Skip to main content
SpringerLink
Log in

BacMam-Mediated Gene Delivery into Multipotent Mesenchymal Stromal Cells

  • Protocol
  • First Online:
Mesenchymal Stem Cell Assays and Applications

Part of the book series: Methods in Molecular Biology ((MIMB,volume 698))

Abstract

Baculoviruses have been used over the last several decades for high-level protein production in insect cells. Recently, modified baculovirus containing a mammalian promoter, known as BacMam virus, has been shown to give high transduction efficiencies across several cell types with minimal cytopathic effects. Cell types amenable to BacMam transduction include primary and adult stem cells. The shuttle vectors used in the construction of BacMam viruses can hold gene fragments up to 38 kb in size, and multiple BacMam viruses can be used in a single transduction for the delivery of more than one gene. BacMam technology has been used in the delivery and expression of targeted fluorescent protein cellular markers, small interfering RNAi, and extensively in the development of cell-based assays. BacMam offers an ideal method for the delivery and expression of large genes in hard-to-transfect cells such as primary and adult stem cells. In this chapter, we describe methods of generating high titer stocks of BacMam for transducing MSC and their derivatives.

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

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 149.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 189.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 219.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Caplan AI. (1991). Mesenchymal stem cells. Journal of Orthopaedic Research 9:641–650.

    Article  PubMed  CAS  Google Scholar 

  2. Noth U, AM Osyczka, R Tuli, NJ Hickok, KG Danielson and RS Tuan. (2002). Multilineage mesenchymal differentiation potential of human trabecular bone-derived cells. Journal of Orthopaedic Research 20:1060–1069.

    Article  PubMed  Google Scholar 

  3. Sarugaser R, D Lickorish, D Baksh, MM Hosseini and JE Davies. (2005). Human umbilical cord perivascular (HUCPV) cells: a source of mesenchymal progenitors. Stem Cells 23:220–229.

    Article  PubMed  Google Scholar 

  4. Tuan RS, G Boland and R Tuli. (2003). Adult mesenchymal stem cells and cell-based tissue engineering. Arthritis Research & Therapy 5:32–45.

    Article  CAS  Google Scholar 

  5. Awad HA, DL Butler, GP Boivin, FN Smith, P Malaviya, B Huibregtse and AI Caplan. (1999). Autologous mesenchymal stem cell-mediated repair of tendon. Tissue Engineering 5:267–277.

    Article  PubMed  CAS  Google Scholar 

  6. Bruder SP, N Jaiswal and SE Haynesworth. (1997). Growth kinetics, self-renewal, and the osteogenic potential of purified human mesenchymal stem cells during extensive subcultivation and following cryopreservation. Journal of Cellular Biochemistry 64:278–294.

    Article  PubMed  CAS  Google Scholar 

  7. Bruder SP, AA Kurth, M Shea, WC Hayes, N Jaiswal and S Kadiyala. (1998). Bone regeneration by implantation of purified, culture-expanded human mesenchymal stem cells. Journal of Orthopaedic Research 16:155–162.

    Article  PubMed  CAS  Google Scholar 

  8. Dennis JE, A Merriam, A Awadallah, JU Yoo, B Johnstone and AI Caplan. (1999). A quadripotential mesenchymal progenitor cell isolated from the marrow of an adult mouse. Journal of Bone and Mineral Research 14:700–709.

    Article  PubMed  CAS  Google Scholar 

  9. Ferrari G, G Cusella-De Angelis, M Coletta, E Paolucci, A Stornaiuolo, G Cossu and F Mavilio. (1998). Muscle regeneration by bone marrow-derived myogenic progenitors. Science 279:1528–1530.

    Article  PubMed  CAS  Google Scholar 

  10. Galmiche MC, VE Koteliansky, J Briere, P Herve and P Charbord. (1993). Stromal cells from human long-term marrow cultures are mesenchymal cells that differentiate following a vascular smooth muscle differentiation pathway. Blood 82:66–76.

    PubMed  CAS  Google Scholar 

  11. Pittenger MF, AM Mackay, SC Beck, RK Jaiswal, R Douglas, JD Mosca, MA Moorman, DW Simonetti, S Craig and DR Marshak. (1999). Multilineage potential of adult human mesenchymal stem cells. Science 284:143–147.

    Article  PubMed  CAS  Google Scholar 

  12. Young RG, DL Butler, W Weber, AI Caplan, SL Gordon and DJ Fink. (1998). Use of mesenchymal stem cells in a collagen matrix for Achilles tendon repair. Journal of Orthopaedic Research 16:406–413.

