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Generation of Stable Cell Lines Expressing Golgi Reassembly Stacking Proteins (GRASPs) by Viral Transduction

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 2557))

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Abstract

Stable cell lines that express a gene of specific interest provide an advantage over transient gene expression by reducing variations in transfection efficiency between experiments, sustaining expression for long-term studies, and controlling expression levels in particular if a clonal population is selected. Transient transfection requires introduction of an exogenous gene into host cells via typically harsh chemicals or conditions that permeabilize the cell membrane, which does not normally integrate into the target cell genome. Here, we describe the method of using retroviral transduction to stably express Golgi proteins fused to a promiscuous biotin ligase (TurboID) in HeLa cells, thus creating cell lines that can be leveraged in studies of the proximome/interactome. We also demonstrate a similar protocol for stable expression of a Golgi protein fused to a fluorescent tag via lentiviral transduction. These methods can be further adapted to establish other cell lines with different sub-cellular markers or fusion tags. Viral transduction is a convenient method to create stable cell lines in cell-based studies.

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References

  1. Wang D, Tai P, Gao G (2019) Adeno-associated virus vector as a platform for gene therapy delivery. Nat Rev Drug Discov 18(5). https://doi.org/10.1038/s41573-019-0012-9

  2. Milone MC, O’Doherty U (2018) Clinical use of lentiviral vectors. Leukemia 32(7):1529–1541. https://doi.org/10.1038/s41375-018-0106-0

    Article  CAS  Google Scholar 

  3. Bekier M, Wang L, Li J, Huang H, Tang D, Zhang X, Wang Y (2017) Knockout of the Golgi stacking proteins GRASP55 and GRASP65 impairs Golgi structure and function. Mol Biol Cell 28(21). https://doi.org/10.1091/mbc.E17-02-0112

  4. Hindmarsh P, Leis J (1999) Retroviral DNA integration. Microbiol Mol Biol Rev 63(4). https://doi.org/10.1128/MMBR.63.4.836-843.1999

  5. Bouard D, Alazard-Dany D, Cosset F (2009) Viral vectors: from virology to transgene expression. Br J Pharmacol 157(2). https://doi.org/10.1038/bjp.2008.349

  6. Joglekar A, Sandoval S (2017) Pseudotyped lentiviral vectors: one vector, many guises. Hum Gene Ther Methods 28(6). https://doi.org/10.1089/hgtb.2017.084

  7. Balvay L, Lopez LM, Sargueil B, Darlix J, Ohlmann T (2007) Translational control of retroviruses. Nat Rev Microbiol 5(2). https://doi.org/10.1038/nrmicro1599

  8. Bekier ME 2nd, Wang L, Li J, Huang H, Tang D, Zhang X, Wang Y (2017) Knockout of the Golgi stacking proteins GRASP55 and GRASP65 impairs Golgi structure and function. Mol Biol Cell 28(21):2833–2842. https://doi.org/10.1091/mbc.E17-02-0112

    Article  CAS  Google Scholar 

  9. Davis H, Morgan J, Yarmush M (2002) Polybrene increases retrovirus gene transfer efficiency by enhancing receptor-independent virus adsorption on target cell membranes. Biophys Chem 97(2–3). https://doi.org/10.1016/s0301-4622(02)00057-1

  10. Moore J, Trkola A, Dragic T (1997) Co-receptors for HIV-1 entry. Curr Opin Immunol 9(4). https://doi.org/10.1016/s0952-7915(97)80110-0

  11. Kafri T, Blömer U, Peterson D, Gage F, Verma I (1997) Sustained expression of genes delivered directly into liver and muscle by lentiviral vectors. Nat Genet 17(3). https://doi.org/10.1038/ng1197-314

  12. Peng K, Pham L, Ye H, Zufferey R, Trono D, Cosset F, Russell S (2001) Organ distribution of gene expression after intravenous infusion of targeted and untargeted lentiviral vectors. Gene Ther 8(19). https://doi.org/10.1038/sj.gt.3301552

