Leishmania pp 123-140 | Cite as

Cosmid Library Construction and Functional Cloning

  • Joachim ClosEmail author
  • Dorothea Zander-Dinse
Part of the Methods in Molecular Biology book series (MIMB, volume 1971)


Cosmid libraries can represent an entire genome in a library of circular DNA molecules, allowing for the faithful amplification, cloning and isolation of large genomic DNA fragments. Moreover, using the so-called shuttle cosmid vectors, genomic DNA may be propagated in bacteria and in eukaryotic cells, which is a prerequisite for classic functional cloning and for the newly described Cos-Seq strategies.

Key words

High-quality gDNA extraction Cosmid library preparation Cosmid library evaluation Functional cloning 



We thank former coworkers Cornelia Hoyer, Katja Mellenthin, Andrea Nühs, Paloma Tejera Nevado, and Eugenia Bifeld for developing the technologies, John Kelly for the gift of pcosTL cosmid, and former and current members of the research group for discussions and suggestions.


  1. 1.
    Sollelis L, Ghorbal M, MacPherson CR, Martins RM, Kuk N, Crobu L, Bastien P, Scherf A, Lopez-Rubio JJ, Sterkers Y (2015) First efficient CRISPR-Cas9-mediated genome editing in Leishmania parasites. Cell Microbiol 17(10):1405–1412. Scholar
  2. 2.
    Zhang WW, Matlashewski G (2015) CRISPR-Cas9-mediated genome editing in Leishmania donovani. MBio 6(4):e00861. Scholar
  3. 3.
    Duncan SM, Myburgh E, Philipon C, Brown E, Meissner M, Brewer J, Mottram JC (2016) Conditional gene deletion with DiCre demonstrates an essential role for CRK3 in Leishmania mexicana cell cycle regulation. Mol Microbiol 100(6):931–944. Scholar
  4. 4.
    Ngo H, Tschudi C, Gull K, Ullu E (1998) Double-stranded RNA induces mRNA degradation in Trypanosoma brucei. Proc Natl Acad Sci U S A 95(25):14687–14692CrossRefGoogle Scholar
  5. 5.
    Ullu E, Tschudi C, Chakraborty T (2004) RNA interference in protozoan parasites. Cell Microbiol 6(6):509–519CrossRefGoogle Scholar
  6. 6.
    Lye LF, Owens K, Shi H, Murta SM, Vieira AC, Turco SJ, Tschudi C, Ullu E, Beverley SM (2010) Retention and loss of RNA interference pathways in trypanosomatid protozoans. PLoS Pathog 6(10):e1001161. Scholar
  7. 7.
    LeBowitz JH, Coburn CM, McMahon-Pratt D, Beverley SM (1990) Development of a stable Leishmania expression vector and application to the study of parasite surface antigen genes. Proc Natl Acad Sci U S A 87:9736–9740CrossRefGoogle Scholar
  8. 8.
    Sanderson SJ, Pollock KG, Hilley JD, Meldal M, Hilaire PS, Juliano MA, Juliano L, Mottram JC, Coombs GH (2000) Expression and characterization of a recombinant cysteine proteinase of Leishmania mexicana. Biochem J 347(Pt 2):383–388CrossRefGoogle Scholar
  9. 9.
    Chen DQ, Kolli BK, Yadava N, Lu HG, Gilman-Sachs A, Peterson DA, Chang KP (2000) Episomal expression of specific sense and antisense mRNAs in Leishmania amazonensis: modulation of gp63 level in promastigotes and their infection of macrophages in vitro. Infect Immun 68(1):80–86CrossRefGoogle Scholar
  10. 10.
    Kelly JM, Das P, Tomás AM (1994) An approach to functional complementation by introduction of large DNA fragments into Trypanosoma cruzi and Leishmania donovani using a cosmid shuttle vector. Mol Biochem Parasitol 65:51–62CrossRefGoogle Scholar
  11. 11.
    Ryan KA, Dasgupta S, Beverley SM (1993) Shuttle cosmid vectors for the trypanosomatid parasite Leishmania. Gene 131(1):145–150CrossRefGoogle Scholar
  12. 12.
    Ryan KA, Garraway LA, Descoteaux A, Turco SJ, Beverley SM (1993) Isolation of virulence genes directing surface glycosyl-phosphatidylinositol synthesis by functional complementation of Leishmania. Proc Natl Acad Sci U S A 90(18):8609–8613CrossRefGoogle Scholar
  13. 13.
    Turco S, Descoteaux A, Ryan K, Garraway L, Beverley S (1994) Isolation of virulence genes directing GPI synthesis by functional complementation of Leishmania. Braz J Med Biol Res 27(2):133–138PubMedGoogle Scholar
  14. 14.
    Beverley SM, Turco SJ (1995) Identification of genes mediating lipophosphoglycan biosynthesis by functional complementation of Leishmania donovani mutants. Ann Trop Med Parasitol 89(Suppl 1):11–17CrossRefGoogle Scholar
  15. 15.
    Descoteaux A, Luo Y, Turco SJ, Beverley SM (1995) A specialized pathway affecting virulence glycoconjugates of Leishmania. Science 269(5232):1869–1872CrossRefGoogle Scholar
  16. 16.
    Descoteaux A, Avila HA, Zhang K, Turco SJ, Beverley SM (2002) Leishmania LPG3 encodes a GRP94 homolog required for phosphoglycan synthesis implicated in parasite virulence but not viability. EMBO J 21(17):4458–4469CrossRefGoogle Scholar
  17. 17.
    Sambrook J, Russell DW (2001) Molecular cloning, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NYGoogle Scholar
  18. 18.
    Hübel A, Krobitsch S, Horauf A, Clos J (1997) Leishmania major Hsp100 is required chiefly in the mammalian stage of the parasite. Mol Cell Biol 17(10):5987–5995CrossRefGoogle Scholar
  19. 19.
    Reiling L, Chrobak M, Schmetz C, Clos J (2010) Overexpression of a single Leishmania major gene is sufficient to enhance parasite infectivity in vivo and in vitro. Mol Microbiol 76(5):1175–1190CrossRefGoogle Scholar
  20. 20.
    Nuhs A, Schafer C, Zander D, Trube L, Tejera Nevado P, Schmidt S, Arevalo J, Adaui V, Maes L, Dujardin JC, Clos J (2014) A novel marker, ARM58, confers antimony resistance to Leishmania spp. Int J Parasitol Drugs Drug Resist 4(1):37–47. Scholar
  21. 21.
    Laban A, Wirth DF (1989) Transfection of Leishmania enriettii and expression of chloramphenicol acetyltransferase gene. Proc Natl Acad Sci U S A 86:9119–9123CrossRefGoogle Scholar
  22. 22.
    Clos J, Hübel A, Brandau S, Dresel A, Hörauf A (1997) LeishmaniaHsp100. In: Gething M-J (ed) Guidebook to molecular chaperones and protein folding catalysts. Sambrook and Tooze, Oxford, pp 259–261Google Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Bernhard Nocht Institute for Tropical MedicineHamburgGermany

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