    Article  PubMed  CAS  Google Scholar 

  13. Horwitz EM, PL Gordon, WK Koo, JC Marx, MD Neel, RY McNall, L Muul and T Hofmann. (2002). Isolated allogeneic bone marrow-derived mesenchymal cells engraft and stimulate growth in children with osteogenesis imperfecta: implications for cell therapy of bone. Proceedings of the National Academy of Sciences of the United States of America 99:8932–8937.

    Article  PubMed  CAS  Google Scholar 

  14. Koc ON, SL Gerson, BW Cooper, SM Dyhouse, SE Haynesworth, AI Caplan and HM Lazarus. (2000). Rapid hematopoietic recovery after coinfusion of autologous-blood stem cells and culture-expanded marrow mesenchymal stem cells in advanced breast cancer patients receiving high-dose chemotherapy. Journal of Clinical Oncology 18:307–316.

    PubMed  CAS  Google Scholar 

  15. Petite H, V Viateau, W Bensaid, A Meunier, C de Pollak, M Bourguignon, K Oudina, L Sedel and G Guillemin. (2000). Tissue-engineered bone regeneration. Nature Biotechnology 18:959–963.

    Article  PubMed  CAS  Google Scholar 

  16. Hamada H, M Kobune, K Nakamura, Y Kawano, K Kato, O Honmou, K Houkin, T Matsunaga and Y Niitsu. (2005). Mesenchymal stem cells (MSC) as therapeutic cytoreagents for gene therapy. Cancer Science 96:149–156.

    Article  PubMed  CAS  Google Scholar 

  17. Ferreira E, E Potier, D Logeart-Avramoglou, S Salomskaite-Davalgiene, LM Mir and H Petite. (2008). Optimization of a gene electrotransfer method for mesenchymal stem cell transfection. Gene Therapy 15:537–544.

    Article  PubMed  CAS  Google Scholar 

  18. Bosch P and SL Stice. (2007). Adenoviral transduction of mesenchymal stem cells. Methods in Molecular Biology 407:265–274.

    Article  PubMed  CAS  Google Scholar 

  19. Kallifatidis G, BM Beckermann, A Groth, M Schubert, A Apel, A Khamidjanov, E Ryschich, T Wenger, W Wagner, A Diehlmann, R Saffrich, U Krause, V Eckstein, J Mattern, M Chai, G Schutz, AD Ho, MM Gebhard, MW Buchler, H Friess, P Buchler and I Herr. (2008). Improved lentiviral transduction of human mesenchymal stem cells for therapeutic intervention in pancreatic cancer. Cancer Gene Therapy 15:231–240.

    Article  PubMed  CAS  Google Scholar 

  20. Zhang XY, VF La Russa and J Reiser. (2004). Transduction of bone-marrow-derived mesenchymal stem cells by using lentivirus vectors pseudotyped with modified RD114 envelope glycoproteins. Journal of virology 78:1219–1229.

    Article  PubMed  CAS  Google Scholar 

  21. Uchibori R, T Okada, T Ito, M Urabe, H Mizukami, A Kume and K Ozawa. (2009). Retroviral vector-producing mesenchymal stem cells for targeted suicide cancer gene therapy. The Journal of Gene Medicine 11:373–381.

    Article  PubMed  CAS  Google Scholar 

  22. Lee HP, YC Ho, SM Hwang, LY Sung, HC Shen, HJ Liu and YC Hu. (2007). Variation of baculovirus-harbored transgene transcription among mesenchymal stem cell-derived progenitors leads to varied expression. Biotechnology and Bioengineering 97:649–655.

    Article  PubMed  CAS  Google Scholar 

  23. Kost TA and JP Condreay. (1999). Recombinant baculoviruses as expression vectors for insect and mammalian cells. Current Opinion in Biotechnology 10:428–433.

    Article  PubMed  CAS  Google Scholar 

  24. Summers MD. (2006). Milestones leading to the genetic engineering of baculoviruses as expression vector systems and viral pesticides. Advances in Virus Research 68:3–73.

    Article  PubMed  CAS  Google Scholar 

  25. Cheshenko N, N Krougliak, RC Eisensmith and VA Krougliak. (2001). A novel system for the production of fully deleted adenovirus vectors that does not require helper adenovirus. Gene Therapy 8:846–854.

    Article  PubMed  CAS  Google Scholar 

  26. Condreay JP, SM Witherspoon, WC Clay and TA Kost. (1999). Transient and stable gene expression in mammalian cells transduced with a recombinant baculovirus vector. Proceedings of the National Academy of Sciences of the United States of America 96:127–132.

