Skip to main content
SpringerLink
Log in

Generation of SV40-transformed rabbit tracheal-epithelial-cell-derived blastocyst by somatic cell nuclear transfer

  • Regular Article
  • Published:
Cell and Tissue Research Aims and scope Submit manuscript

Abstract

The prospect of developing large animal models for the study of inherited diseases, such as cystic fibrosis (CF), through somatic cell nuclear transfer (SCNT) has opened up new opportunities for enhancing our understanding of disease pathology and for identifying new therapies. Thus, the development of species-specific in vitro cell systems that will provide broader insight into organ- and cell-type-specific functions relevant to the pathology of the disease is crucial. Studies have been undertaken to establish transformed rabbit airway epithelial cell lines that display differentiated features characteristic of the primary airway epithelium. This study describes the successful establishment and characterization of two SV40-transformed rabbit tracheal epithelial cell lines. These cell lines, 5RTEo- and 9RTEo-, express the CF transmembrane conductance regulator (CFTR) gene, retain epithelial-specific differentiated morphology and show CFTR-based cAMP-dependent Cl− ion transport across the apical membrane of a confluent monolayer. Immunocytochemical analysis indicates the presence of airway cytokeratins and tight-junction proteins in the 9RTEo- cell line after multiple generations. However, the tight junctions appear to diminish in their efficacy in both cell lines after at  least 100 generations. Initial SCNT studies with the 9RTEo- cells have revealed that SV40-transformed rabbit airway epithelial donor cells can be used to generate blastocysts. These cell systems provide valuable models for studying the developmental and metabolic modulation of CFTR gene expression and rabbit airway epithelial cell biology.

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
Fig. 6

Similar content being viewed by others

References

  • Bosze Z, Hiripi L, Carnwath JW, Niemann H (2003) The transgenic rabbit as model for human diseases and as a source of biologically active recombinant proteins. Transgenic Res 12:541–553

    Article  PubMed  CAS  Google Scholar 

  • Burns KD, Regnier L, Roczniak A, Hebert RL (1996) Immortalized rabbit cortical collecting duct cells express AT1 angiotensin II receptors. Am J Physiol 271:F1147–F1157

    PubMed  CAS  Google Scholar 

  • Cervera RP, Garcia-Ximenez F (2003) Oocyte age and nuclear donor cell type affect the technical efficiency of somatic cloning in rabbits. Zygote 11:151–158

    Article  PubMed  CAS  Google Scholar 

  • Challah-Jacques M, Chesne P, Renard JP (2003) Production of cloned rabbits by somatic nuclear transfer. Cloning Stem Cells 5:295–299

    Article  PubMed  CAS  Google Scholar 

  • Chen JM, Cutler C, Jacques C, Boeuf G, Denamur E, Lecointre G, Mercier B, Cramb G, Ferec C (2001) A combined analysis of the cystic fibrosis transmembrane conductance regulator: implications for structure and disease models. Mol Biol Evol 18:1771–1788

    Article  PubMed  CAS  Google Scholar 

  • 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 20:366–369

    Article  PubMed  CAS  Google Scholar 

  • Cozens AL, Yezzi MJ, Chin L, Simon EM, Finkbeiner WE, Wagner JA, Gruenert DC (1992a) Characterization of immortal cystic fibrosis tracheobronchial gland epithelial cells. Proc Natl Acad Sci USA 89:5171–5175

    Article  PubMed  CAS  Google Scholar 

  • Cozens AL, Yezzi MJ, Yamaya M, Steiger D, Wagner JA, Garber SS, Chin L, Simon EM, Cutting GR, Gardner P et al (1992b) A transformed human epithelial cell line that retains tight junctions post crisis. In Vitro Cell Dev Biol 28A:735–744

    Article  PubMed  CAS  Google Scholar 

  • Cozens AL, Yezzi MJ, Kunzelmann K, Ohrui T, Chin L, Eng K, Finkbeiner WE, Widdicombe JH, Gruenert DC (1994) CFTR expression and chloride secretion in polarized immortal human bronchial epithelial cells. Am J Respir Cell Mol Biol 10:38–47

    PubMed  CAS  Google Scholar 

  • Cui W, Wylie D, Aslam S, Dinnyes A, King T, Wilmut I, Clark AJ (2003) Telomerase-immortalized sheep fibroblasts can be reprogrammed by nuclear transfer to undergo early development. Biol Reprod 69:15–21

