Molecular Genetics and Genomics

, Volume 287, Issue 4, pp 313–324 | Cite as

Triple mammalian/yeast/bacterial shuttle vectors for single and combined Lentivirus- and Sindbis virus-mediated infections of neurons

  • Lidia Bakota
  • Roland Brandt
  • Jürgen J. Heinisch
Original Paper


Today, a large variety of viral vectors is available for ectopic gene expression in mammalian cell cultures or in vivo. Among them, infection with Sindbis virus- or Lentivirus-derived constructs is often used to address biological questions or for applications in neuronal therapies. However, cloning of genes of interest is time consuming, since it relies on restriction and ligation, frequently of PCR-generated DNA fragments with suitable restriction sites introduced by the primers employed. We here take advantage of the unusually high capacity for homologous recombination in Saccharomyces cerevisiae to circumvent this problem, and introduce a new set of triple shuttle vectors, which can be shuffled between E. coli, yeast, and mammalian cells. The system allows the introduction of genes of interest largely independent of the target site in the vectors. It also allows the removal of the yeast selection marker by Cre-recombinase directed recombination in E. coli, if vector size limits transfection efficiency in the mammalian cells. We demonstrate the expression of genes encoding fluorescent proteins (EGFP and mCherry) both separately and in combination, using two different viral systems in mammalian cell lines, primary neurons and organotypic slices.


Saccharomyces cerevisiae In vivo recombination Viral vectors Fluorescent protein fusions 



We thank Andrea Murra for excellent technical assistance. We are also indebted to Sondra Schlesinger (Washington, MO), Knut Jahreis (Osnabrück, Germany), Michael Knop (Heidelberg, Germany), Pavel Osten (Chicago, IL) and Roel Nusse (Stanford, CA) for providing vectors and plasmids. Funds have been provided by the Deutsche Forschungsgemeinschaft (SFB 944) to R.B. and J.J.H., and (DFG BR1192/11-2) to R.B.