  13. Sakuma T, De Ravin S, Tonne J, Thatava T, Ohmine S, Takeuchi Y, Malech H, Ikeda Y (2010) Characterization of retroviral and lentiviral vectors pseudotyped with xenotropic murine leukemia virus-related virus envelope glycoprotein. Hum Gene Ther 21(12). https://doi.org/10.1089/hum.2010.063

  14. Diaz R, Bateman A, Emiliusen L, Fielding A, Trono D, Russell S, Vile R (2000) A lentiviral vector expressing a fusogenic glycoprotein for cancer gene therapy. Gene Ther 7(19). https://doi.org/10.1038/sj.gt.3301277

  15. Jang J, Shaw K, Yu X, Petersen D, Pepper K, Lutzko C, Kohn D (2006) Specific and stable gene transfer to human embryonic stem cells using pseudotyped lentiviral vectors. Stem Cells Dev 15(1). https://doi.org/10.1089/scd.2006.15.109

  16. Bell A, Fegen D, Ward M, Bank A (2010) RD114 envelope proteins provide an effective and versatile approach to pseudotype lentiviral vectors. Exp Biol Med (Maywood, NJ) 235(10). https://doi.org/10.1258/ebm.2010.010053

  17. Hale C, Majumdar S, Elmore J, Pfister N, Compton M, Olson S, Resch A, Glover C, Graveley B, Terns R, Terns M (2012) Essential features and rational design of CRISPR RNAs that function with the Cas RAMP module complex to cleave RNAs. Mol Cell 45(3). https://doi.org/10.1016/j.molcel.2011.10.023

  18. Schambach A, Galla M, Modlich U, Will E, Chandra S, Reeves L, Colbert M, Williams D, von Kalle C, Baum C (2006) Lentiviral vectors pseudotyped with murine ecotropic envelope: increased biosafety and convenience in preclinical research. Exp Hematol 34(5). https://doi.org/10.1016/j.exphem.2006.02.005

  19. Hanawa H, Kelly P, Nathwani A, Persons D, Vandergriff J, Hargrove P, Vanin E, Nienhuis A (2002) Comparison of various envelope proteins for their ability to pseudotype lentiviral vectors and transduce primitive hematopoietic cells from human blood. Mol Ther 5(3). https://doi.org/10.1006/mthe.2002.0549

  20. Janssens W, Chuah M, Naldini L, Follenzi A, Collen D, Saint-Remy J, VandenDriessche T (2003) Efficiency of onco-retroviral and lentiviral gene transfer into primary mouse and human B-lymphocytes is pseudotype dependent. Hum Gene Ther 14(3). https://doi.org/10.1089/10430340360535814

  21. Levy C, Fusil F, Amirache F, Costa C, Girard-Gagnepain A, Negre D, Bernadin O, Garaulet G, Rodriguez A, Nair N, Vandendriessche T, Chuah M, Cosset F, Verhoeyen E (2016) Baboon envelope pseudotyped lentiviral vectors efficiently transduce human B cells and allow active factor IX B cell secretion in vivo in NOD/SCIDγc −/− mice. J Thromb Haemost 14(12). https://doi.org/10.1111/jth.13520

  22. Uhlig KM, Schülke S, Scheuplein VAM, Malczyk AH, Reusch J, Kugelmann S, Muth A, Koch V, Hutzler S, Bodmer BS, Schambach A, Buchholz CJ, Waibler Z, Scheurer S, Mühlebach MD, Kirchhoff F (2015) Lentiviral protein transfer vectors are an efficient vaccine platform and induce a strong antigen-specific xytotoxic T cell response. J Virol 89(17):9044–9060. https://doi.org/10.1128/JVI.00844-15

    Article  CAS  Google Scholar 

  23. Zhou Q, Schneider I, Gallet M, Kneissl S, Buchholz C (2011) Resting lymphocyte transduction with measles virus glycoprotein pseudotyped lentiviral vectors relies on CD46 and SLAM. Virology 413(2). https://doi.org/10.1016/j.virol.2011.02.010

  24. Witting SR, Vallanda P, Gamble AL (2013) Characterization of a third generation lentiviral vector pseudotyped with Nipah virus envelope proteins for endothelial cell transduction. Gene Therapy 20(10):997–1005. https://doi.org/10.1038/gt.2013.23

    Article  CAS  Google Scholar 

  25. Palomares K, Vigant F, Handel BV, Pernet O, Chikere K, Hong P, Sherman SP, Patterson M, An DS, Lowry WE, Mikkola HKA, Morizono K, Pyle AD, Lee B (2013) Nipah virus envelope-pseudotyped lentiviruses efficiently target ephrinB2-positive stem cell populations in vitro and bypass the liver sink when administered in vivo. J Virol 87(4):2094–2108. https://doi.org/10.1128/JVI.02032-12

    Article  CAS  Google Scholar 

  26. Carpentier DCJ, Vevis K, Trabalza A, Georgiadis C, Ellison SM, Asfahani RI, Mazarakis ND (2011) Enhanced pseudotyping efficiency of HIV-1 lentiviral vectors by a rabies/vesicular stomatitis virus chimeric envelope glycoprotein. Gene Therapy 19(7):761–774. https://doi.org/10.1038/gt.2011.124

    Article  CAS  Google Scholar 

  27. Cronin J, Zhang X, Reiser J (2005) Altering the tropism of lentiviral vectors through pseudotyping. Curr Gene Ther 5(4). https://doi.org/10.2174/1566523054546224

  28. Kato S, Kobayashi K, Kobayashi K (2014) Improved transduction efficiency of a lentiviral vector for neuron-specific retrograde gene transfer by optimizing the junction of fusion envelope glycoprotein, J Neurosci Methods 227. https://doi.org/10.1016/j.jneumeth.2014.02.015

  29. Hachiya A, Sriwiriyanont P, Patel A, Saito N, Ohuchi A, Kitahara T, Takema Y, Tsuboi R, Boissy RE, Visscher MO, James WM, Kobinger GP (2007) Gene transfer in human skin with different pseudotyped HIV-based vectors. Gene Ther 14(8):648–656. https://doi.org/10.1038/sj.gt.3302915

    Article  CAS  Google Scholar 

  30. Colin A, Faideau M, Dufour N, Auregan G, Hassig R, Andrieu T, Brouillet E, Hantraye P, Bonvento G, Déglon N (2009) Engineered lentiviral vector targeting astrocytes in vivo. Glia 57(6). https://doi.org/10.1002/glia.20795

  31. Sinn P, Coffin J, Ayithan N, Holt K, Maury W (2017) Lentiviral Vectors Pseudotyped with Filoviral Glycoproteins. Methods in molecular biology (Clifton, NJ) 1628. https://doi.org/10.1007/978-1-4939-7116-9_5

  32. Dylla DE, Xie L, Michele DE, Kunz S, McCray PB (2011) Altering α-dystroglycan receptor affinity of LCMV pseudotyped lentivirus yields unique cell and tissue tropism. Genet Vaccines Ther 9(1):1–13. https://doi.org/10.1186/1479-0556-9-8

    Article  CAS  Google Scholar 

  33. Kumar M, Bradow BP, Zimmerberg J (2004) Large-scale production of pseudotyped lentiviral vectors using baculovirus GP64. https://home.liebertpub.com/hum. https://doi.org/10.1089/10430340360464723

  34. Kahl CA, Marsh J, Fyffe J, Sanders DA, Cornetta K (2004) Human immunodeficiency virus type 1-derived lentivirus vectors pseudotyped with envelope glycoproteins derived from Ross River virus and Semliki Forest virus. J Virol 78(3):1421–1430. https://doi.org/10.1128/JVI.78.3.1421-1430.2004