    Article  PubMed  CAS  Google Scholar 

  • de Semir D, Maurisse R, Vock E, Gruenert DC (2008) Immortalization strategies for epithelial cells in primary culture. In: Ehrhardt C, Kim K-J (eds) Biotechnology: pharmeceutical aspects; drug absorption studies—in situ, in vitro and in silico tools. Springer, New York, pp 616–639

    Google Scholar 

  • Diamond G, Scanlin TF, Zasloff MA, Bevins CL (1991) A cross-species analysis of the cystic fibrosis transmembrane conductance regulator. Potential functional domains and regulatory sites. J Biol Chem 266:22761–22769

    PubMed  CAS  Google Scholar 

  • Du F, Giles JR, Foote RH, Graves KH, Yang X, Moreadith RW (1995) Nuclear transfer of putative rabbit embryonic stem cells leads to normal blastocyst development. J Reprod Fertil 104:219–223

    Article  PubMed  CAS  Google Scholar 

  • Du F, Shen PC, Xu J, Sung LY, Jeong BS, Lucky Nedambale T, Riesen J, Cindy Tian X, Cheng WT, Lee SN, Yang X (2006) The cell agglutination agent, phytohemagglutinin-L, improves the efficiency of somatic nuclear transfer cloning in cattle (Bos taurus). Theriogenology 65:642–657

    Article  PubMed  CAS  Google Scholar 

  • Du F, Xu J, Zhang J, Gao S, Carter MG, He C, Sung LY, Chaubal S, Fissore RA, Tian XC, Yang X, Chen YE (2009) Beneficial effect of young oocytes for rabbit somatic cell nuclear transfer. Cloning Stem Cells 11:131–140

    Article  PubMed  CAS  Google Scholar 

  • Ehrhardt C, Collnot EM, Baldes C, Becker U, Laue M, Kim KJ, Lehr CM (2006) Towards an in vitro model of cystic fibrosis small airway epithelium: characterisation of the human bronchial epithelial cell line CFBE41o. Cell Tissue Res 323:405–415

    Article  PubMed  CAS  Google Scholar 

  • Fan J, Watanabe T (2003) Transgenic rabbits as therapeutic protein bioreactors and human disease models. Pharmacol Ther 99:261–282

    Article  PubMed  CAS  Google Scholar 

  • Fang ZF, Gai H, Huang YZ, Li SG, Chen XJ, Shi JJ, Wu L, Liu A, Xu P, Sheng HZ (2006) Rabbit embryonic stem cell lines derived from fertilized, parthenogenetic or somatic cell nuclear transfer embryos. Exp Cell Res 312:3669–3682

    Article  PubMed  CAS  Google Scholar 

  • Gomez MC, Pope CE, Dresser BL (2006) Nuclear transfer in cats and its application. Theriogenology 66:72–81

    Article  PubMed  CAS  Google Scholar 

  • Goncz KK, Feeney L, Gruenert DC (1999) Differential sensitivity of normal and cystic fibrosis airway epithelial cells to epinephrine. Br J Pharmacol 128:227–233

    Article  PubMed  CAS  Google Scholar 

  • Gong G, Dai Y, Zhu H, Wang H, Wang L, Li R, Wan R, Liu Y, Li N (2004) Generation of cloned calves from different types of somatic cells. Sci China C Life Sci 47:470–476

    Article  PubMed  Google Scholar 

  • Graves KH, Moreadith RW (1993) Derivation and characterization of putative pluripotential embryonic stem cells from preimplantation rabbit embryos. Mol Reprod Dev 36:424–433

    Article  PubMed  CAS  Google Scholar 

  • Grubb BR, Boucher RC (1999) Pathophysiology of gene-targeted mouse models for cystic fibrosis. Physiol Rev 79:S193–S214

    PubMed  CAS  Google Scholar 

  • Gruenert DC (1987) Differentiated properties of human epithelial cells transformed in vitro. Biotechniques 5:740–749

    CAS  Google Scholar 

  • Gruenert DC, Basbaum CB, Welsh MJ, Li M, Finkbeiner WE, Nadel JA (1988) Characterization of human tracheal epithelial cells transformed by an origin-defective simian virus 40. Proc Natl Acad Sci USA 85:5951–5955

    Article  PubMed  CAS  Google Scholar 

  • Gruenert DC, Basbaum CB, Widdicombe JH (1990) Long-term culture of normal and cystic fibrosis epithelial cells grown under serum-free conditions. In Vitro Cell Dev Biol 26:411–418

    Article  PubMed  CAS  Google Scholar 

  • Gruenert DC, Finkbeiner WE, Widdicombe JH (1995) Culture and transformation of human airway epithelial cells. Am J Physiol 268:L347–L360

