25. If you count more than 300 cells, then a dilution is recommended. If you count less than 100 cells, the cell suspension
must be concentrated (e.g., by centrifugation and resuspension
of the remaining pellet in a smaller volume).
26. Trypan blue is toxic. Wear protective gloves, clothing, eye- and
face protection, when working with trypan blue.
27. Cells should be between 70% and 80% confluent to achieve an
optimal transduction.
28. Vortexing is not recommended.
29. If the virus titer is not determined, as rule of thumb the
required volume of the pseudotyped BV (P2 viral stock) solution is 5% (v/v) of the cell culture medium (e.g., 1 mL virus
solution per 20 mL cell culture medium).
Acknowledgments
F.E. was supported by the Heinrich Bo ¨ll Foundation. The authors
would like to thank the Hessen State Ministry of Higher Education, Research and the Arts for the financial support within the
Hessen initiative for scientific and economic excellence (LOEWEProgram, LOEWE ZIB (Center for Insect Biotechnology and
Bioresources). The authors acknowledge Catherine MeckelOschmann and Keven Lothert for revising the chapter.
References
1. Ginn SL, Amaya AK, Alexander IE et al
(2018) Gene therapy clinical trials worldwide
to 2017: an update. J Gene Med 20(5):
e3015. https://doi.org/10.1002/jgm.3015
2. Wirth T, Parker N, Yl€ a-Herttuala S (2013)
History of gene therapy. Gene 525
(2):162–169.
https://doi.org/10.1016/j.
gene.2013.03.137
3. Gardlı ´k R, Pa ´lffy R, Hodosy J et al (2005)
Vectors and delivery systems in gene therapy.
Med Sci Monit 11(4):RA110–RA121
4. Ibraheem D, Elaissari A, Fessi H (2014) Gene
therapy and DNA delivery systems. Int J
Pharm 459(1–2):70–83. https://doi.org/
10.1016/j.ijpharm.2013.11.041
5. Somia N, Verma IM (2000) Gene therapy:
trials and tribulations. Nat Rev Genet 1
(2):91–99.
https://doi.org/10.1038/
35038533
6. Wold WSM, Toth K (2013) Adenovirus vectors for gene therapy, vaccination and cancer
gene therapy. Curr Gene Ther 13
(6):421–433
7. Naso MF, Tomkowicz B, Perry WL et al
(2017) Adeno-associated virus (AAV) as a
vector for gene therapy. BioDrugs 31
(4):317–334.
https://doi.org/10.1007/
s40259-017-0234-5
8. Yi Y, Jong Noh M, Hee Lee K (2011) Current
advances in retroviral gene therapy. Curr Gene
Ther 11(3):218–228. https://doi.org/10.
2174/156652311795684740
9. 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
10. Merten O-W, Gaillet B (2016) Viral vectors
for gene therapy and gene modification
approaches. Biochem Eng J 108:98–115.
https://doi.org/10.1016/j.bej.2015.09.005
11. Ayres MD, Howard SC, Kuzio J et al (1994)
The complete DNA sequence of Autographa
californica nuclear polyhedrosis virus. Virology 202(2):586–605. https://doi.org/10.
1006/viro.1994.1380
Baculovirus-Mediated Gene Transfer to Stem Cells
385
must be concentrated (e.g., by centrifugation and resuspension
of the remaining pellet in a smaller volume).
26. Trypan blue is toxic. Wear protective gloves, clothing, eye- and
face protection, when working with trypan blue.
27. Cells should be between 70% and 80% confluent to achieve an
optimal transduction.
28. Vortexing is not recommended.
29. If the virus titer is not determined, as rule of thumb the
required volume of the pseudotyped BV (P2 viral stock) solution is 5% (v/v) of the cell culture medium (e.g., 1 mL virus
solution per 20 mL cell culture medium).
Acknowledgments
F.E. was supported by the Heinrich Bo ¨ll Foundation. The authors
would like to thank the Hessen State Ministry of Higher Education, Research and the Arts for the financial support within the
Hessen initiative for scientific and economic excellence (LOEWEProgram, LOEWE ZIB (Center for Insect Biotechnology and
Bioresources). The authors acknowledge Catherine MeckelOschmann and Keven Lothert for revising the chapter.
References
1. Ginn SL, Amaya AK, Alexander IE et al
(2018) Gene therapy clinical trials worldwide
to 2017: an update. J Gene Med 20(5):
e3015. https://doi.org/10.1002/jgm.3015
2. Wirth T, Parker N, Yl€ a-Herttuala S (2013)
History of gene therapy. Gene 525
(2):162–169.
https://doi.org/10.1016/j.
gene.2013.03.137
3. Gardlı ´k R, Pa ´lffy R, Hodosy J et al (2005)
Vectors and delivery systems in gene therapy.
Med Sci Monit 11(4):RA110–RA121
4. Ibraheem D, Elaissari A, Fessi H (2014) Gene
therapy and DNA delivery systems. Int J
Pharm 459(1–2):70–83. https://doi.org/
10.1016/j.ijpharm.2013.11.041
5. Somia N, Verma IM (2000) Gene therapy:
trials and tribulations. Nat Rev Genet 1
(2):91–99.
https://doi.org/10.1038/
35038533
6. Wold WSM, Toth K (2013) Adenovirus vectors for gene therapy, vaccination and cancer
gene therapy. Curr Gene Ther 13
(6):421–433
7. Naso MF, Tomkowicz B, Perry WL et al
(2017) Adeno-associated virus (AAV) as a
vector for gene therapy. BioDrugs 31
(4):317–334.
https://doi.org/10.1007/
s40259-017-0234-5
8. Yi Y, Jong Noh M, Hee Lee K (2011) Current
advances in retroviral gene therapy. Curr Gene
Ther 11(3):218–228. https://doi.org/10.
2174/156652311795684740
9. 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
10. Merten O-W, Gaillet B (2016) Viral vectors
for gene therapy and gene modification
approaches. Biochem Eng J 108:98–115.
https://doi.org/10.1016/j.bej.2015.09.005
11. Ayres MD, Howard SC, Kuzio J et al (1994)
The complete DNA sequence of Autographa
californica nuclear polyhedrosis virus. Virology 202(2):586–605. https://doi.org/10.
1006/viro.1994.1380
Baculovirus-Mediated Gene Transfer to Stem Cells
385