    Article  CAS  Google Scholar 

  35. Salvador B, Zhou Y, Michault A, Muench M, Simmons G (2009) Characterization of Chikungunya pseudotyped viruses: identification of refractory cell lines and demonstration of cellular tropism differences mediated by mutations in E1 glycoprotein. Virology 393(1). https://doi.org/10.1016/j.virol.2009.07.013

  36. Kang Y, Stein C, Heth J, Sinn P, Penisten A, Staber P, Ratliff K, Shen H, Barker C, Martins I, Sharkey C, Sanders D, McCray P, Davidson B (2002) In vivo gene transfer using a nonprimate lentiviral vector pseudotyped with Ross River Virus glycoproteins. J Virol 76(18). https://doi.org/10.1128/jvi.76.18.9378-9388.2002

  37. Poluri A, Ainsworth R, Weaver SC, Sutton RE (2008) Functional pseudotyping of human immunodeficiency virus type 1 vectors by western equine encephalitis virus envelope glycoprotein. https://doi.org/10.1128/JVI.01503-08

  38. McKay T, Patel M, Pickles RJ, Johnson LG, Olsen JC (2006) Influenza M2 envelope protein augments avian influenza hemagglutinin pseudotyping of lentiviral vectors. Gene Ther 13(8):715–724. https://doi.org/10.1038/sj.gt.3302715

    Article  CAS  Google Scholar 

  39. Patel M, Giddings A, Sechelski J, Olsen J (2013) High efficiency gene transfer to airways of mice using influenza hemagglutinin pseudotyped lentiviral vectors. J Gene Med 15(1). https://doi.org/10.1002/jgm.2695

  40. Burns JC, Friedmann T, Driever W, Burrascano M, Yee JK (1993) Vesicular stomatitis virus G glycoprotein pseudotyped retroviral vectors: concentration to very high titer and efficient gene transfer into mammalian and nonmammalian cells. Proc Natl Acad Sci U S A 90(17):8033–8037. https://doi.org/10.1073/pnas.90.17.8033

    Article  CAS  Google Scholar 

  41. Wong L, Azzouz M, Walmsley L, Askham Z, Wilkes F, Mitrophanous K, Kingsman S, Mazarakis N (2004) Transduction patterns of pseudotyped lentiviral vectors in the nervous system. Mol Ther 9(1). https://doi.org/10.1016/j.ymthe.2003.09.017

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Authors and Affiliations

  1. Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, MI, USA

    Sarah Bui, Jie Li, Drew Stark, Ali Houmani & Yanzhuang Wang

  2. Department of Neurology, University of Michigan School of Medicine, Ann Arbor, MI, USA

    Yanzhuang Wang

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  1. Sarah Bui
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  2. Jie Li
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  3. Drew Stark
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  4. Ali Houmani
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  5. Yanzhuang Wang
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Correspondence to Yanzhuang Wang .

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Editors and Affiliations

  1. Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, MI, USA

    Yanzhuang Wang

  2. Department of Physiology and Cell Biology, University of Arkansas for Medical Sciences, Little Rock, AR, USA

    Vladimir V Lupashin

  3. Department of Biological Sciences, Vanderbilt University, Nashville, TN, USA

    Todd R. Graham

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Bui, S., Li, J., Stark, D., Houmani, A., Wang, Y. (2023). Generation of Stable Cell Lines Expressing Golgi Reassembly Stacking Proteins (GRASPs) by Viral Transduction. In: Wang, Y., Lupashin, V.V., Graham, T.R. (eds) Golgi. Methods in Molecular Biology, vol 2557. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2639-9_24

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  • DOI: https://doi.org/10.1007/978-1-0716-2639-9_24

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2638-2

  • Online ISBN: 978-1-0716-2639-9

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