    PubMed  CAS  Google Scholar 

  • Gruenert DC, Willems M, Cassiman JJ, Frizzell RA (2004) Established cell lines used in cystic fibrosis research. J Cyst Fibros 3 (Suppl 2):191–196

    Article  PubMed  CAS  Google Scholar 

  • Guilbault C, Saeed Z, Downey GP, Radzioch D (2007) Cystic fibrosis mouse models. Am J Respir Cell Mol Biol 36:1–7

    Article  PubMed  CAS  Google Scholar 

  • Hatoya S, Torii R, Kondo Y, Okuno T, Kobayashi K, Wijewardana V, Kawate N, Tamada H, Sawada T, Kumagai D, Sugiura K, Inaba T (2006) Isolation and characterization of embryonic stem-like cells from canine blastocysts. Mol Reprod Dev 73:298–305

    Article  PubMed  CAS  Google Scholar 

  • Hayflick L (1974) The longevity of cultured human cells. J Am Geriatr Soc 22:1–12

    PubMed  CAS  Google Scholar 

  • Hayflick L (1998) A brief history of the mortality and immortality of cultured cells. Keio J Med 47:174–182

    Article  PubMed  CAS  Google Scholar 

  • Illek B, Fischer H, Machen TE (1996) Alternate stimulation of apical CFTR by genistein in epithelia. Am J Physiol 270:C265–C275

    PubMed  CAS  Google Scholar 

  • Illek B, Lei D, Fischer H, Gruenert DC (2010) Sensitivity of chloride efflux vs. transepithelial measurements in mixed CF and normal airway epithelial cell populations. Cell Physiol Biochem 26:983–990

    Article  PubMed  CAS  Google Scholar 

  • Kang SS, Wang L, Kao WW, Reinach PS, Lu L (2001) Control of SV-40 transformed RCE cell proliferation by growth-factor-induced cell cycle progression. Curr Eye Res 23:397–405

    Article  PubMed  CAS  Google Scholar 

  • Kasinathan P, Knott JG, Wang Z, Jerry DJ, Robl JM (2001) Production of calves from G1 fibroblasts. Nat Biotechnol 19:1176–1178

    Article  PubMed  CAS  Google Scholar 

  • Kishigami S, Wakayama T (2009) Somatic cell nuclear transfer in the mouse. Methods Mol Biol 518:207–218

    Article  PubMed  CAS  Google Scholar 

  • Li J, Villemoes K, Zhang Y, Du Y, Kragh PM, Purup S, Xue Q, Pedersen AM, Jorgensen AL, Jakobsen JE, Bolund L, Yang H, Vajta G (2009) Efficiency of two enucleation methods connected to handmade cloning to produce transgenic porcine embryos. Reprod Domest Anim 44:122–127

    Article  PubMed  CAS  Google Scholar 

  • Li M, Zhang D, Hou Y, Jiao L, Zheng X, Wang WH (2003) Isolation and culture of embryonic stem cells from porcine blastocysts. Mol Reprod Dev 65:429–434

    Article  PubMed  CAS  Google Scholar 

  • Li Z, Sun X, Chen J, Liu X, Wisely SM, Zhou Q, Renard JP, Leno GH, Engelhardt JF (2006) Cloned ferrets produced by somatic cell nuclear transfer. Dev Biol 293:439–448

    Article  PubMed  CAS  Google Scholar 

  • Lotan R, Pieniazek J, George MD, Jetten AM (1992) Identification of a new squamous cell differentiation marker and its suppression by retinoids. J Cell Physiol 151:94–102

    Article  PubMed  CAS  Google Scholar 

  • MacDonald C, Watts P, Stuart B, Kreuzburg-Duffy U, Scott DM, Kinne RK (1991) Studies on the phenotype and karyotype of immortalized rabbit kidney epithelial cell lines. Exp Cell Res 195:458–461

    Article  PubMed  CAS  Google Scholar 

  • Matsuda J, Takahashi S, Ohkoshi K, Kaminaka K, Kaminaka S, Nozaki C, Maeda H, Tokunaga T (2002) Production of transgenic chimera rabbit fetuses using somatic cell nuclear transfer. Cloning Stem Cells 4:9–19

    Article  PubMed  CAS  Google Scholar 

  • Maurisse R, de Semir D, Emamekhoo H, Bedayat B, Abdolmohammadi A, Parsi H, Gruenert DC (2010) Comparative transfection of DNA into primary and transformed mammalian cells from different lineages. BMC Biotechnol 10:9