  1. Breckpot K, Emeagi PU, Thielemans K (2008) Lentiviral vectors for anti-tumor immunotherapy. Curr Gene Ther 8(6):438–448PubMedCrossRefGoogle Scholar
  2. Cockrell AS, Kafri T (2007) Gene delivery by Lentivirus vectors. Mol Biotechnol 36(3):184–204 (pii:MB:36:3:184)PubMedCrossRefGoogle Scholar
  3. Dittgen T, Nimmerjahn A, Komai S, Licznerski P, Waters J, Margrie TW, Helmchen F, Denk W, Brecht M, Osten P (2004) Lentivirus-based genetic manipulations of cortical neurons and their optical and electrophysiological monitoring in vivo. Proc Natl Acad Sci USA 101(52):18206–18211. doi: 10.1073/pnas.0407976101 PubMedCrossRefGoogle Scholar
  4. Fuerer C, Nusse R (2010) Lentiviral vectors to probe and manipulate the Wnt signaling pathway. PLoS One 5(2):e9370. doi: 10.1371/journal.pone.0009370 PubMedCrossRefGoogle Scholar
  5. Heinisch JJ (1993) PFK2, ISP42, ERG2 and RAD14 are located on the right arm of chromosome XIII. Yeast 9(10):1103–1105PubMedCrossRefGoogle Scholar
  6. Heinisch JJ, Buchwald U, Gottschlich A, Heppeler N, Rodicio R (2010) A tool kit for molecular genetics of Kluyveromyces lactis comprising a congenic strain series and a set of versatile vectors. FEMS Yeast Res 10(3):333–342. doi: 10.1111/j.1567-1364.2009.00604.x PubMedCrossRefGoogle Scholar
  7. Hill JE, Myers AM, Koerner TJ, Tzagoloff A (1986) Yeast/E. coli shuttle vectors with multiple unique restriction sites. Yeast 2(3):163–167. doi: 10.1002/yea.320020304 PubMedCrossRefGoogle Scholar
  8. Hua SB, Qiu M, Chan E, Zhu L, Luo Y (1997) Minimum length of sequence homology required for in vivo cloning by homologous recombination in yeast. Plasmid 38(2):91–96. doi: 10.1006/plas.1997.1305 PubMedCrossRefGoogle Scholar
  9. Kirchrath L, Lorberg A, Schmitz HP, Gengenbacher U, Heinisch JJ (2000) Comparative genetic and physiological studies of the MAP kinase Mpk1p from Kluyveromyces lactis and Saccharomyces cerevisiae. J Mol Biol 300(4):743–758PubMedCrossRefGoogle Scholar
  10. Krupka N, Strappe P, Gotz J, Ittner LM (2010) Gateway-compatible lentiviral transfer vectors for ubiquitin promoter driven expression of fluorescent fusion proteins. Plasmid 63(3):155–160. doi: 10.1016/j.plasmid.2010.01.002 PubMedCrossRefGoogle Scholar
  11. Leschik J, Welzel A, Weissmann C, Eckert A, Brandt R (2007) Inverse and distinct modulation of tau-dependent neurodegeneration by presenilin 1 and amyloid-beta in cultured cortical neurons: evidence that tau phosphorylation is the limiting factor in amyloid-beta-induced cell death. J Neurochem 101(5):1303–1315. doi: 10.1111/j.1471-4159.2006.04435.x PubMedCrossRefGoogle Scholar
  12. Lundberg C, Bjorklund T, Carlsson T, Jakobsson J, Hantraye P, Deglon N, Kirik D (2008) Applications of lentiviral vectors for biology and gene therapy of neurological disorders. Curr Gene Ther 8(6):461–473PubMedCrossRefGoogle Scholar
  13. Ma H, Kunes S, Schatz PJ, Botstein D (1987) Plasmid construction by homologous recombination in yeast. Gene 58:201–216PubMedCrossRefGoogle Scholar
  14. Maeder CI, Hink MA, Kinkhabwala A, Mayr R, Bastiaens PI, Knop M (2007) Spatial regulation of Fus3 MAP kinase activity through a reaction-diffusion mechanism in yeast pheromone signalling. Nat Cell Biol 9(11):1319–1326. doi: 10.1038/ncb1652 PubMedCrossRefGoogle Scholar
  15. Marfori M, Mynott A, Ellis JJ, Mehdi AM, Saunders NF, Curmi PM, Forwood JK, Boden M, Kobe B (2011) Molecular basis for specificity of nuclear import and prediction of nuclear localization. Biochim Biophys Acta 1813(9):1562–1577. doi: 10.1016/j.bbamcr.2010.10.013 PubMedCrossRefGoogle Scholar
  16. Martinez E, Bartolome B, de la Cruz F (1988) pACYC184-derived cloning vectors containing the multiple cloning site and lacZ alpha reporter gene of pUC8/9 and pUC18/19 plasmids. Gene 68(1):159–162 (pii:0378-1119(88)90608-7)PubMedCrossRefGoogle Scholar
  17. Naldini L, Blomer U, Gallay P, Ory D, Mulligan R, Gage FH, Verma IM, Trono D (1996) In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science 272(5259):263–267PubMedCrossRefGoogle Scholar
  18. Osten P, Dittgen T, Licznerski P (2006) Lentivirus-based genetic manipulations in neurons in vivo. In: Kittler JT, Moss SJ (eds) The dynamic synapse: molecular methods in ionotropic receptor biology. CRC Press, Boca RatonGoogle Scholar
  19. Prado F, Aguilera A (1994) New in vivo cloning methods by homologous recombination in yeast. Curr Genet 25:180–183PubMedCrossRefGoogle Scholar
  20. Rheme C, Ehrengruber MU, Grandgirard D (2005) Alphaviral cytotoxicity and its implication in vector development. Exp Physiol 90(1):45–52. doi: 10.1113/expphysiol.2004.028142 PubMedCrossRefGoogle Scholar
  21. Rodicio R, Koch S, Schmitz HP, Heinisch JJ (2006) KlRHO1 and KlPKC1 are essential for cell integrity signalling in Kluyveromyces lactis. Microbiology 152(Pt 9):2635–2649PubMedCrossRefGoogle Scholar
  22. Rothstein RJ (1983) One-step gene disruption in yeast. Methods Enzymol 101:202–211PubMedCrossRefGoogle Scholar
  23. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning—a laboratory manual. Cold Spring Harbor Laboratory Press, New YorkGoogle Scholar
  24. Shahani N, Subramaniam S, Wolf T, Tackenberg C, Brandt R (2006) Tau aggregation and progressive neuronal degeneration in the absence of changes in spine density and morphology after targeted expression of Alzheimer’s disease-relevant tau constructs in organotypic hippocampal slices. J Neurosci 26(22):6103–6114. doi: 26/22/6103[pii]10.1523/JNEUROSCI.4245-05.2006 PubMedCrossRefGoogle Scholar
  25. Shaner NC, Steinbach PA, Tsien RY (2005) A guide to choosing fluorescent proteins. Nat Methods 2(12):905–909. doi: 10.1038/nmeth819 PubMedCrossRefGoogle Scholar
  26. Shao Z, Zhao H, Zhao H (2009) DNA assembler, an in vivo genetic method for rapid construction of biochemical pathways. Nucleic Acids Res 37(2):e16. doi: 10.1093/nar/gkn991 PubMedCrossRefGoogle Scholar
  27. Sherman F, Fink GR, Hicks JB (1986) Laboratory course manual for methods in yeast genetics. Cold Spring Harbor Laboratory, New YorkGoogle Scholar
  28. Stoppini L, Buchs PA, Muller D (1991) A simple method for organotypic cultures of nervous tissue. J Neurosci Methods 37(2):173–182 (pii:0165-0270(91)90128-M)PubMedCrossRefGoogle Scholar
  29. Tackenberg C, Brandt R (2009) Divergent pathways mediate spine alterations and cell death induced by amyloid-beta, wild-type tau, and R406W tau. J Neurosci 29(46):14439–14450. doi: 10.1523/JNEUROSCI.3590-09.2009 PubMedCrossRefGoogle Scholar
  30. Weissmann C, Reyher HJ, Gauthier A, Steinhoff HJ, Junge W, Brandt R (2009) Microtubule binding and trapping at the tip of neurites regulate tau motion in living neurons. Traffic 10(11):1655–1668. doi: 10.1111/j.1600-0854.2009.00977.x PubMedCrossRefGoogle Scholar
  31. Wendland J (2003) PCR-based methods facilitate targeted gene manipulations and cloning procedures. Curr Genet 44(3):115–123. doi: 10.1007/s00294-003-0436-x PubMedCrossRefGoogle Scholar
  32. Zhu ZH, Chen SS, Huang AS (1990) Phenotypic mixing between human immunodeficiency virus and vesicular stomatitis virus or herpes simplex virus. J Acquir Immune Defic Syndr 3(3):215–219PubMedGoogle Scholar
  33. Zufferey R, Nagy D, Mandel RJ, Naldini L, Trono D (1997) Multiply attenuated lentiviral vector achieves efficient gene delivery in vivo. Nat Biotechnol 15(9):871–875. doi: 10.1038/nbt0997-871 PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Lidia Bakota
    • 1
  • Roland Brandt
    • 1
  • Jürgen J. Heinisch
    • 2
  1. 1.Department of Neurobiology, Faculty of Biology/ChemistryUniversity of OsnabrückOsnabrückGermany
  2. 2.Department of Genetics, Faculty of Biology/ChemistryUniversity of OsnabrückOsnabrückGermany

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