during cell culture to ensure sufficient numbers of cells to
conduct the transfection experiments across all the associated
vectors and controls.
11. MOI indicates the ratio of bacteria to macrophage.
12. Due to the risk associated with disturbing the cells, we recommend removing the media via gentle aspiration. This can be
achieved by attaching a sterile glass pipette to a vacuum line
and carefully removing the media from a corner of the well or a
similar location that is away from the majority of cells.
13. With a higher MOI, reduced macrophage viability is expected
due to the greater bacterial load negatively influencing the
coculture environment. Thus, through LyE expression, the
experiment will reveal to what extent attenuation of the bacterial vectors affects this trend.
Acknowledgments
The authors recognize support from NIH awards AI088485 and
AI117309 and a grant from the Technology Accelerator Fund from
the State University of New York Research Foundation (all to
BAP).
References
1. Courvalin P, Goussard S, Grillot-Courvalin C
et al (1995) Gene transfer from bacteria to
mammalian cells. C R Acad Sci III
318:1207–1212
2. Grillot-Courvalin C et al (1998) Functional
gene transfer from intracellular bacteria to
mammalian cells. Nat Biotechnol 16(9):862
3. Sizemore DR, Branstrom A, Sadoff J (1995)
Attenuated Shigella as a DNA delivery vehicle
for DNA-mediated immunization. Science
270:299–302
4. Grillot-Courvalin C, Goussard S, Courvalin P
(2002) Wild-type intracellular bacteria deliver
DNA into mammalian cells. Cell Microbiol
4:177–186
5. Al-Mariri A, Tibor A et al (2002) Yersinia
enterocolitica as a vehicle for a naked DNA
vaccine encoding Brucella abortus bacterioferritin or P39 antigen. Infect Immun
70:1915–1923
6. Gardlik R, Behuliak M, Palffy R, Celec P, Li C
(2011) Gene therapy for cancer: bacteriamediated anti-angiogenesis therapy. Gene
Ther 18:425–431
7. Steinman RM (2001) Dendritic cells and the
control of immunity: enhancing the efficiency
of antigen presentation. Mt Sinai J Med
68:160–166
8. Birmingham CL, Canadien V, Kaniuk NA et al
(2008) Listeriolysin O allows Listeria monocytogenes replication in macrophage vacuoles.
Nature 451:350–354
9. Palffy R, Gardlik R, Hodosy J et al (2006)
Bacteria in gene therapy: bactofection versus
alternative gene therapy. Gene Ther
13:101–105
10. Chung TC, Jones CH, Gollakota A et al
(2015) Improved Escherichia coli Bactofection
and cytotoxicity by heterologous expression of
bacteriophage PhiX174 lysis gene E. Mol
Pharm 12:1691–1700
11. Parsa S, Wang Y, Fuller J (2008) A comparison
between polymeric microsphere and bacterial
vectors for macrophage P388D1 gene delivery.
Pharm Res 25:1202–1208
14
Dongwon Park et al.
conduct the transfection experiments across all the associated
vectors and controls.
11. MOI indicates the ratio of bacteria to macrophage.
12. Due to the risk associated with disturbing the cells, we recommend removing the media via gentle aspiration. This can be
achieved by attaching a sterile glass pipette to a vacuum line
and carefully removing the media from a corner of the well or a
similar location that is away from the majority of cells.
13. With a higher MOI, reduced macrophage viability is expected
due to the greater bacterial load negatively influencing the
coculture environment. Thus, through LyE expression, the
experiment will reveal to what extent attenuation of the bacterial vectors affects this trend.
Acknowledgments
The authors recognize support from NIH awards AI088485 and
AI117309 and a grant from the Technology Accelerator Fund from
the State University of New York Research Foundation (all to
BAP).
References
1. Courvalin P, Goussard S, Grillot-Courvalin C
et al (1995) Gene transfer from bacteria to
mammalian cells. C R Acad Sci III
318:1207–1212
2. Grillot-Courvalin C et al (1998) Functional
gene transfer from intracellular bacteria to
mammalian cells. Nat Biotechnol 16(9):862
3. Sizemore DR, Branstrom A, Sadoff J (1995)
Attenuated Shigella as a DNA delivery vehicle
for DNA-mediated immunization. Science
270:299–302
4. Grillot-Courvalin C, Goussard S, Courvalin P
(2002) Wild-type intracellular bacteria deliver
DNA into mammalian cells. Cell Microbiol
4:177–186
5. Al-Mariri A, Tibor A et al (2002) Yersinia
enterocolitica as a vehicle for a naked DNA
vaccine encoding Brucella abortus bacterioferritin or P39 antigen. Infect Immun
70:1915–1923
6. Gardlik R, Behuliak M, Palffy R, Celec P, Li C
(2011) Gene therapy for cancer: bacteriamediated anti-angiogenesis therapy. Gene
Ther 18:425–431
7. Steinman RM (2001) Dendritic cells and the
control of immunity: enhancing the efficiency
of antigen presentation. Mt Sinai J Med
68:160–166
8. Birmingham CL, Canadien V, Kaniuk NA et al
(2008) Listeriolysin O allows Listeria monocytogenes replication in macrophage vacuoles.
Nature 451:350–354
9. Palffy R, Gardlik R, Hodosy J et al (2006)
Bacteria in gene therapy: bactofection versus
alternative gene therapy. Gene Ther
13:101–105
10. Chung TC, Jones CH, Gollakota A et al
(2015) Improved Escherichia coli Bactofection
and cytotoxicity by heterologous expression of
bacteriophage PhiX174 lysis gene E. Mol
Pharm 12:1691–1700
11. Parsa S, Wang Y, Fuller J (2008) A comparison
between polymeric microsphere and bacterial
vectors for macrophage P388D1 gene delivery.
Pharm Res 25:1202–1208
14
Dongwon Park et al.