    Article  PubMed  Google Scholar 

  • Nachtigal M, Legrand A, Nagpal ML, Nachtigal SA, Greenspan P (1990) Transformation of rabbit vascular smooth muscle cells by transfection with the early region of SV40 DNA. Am J Pathol 136:297–306

    PubMed  CAS  Google Scholar 

  • Pilewski JM, Frizzell RA (1999) Role of CFTR in airway disease. Physiol Rev 79:S215–S255

    PubMed  CAS  Google Scholar 

  • Rogers CS, Abraham WM, Brogden KA, Engelhardt JF, Fisher JT, McCray PB Jr, McLennan G, Meyerholz DK, Namati E, Ostedgaard LS, Prather RS, Sabater JR, Stoltz DA, Zabner J, Welsh MJ (2008a) The porcine lung as a potential model for cystic fibrosis. Am J Physiol Lung Cell Mol Physiol 295:L240–L263

    Article  PubMed  CAS  Google Scholar 

  • Rogers CS, Hao Y, Rokhlina T, Samuel M, Stoltz DA, Li Y, Petroff E, Vermeer DW, Kabel AC, Yan Z, Spate L, Wax D, Murphy CN, Rieke A, Whitworth K, Linville ML, Korte SW, Engelhardt JF, Welsh MJ, Prather RS (2008b) Production of CFTR-null and CFTR-DeltaF508 heterozygous pigs by adeno-associated virus-mediated gene targeting and somatic cell nuclear transfer. J Clin Invest 118:1571–1577

    Article  PubMed  CAS  Google Scholar 

  • Rogers CS, Stoltz DA, Meyerholz DK, Ostedgaard LS, Rokhlina T, Taft PJ, Rogan MP, Pezzulo AA, Karp PH, Itani OA, Kabel AC, Wohlford-Lenane CL, Davis GJ, Hanfland RA, Smith TL, Samuel M, Wax D, Murphy CN, Rieke A, Whitworth K, Uc A, Starner TD, Brogden KA, Shilyansky J, McCray PB Jr, Zabner J, Prather RS, Welsh MJ (2008c) Disruption of the CFTR gene produces a model of cystic fibrosis in newborn pigs. Science 321:1837–1841

    Article  PubMed  CAS  Google Scholar 

  • Shi LH, Ai JS, Ouyang YC, Huang JC, Lei ZL, Wang Q, Yin S, Han ZM, Sun QY, Chen DY (2008) Trichostatin A and nuclear reprogramming of cloned rabbit embryos. J Anim Sci 86:1106–1113

    Article  PubMed  CAS  Google Scholar 

  • Shiffman ML, Spitzer RE, Swender PT, Galey WR (1983) Altered bicarbonate reabsorption in the pancreas of reserpine-treated rabbits—a model for cystic fibrosis. Pediatr Res 17:486–490

    Article  PubMed  CAS  Google Scholar 

  • Shimada A, Nakano H, Takahashi T, Imai K, Hashizume K (2001) Isolation and characterization of a bovine blastocyst-derived trophoblastic cell line, BT-1: development of a culture system in the absence of feeder cell. Placenta 22:652–662

    Article  PubMed  CAS  Google Scholar 

  • Small MB, Gluzman Y, Ozer HL (1982) Enhanced transformation of human fibroblasts by origin-defective simian virus 40. Nature 296:671–672

    Article  PubMed  CAS  Google Scholar 

  • Sun X, Yan Z, Yi Y, Li Z, Lei D, Rogers CS, Chen J, Zhang Y, Welsh MJ, Leno GH, Engelhardt JF (2008) Adeno-associated virus-targeted disruption of the CFTR gene in cloned ferrets. J Clin Invest 118:1578–1583

    Article  PubMed  CAS  Google Scholar 

  • Taub M, Han HJ, Rajkhowa T, Allen C, Park JH (2002) Clonal analysis of immortalized renal proximal tubule cells: Na(+)/glucose cotransport system levels are maintained despite a decline in transport function. Exp Cell Res 281:205–212

    Article  PubMed  CAS  Google Scholar 

  • Thenet S, Benya PD, Demignot S, Feunteun J, Adolphe M (1992) SV40-immortalization of rabbit articular chondrocytes: alteration of differentiated functions. J Cell Physiol 150:158–167

    Article  PubMed  CAS  Google Scholar 

  • Van Goor F, Straley KS, Cao D, Gonzalez J, Hadida S, Hazlewood A, Joubran J, Knapp T, Makings LR, Miller M, Neuberger T, Olson E, Panchenko V, Rader J, Singh A, Stack JH, Tung R, Grootenhuis PD, Negulescu P (2006) Rescue of DeltaF508-CFTR trafficking and gating in human cystic fibrosis airway primary cultures by small molecules. Am J Physiol Lung Cell Mol Physiol 290:L1117–L1130

    Article  PubMed  Google Scholar 

  • Vuillaumier S, Kaltenboeck B, Lecointre G, Lehn P, Denamur E (1997) Phylogenetic analysis of cystic fibrosis transmembrane conductance regulator gene in mammalian species argues for the development of a rabbit model for cystic fibrosis. Mol Biol Evol 14:372–380

    PubMed  CAS  Google Scholar 

  • Widdicombe JH, Welsh MJ, Finkbeiner WE (1985) Cystic fibrosis decreases the apical membrane chloride permeability of monolayers cultured from cells of tracheal epithelium. Proc Natl Acad Sci USA 82:6167–6171

    Article  PubMed  CAS  Google Scholar 

  • Yang F, Hao R, Kessler B, Brem G, Wolf E, Zakhartchenko V (2007) Rabbit somatic cell cloning: effects of donor cell type, histone acetylation status and chimeric embryo complementation. Reproduction 133:219–230

    Article  PubMed  CAS  Google Scholar 

  • Zakhartchenko V, Durcova-Hills G, Schernthaner W, Stojkovic M, Reichenbach HD, Mueller S, Steinborn R, Mueller M, Wenigerkind H, Prelle K, Wolf E, Brem G (1999) Potential of fetal germ cells for nuclear transfer in cattle. Mol Reprod Dev 52:421–426

    Article  PubMed  CAS  Google Scholar 

  • Zeitlin PL, Crawford I, Lu L, Woel S, Cohen ME, Donowitz M, Montrose MH, Hamosh A, Cutting GR, Gruenert D et al (1992) CFTR protein expression in primary and cultured epithelia. Proc Natl Acad Sci USA 89:344–347

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgements

We thank Dr. Michael Yezzi, Janet Nguyen, Judy Cheung and Lindsay Juarez for their technical assistance and Drs. A. Abdolmohammadi and B. Bedayat for their input and commentary.

Author information

Author notes

  1. R. Maurisse

    Present address: Medicen, 6 Rue Alexandre Cabanel, 75015, Paris, France

  2. F. Du & J. Xu

    Present address: Renova Life, 387 Technology Drive, College Park, Md., USA

Authors and Affiliations

  1. Department of Otolaryngology—Head and Neck Surgery, University of California, San Francisco, Mount Zion Cancer Center, 2340 Sutter Street, Box 1330, San Francisco, CA 94115, USA

    D. C. Gruenert

  2. Department of Laboratory Medicine, Helen Diller Family Comprehensive Cancer Center, Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research, Institute for Human Genetics, University of California, San Francisco, USA

    D. C. Gruenert

  3. California Pacific Medical Center Research Institute, San Francisco, Calif., USA

    D. de Semir & R. Maurisse

  4. Evergen Biotechnologies, 1392 Storrs Road, Unit 4213, Storrs, Conn., USA

    F. Du & J. Xu

  5. Institute for Regenerative Medicine, University of Connecticut, Storrs, Conn., USA

    X. Yang

  6. Children’s Hospital Oakland Research Institute, Oakland, Calif., USA

    B. Illek

  7. Department of Pediatrics, University of Vermont College of Medicine, Burlington, Vt., USA

    D. C. Gruenert

  8. Renova Life, 387 Technology Drive, College Park, Md., USA

    R. Maurisse

Authors
  1. D. de Semir
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. R. Maurisse
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. F. Du
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. J. Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. X. Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. B. Illek
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. D. C. Gruenert
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to D. C. Gruenert.

Additional information

We dedicate this manuscript to Dr. Xiangzhong Yang (deceased) whose enthusiasm and support was an inspiration during the work described in this manuscript.

This study was supported by NIH grant HL80814 and grants from the Cystic Fibrosis Foundation and from Pennsylvania Cystic Fibrosis. B.I. was also the recipient of a donation from the Folger Foundation. VRT-532, CFTR-inh172 and GlyH-101 were kindly provided to B.I. by the CFTR Compound Distribution Program of Cystic Fibrosis Foundation Therapeutics.

Rights and permissions

Reprints and permissions

About this article

Cite this article

de Semir, D., Maurisse, R., Du, F. et al. Generation of SV40-transformed rabbit tracheal-epithelial-cell-derived blastocyst by somatic cell nuclear transfer. Cell Tissue Res 347, 357–367 (2012). https://doi.org/10.1007/s00441-011-1296-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00441-011-1296-1

Keywords

Search

Navigation