

We are grateful to Dr. Yoko Kosaka for assistance with Flow cytometric analysis and to Mr. Kikuchi and Dr. He for training S. Montanari in microsurgery. We also thank Dr. Takashi Murakami for assistance in Southern Blot design. This research was supported by the Orsino Translational Research Laboratory at Princess Margaret Hospital and by the Gloria and Seymour Epstein Chair in Cell Therapy and Transplantation at University Health Network and the University of Toronto to AK.

Conflict of interest

No competing financial interests exist for any of the authors.

Author information

Authors and Affiliations

  1. Cell Therapy Program, Princess Margaret Hospital, University Health Network, Toronto, ON, Canada

    Sonia Montanari, Xing-Hua Wang, Gustavo Yannarelli, Victor Dayan, Larissa Zocche, Sowmya Viswanathan & Armand Keating

  2. Institute of Medical Science, University of Toronto, Toronto, ON, Canada

    Sonia Montanari & Armand Keating

  3. The Campbell Family Institute for Breast Cancer Research, University Health Network, Toronto, ON, Canada

    Thorsten Berger

  4. Divisions of Organ Replacement Research and Animal Resource Project, Centre for Molecular Medicine, Jichi Medical School, Tochigi, Japan

    Eiji Kobayashi

  5. Orsino Cell Therapy Translational Research Laboratory, Princess Margaret Cancer Centre, 610 University Avenue, Room 5–303, Toronto, ON, M5G 2M9, Canada

    Sonia Montanari

Authors
  1. Sonia Montanari
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Xing-Hua Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Gustavo Yannarelli
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Victor Dayan
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Thorsten Berger
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Larissa Zocche
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Eiji Kobayashi
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Sowmya Viswanathan
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Armand Keating
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Sonia Montanari.

Electronic supplementary material

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Montanari, S., Wang, XH., Yannarelli, G. et al. Development and characterization of a new inbred transgenic rat strain expressing DsRed monomeric fluorescent protein. Transgenic Res 23, 779–793 (2014). https://doi.org/10.1007/s11248-014-9814-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11248-014-9814-z

Keywords

Search

Navigation