    Article  PubMed  CAS  Google Scholar 

  27. Huser A and C Hofmann. (2003). Baculovirus vectors: novel mammalian cell gene-delivery vehicles and their applications. American Journal of Pharmacogenomics 3:53–63.

    Article  PubMed  CAS  Google Scholar 

  28. Merrihew RV, WC Clay, JP Condreay, SM Witherspoon, WS Dallas and TA Kost. (2001). Chromosomal integration of transduced recombinant baculovirus DNA in mammalian cells. Journal of Virology 75:903–909.

    Article  PubMed  CAS  Google Scholar 

  29. Kost TA and JP Condreay. (2002). Innovations-biotechnology: baculovirus vectors as gene transfer vectors for mammalian cells: biosafety considerations. Applied Biosafety 7:167–169.

    Google Scholar 

  30. Kost TA and JP Condreay. (2002). Recombinant baculoviruses as mammalian cell gene-delivery vectors. Trends in Biotechnology 20:173–180.

    Article  PubMed  CAS  Google Scholar 

  31. Kost TA, JP Condreay and DL Jarvis. (2005). Baculovirus as versatile vectors for protein expression in insect and mammalian cells. Nature Biotechnology 23:567–575.

    Article  PubMed  CAS  Google Scholar 

  32. Hu YC. (2008). Baculoviral vectors for gene delivery: a review. Current Gene Therapy 8:54–65.

    Article  PubMed  CAS  Google Scholar 

  33. Boyce FM and NL Bucher. (1996). Baculovirus-mediated gene transfer into mammalian cells. Proceedings of the National Academy of Sciences of the United States of America 93:2348–2352.

    Article  PubMed  CAS  Google Scholar 

  34. Ernst WJ, RM Grabherr and HW Katinger. (1994). Direct cloning into the Autographa californica nuclear polyhedrosis virus for generation of recombinant baculoviruses. Nucleic Acids Research 22:2855–2856.

    Article  PubMed  CAS  Google Scholar 

  35. Hartig PC, MC Cardon and CY Kawanishi. (1991). Generation of recombinant baculovirus via liposome-mediated transfection. BioTechniques 11:310, 312–313.

    Google Scholar 

  36. Murphy CI, H Piwnica-Worms, S Grunwald, WG Romanow, N Francis and HY Fan. (1997). Maintenance of insect cell cultures and generation of recombinant baculoviruses. Current Protocols in Molecular Biology (Ausubel FM., Brent R., Kingston RE., et al., Eds.) John Wiley & Sons, New York, pp. 16.10.1–16.10.17.

    Google Scholar 

  37. Karkkainen HR, HP Lesch, AI Maatta, PI Toivanen, AJ Mahonen, MM Roschier, KJ Airenne, OH Laitinen and S Yla-Herttuala. (2009). A 96-well format for a high-throughput baculovirus generation, fast titering and recombinant protein production in insect and mammalian cells. BMC Research Notes 2:63.

    Article  PubMed  Google Scholar 

  38. Hu YC. (2005). Baculovirus as a highly efficient expression vector in insect and mammalian cells. Acta Pharmacologica Sinica 26:405–416.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Primary and Stem Cell Systems, Life Technologies, Carlsbad, CA, USA

    Uma Lakshmipathy

Authors
  1. Michael O’Grady
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Robert H. Batchelor
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Kelly Scheyhing
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Christopher W. Kemp
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. George T. Hanson
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Uma Lakshmipathy
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Uma Lakshmipathy .

Editor information

Editors and Affiliations

  1. , Primary and Stem Cell Systems, Life Technologies, Frederick, 21704, USA

    Mohan Vemuri

  2. Cellular Dynamics International, Inc., Science Drive 525, Madison, 53711, Wisconsin, USA

    Lucas G. Chase

  3. Life Technologies, Executive Way 7335, Frederick, 21704, Maryland, USA

    Mahendra S. Rao

Rights and permissions

Reprints and permissions

Copyright information

© 2011 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

O’Grady, M., Batchelor, R.H., Scheyhing, K., Kemp, C.W., Hanson, G.T., Lakshmipathy, U. (2011). BacMam-Mediated Gene Delivery into Multipotent Mesenchymal Stromal Cells. In: Vemuri, M., Chase, L., Rao, M. (eds) Mesenchymal Stem Cell Assays and Applications. Methods in Molecular Biology, vol 698. Humana Press. https://doi.org/10.1007/978-1-60761-999-4_34

Download citation

  • DOI: https://doi.org/10.1007/978-1-60761-999-4_34

  • Published:

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-60761-998-7

  • Online ISBN: 978-1-60761-999-4

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation