Insitu Cell Electroporation Scientific References

Most of the publications cited here are highly relevant in content however have been used on the older design InSitu apparatus. The New Insitu Porator has undergone many design changes to improve the quality of results, and the ability to electroporate a much broader range of cells types. The most important design change is the removal of the top electrode eliminating any disturbance to the adherent cells, created by turbulence. This makes it easy to recover the electroporation solution containing expensive material to use for repeat experiments.

The system has been tested with a large variety of cell types, such as fibroblasts, epithelial cell lines, primary human and mouse lung carcinoma cells, neuronal cells and others. Cells that do not adhere well can be attached with fibronectin, poly-lysine or CellTakTM to be electroporated in situ.

Most Significant Publications

A: In situ electroporation for the study of gap junctional communication

Anagnostopoulou A, Cao J, Vultur A, Firth K and Raptis L. (2007). Examination of gap junctional, intercellular communication by in situ electroporation on two co-planar indium-tin oxide electrodes. Molecular Oncology, 1: 226-231
This is the first report on electroporation without the use of an electrode placed on top of the cells.

Vultur A, Tomai E, Peebles K, Malkinson AM, Grammatikakis N, Forkert PG, and Raptis L (2003).
Gap junctional, intercellular communication in urethane-induced tumors in A/J mice. DNA and Cell Biology 20:33-40.

Grammatikakis, N., Vultur, A., Siganou, A., Schweinfest, V., Watson, D. and Raptis, L. (2002).
The role of Hsp90N, a new member of the Hsp90 family, in signal transduction and neoplastic transformation. Journal of Biological Chemistry 277:8312-8320.
In situ electroporation on a partly conductive slide was used to demonstrate that the Hsp90N oncogene can block gap junctional communication in rat fibroblasts.

Tomai, E., H.L. Brownell, T. Tufescu, K. Reid, B.G. Campling, and L. Raptis. 1999.
Gap junctional, intercellular communication in human lung carcinoma cells.
Lung Cancer 23:223-231.
Electroporation on a partly conductive slide was used to examine gap junctional communication in a large number of lung cancer lines and primary tumors.

Tomai, E., H.L. Brownell, T. Tufescu, K. Reid, S. Raptis, B.G. Campling, and L. Raptis. 1998.
A functional assay for intercellular, junctional communication in cultured human lung carcinoma cells. Laboratory Investigation, 78:639-640.
Electroporation on a partly conductive slide was used to demonstrate the potential for examination of gap junctional communication in human lung carcinoma cells.

Brownell, H.L., Narsimhan, R.P., Corbley, M.J., Mann, V.M., Whitfield, J.J. and Raptis, L.1996.
Ras is involved in gap junction closure in fibroblasts or preadipocytes but not differentiated adipocytes. DNA and Cell Biology, 15:443-451.
Electroporation on a partly conductive slide was used to demonstrate that differentiated adipocytes do not have gap junctions. A Figure from this paper was placed on the cover of this issue.

Ennaji M.M., J.L. Schwartz, L. Belbaraka, C.Parker, M. Parentaux, H. Jouishomme, M. Arella, J.F. Whitfield, and J. Phipps 1995.
Alterations in cell to cell communication in human papillomavirus type 16-transformed rat myoblasts. Cellular and Molecular Biology 41:481-499.

Raptis, L., H.L. Brownell, K.L. Firth, and L.W. MacKenzie. 1994.
A novel technique for the study of intercellular, junctional communication; electroporation of adherent cells on a partly conductive slide. DNA and Cell Biology, 13:963-975.
This is the first report on the examination of gap junctional communication of cells growing on a partly conductive slide. The current was delivered with an aluminum electrode.

B. Electroporation of peptides, nucleotides or other compounds for the study of signal transduction

Raptis L and Firth K. 2007. Electrode assemblies used for electroporation of cultured cells.
In Methods in Molecular Biology. Electroporation protocols. Shulin Li, ed. The Humana Press Inc., Totowa, NJ., Chapter 4, pages 58-73. Review

Raptis L, Vultur A, Brownell HL, Tomai E, Anagnostopoulou A, Arulanandam R, Cao J, and Firth KL. 2007.
Electroporation of adherent cells in situ for the study of signal transduction and gap junctional communication. In: Methods in Molecular Biology. Electroporation protocols. Shulin Li, ed. The Humana Press Inc., Totowa, NJ., Chapter 12, pages 167-183. Review

Anagnostopoulou A, Vultur A, Arulanandam R, Cao J, Turkson J, Jove R, Joon S. Kim, Glenn M, Hamilton AD and Raptis L (2006).
Differential effects of Stat3 inhibition in sparse vs confluent normal and breast cancer cells. Cancer Letters, 242:120-132. Peptides or peptidomimetics were electroporated to examine their effectiveness in inhibiting Stat3 activity.

Raptis, L., Vultur, A., Brownell, H.L., and Firth, K.L. 2006.
Dissecting pathways; in situ electroporation for the study of signal transduction and gap junctional communication. In: Cell Biology, a laboratory Handbook. Third Edition. Celis J, Editor, Academic Press Inc. Chapter 44, pages 341-354. Review

Arulanandam, R., Vultur, A. and Raptis, L (2005).
Transfection techniques affecting Stat3 activity levels. Analytical Biochemistry, 338:83-89.
This paper demonstrates that Calcium-phosphate transfection or liposomes can increase Stat3 activity, possibly due to changes in cell to cell adhesion. On the other hand, in situ electroporation does not affect Stat3, p-tyr705 phosphorylation, DNA binding or transcriptional activity.

Specific inhibition of Growth Factor-stimulated ERK1/2 activation in intact cells by electroporation of a Grb2-SH2 binding peptide. Cell Growth and Differentiation, 11:293-303.
This paper has the first demonstration that transfer of a peptide through gap junctions can inhibit a signal in neighboring, non-electroporated cells. Figure 4A from this paper was placed on the cover of this issue.

Brownell, H.L., N. Lydon, E. Schaefer, T.M. Roberts, and L. Raptis. 1998.
Inhibition of Epidermal Growth Factor­mediated ERK1/2 activation by in situ electroporation of nonpermeant [(alkylamino)methyl]acrylophenone derivatives. DNA and Cell Biology, 17:265-274.
Two compounds were electroporated and following EGF addition, the inhibition of the signal was monitored by staining in situ for p-Erk. In this paper it is also shown that electroporation in situ does not affect the stress pathways.

Nakashima, N., Rose, D. Xiao, S., Egawa, K., Martin, S., Haruta, T., Saltiel, A. R. and Olefsky, J.M. 1999.
The functional role of CrkII in actin cytoskeleton organization and mitogenesis. J. Biol. Chem. 274:3001-3008.
The authors electroporated GST fusion proteins containing either the Crk SH2 domain or the Crk-(N)SH3 domain. They demonstrated that c-CrkII bound to p130cas, but not to paxillin in serum-starved rat-1 fibroblasts overexpressing the human insulin receptor (HIRc cells) in vivo. These data suggest that the complex containing p130cas·c-CrkII may play a crucial role in actin cytoskeleton organization and in anchorage-dependent DNA synthesis.

Bardelli, A., P. Longati, D. Gramaglia, C. Basilico, L. Tamagnone, S. Giordano, D. Ballinari, P. Michieli, and P.M. Comoglio. 1998.
Uncoupling signal transducers from oncogenic MET mutants abrogates cell transformation and inhibits invasive growth. Proc. Nat. Acad. Sci. USA 95:14379­14383.
Using this setup, the authors electroporated double-stranded decoy oligonucleotides, as well as peptides to study HGF signalling.

Induction of epithelial tubules by growth factor HGF depends on the STAT pathway. Nature, 391:285-288.
Using this setup, the authors electroporated double-stranded decoy oligonucleotides, as well as peptides to study HGF signalling.

Marais, R., Spooner, RA, Stribbling, SM, Light, Y., Martin, J. and Springer, CJ. 1997.
A cell surface tethered enzyme improves efficiency in gene-directed enzyme prodrug therapy. Nature Biotechnology 15:1373-1377. The authors introduced various prodrugs.

Giorgetti­Peraldi, S., E. Ottinger, G. Wolf, B. Ye, T.R. Burke, and S.E. Shoelson. 1997.
Cellular effects of phosphotyrosine­binding domain inhibitors on insulin receptor signalling and trafficking. Mol. Cell. Biol. 17:1180­1188.
Tyr960 of the insulin receptor binds both Shc and IRS1, but the requirements for binding are slightly different. Using this setup, the authors electroporated peptides which could inhibit binding of Shc but not IRS1.

Gambarotta, G., C. Boccaccio, S. Giordano, M. Ando, M.C. Stella, and P.M. Comoglio. 1996.
Ets up­regulates met transcription. Oncogene 13:1911­1917.
Using this setup, the authors electroporated double-stranded decoy oligonucleotides, binding sites of the ets factor, to specifically inhibit transcription.

C: Electroporation of Radioactive Nucleotides

These publications used a large surface area of electroporation on the older design that is currently not available on the new InSitu Porator. However, with the continuing improvements in sensitivity of detection it is likely that some of these applications are now usefully transferable to the new InSitu equipment. We are also currently looking at new electrode designs that can be used to electroporate larger surface areas.

Raptis, L., Vultur, A., Tomai, E., Brownell, H.L. and Firth, K.L. 2006.
In situ electroporation of radioactive nucleotides: assessment of Ras activity or 32P labelling of cellular proteins. In: Cell Biology, a laboratory Handbook. Third Edition. Celis J, Editor, Academic Press Inc. Chapter 43, pages 329-339. Review

Egawa, K., P.M. Sharma, M. Nakashima, Y. Huang, E. Huver, G.R. Boss, and J.M. Olefsky. 1999.
Membrane­targeted Phosphatidylinositol kinase mimics insulin actions and induces a state of cellular insulin resistance. J. Biol. Chem. 274:14306­14314
The authors electroporated [a-32P]GTP in 3T3-L1 adipocytes

Haruta, T., A.J. Morris, P. Vollenweider, J. Nelson, D.W. Rose, M. Mueckler, and J.M. Olefsky. 1998.
Ligand­independent Glut4 translocation induced by Guanosine 5'­O­(3­Thiophosphate) involves Tyrosine phosphorylation. Endocrinology 139:358­364.

Raptis, L., H.L. Brownell, Y. Lu, T. Preston, R.P. Narsimhan, E. Schaefer, S. Anderson, and T. Haliotis. 1997.
v­Ras and v­Raf block differentiation of transformable C3H10T½­derived preadipocytes at lower levels than required for neoplastic transformation. Exp. Cell Res., 235:188-197.
Ras activity was measured by electroporation of [α32P]GTP.

Raptis, L., H.L. Brownell, K. Wood, M. Corbley, D. Wang, and T. Haliotis. 1997.
Cellular ras gene activity is required for full neoplastic transformation by Simian Virus 40. Cell Growth. Differ. 8:891­901.
Ras activity was measured by electroporation of [α32P]GTP.

Raptis, L., J. Yang, H.L. Brownell, J. Lai, T. Preston, M.J. Corbley, R.P. Narsimhan, and T. Haliotis. 1997.
Rasleu61 blocks differentiation of transformable 3T3 L1 and C3H10T½­derived preadipocytes in a dose­ and time­dependent manner. Cell Growth. Differ. 8:11­21.
Ras activity was measured by electroporation of [α32P]GTP.

Boussiotis VA, Freeman GJ, Berezovskaya A, Barber DL and Nadler LM (1997).
Maintenance of human T-cell anergy: Blocking of IL-2 transcription by activated Rap1. Science 278:124-128.

Brownell, H.L., K.L. Firth, K. Kawauchi, T.L. Delovitch, and L. Raptis. 1997.
A novel technique for the study of Ras activation; electroporation of [α32P]GTP. DNA and Cell Biology 16:103­110.
For the first time, Ras activity was measured by electroporation of [α32P]GTP, followed by Ras immunoprecipitation and elution of Ras-bound, GTP and GDP. This paper also shows that unlike other methods of cell permeabilisation, such as Streptolysin-O, in situ electroporation does not activate non-specific nucleotidases which degrade GTP.

D: Other publications on in situ electroporation

Anagnostopoulou A, Vultur A, Arulanandam R, Cao J, Turkson J, Jove R, Joon S. Kim, Glenn M, Hamilton AD and Raptis L. 2006.
Role of Stat3 in normal and SV40 transformed cells. Trends in Cancer Research, 2:93-103.

Tomai, E., Vultur, A., Balboa, V., Hsu, T., Brownell, H.L., Firth, K.L. and Raptis, L. 2003.
In situ electroporation of radioactive compounds into adherent cells. DNA and Cell Biology, 22:339-346.

Raptis, L., Balboa, V., Hsu, T., Vultur, A., Turkson, J., Jove, R. and Kevin L. Firth. (2003).
In situ electroporation of large numbers of cells. Analytical Biochemistry, 317(1):124-8.

Raptis, L., Firth, K.L., Tomai, E. and Forkert, P.G. (2001)
Improved procedure for examination of gap junctional, intercellular communication by in situ electroporation on a partly conductive slide. Biotechniques, 29:222-226.

Brownell, H.L. and Raptis, L. 2001. [α32P]
GTP electroporation for the measurement of Ras activation by SVLT. In: Methods in Molecular Biology. SV40 Protocols. The Humana Press Inc., Totowa, NJ., Review

Raptis, L., H.L. Brownell, N.B. Lydon, and T.M. Roberts. 1998.
Inhibition of EGF­induced ERK1 and ERK2 enzyme activation in NIH3T3 cells by in situ electroporation of a nonpermeant inhibitor. Promega Notes 67:12­15. Review

Brownell, H.L., S. Giorgetti­Peraldi, E. Tomai, K.L. Firth, and L. Raptis. 1998.
In situ electroporation in the study of signal transduction, G.G. Skouteris and G.L. Nickolson (eds.), Intermolecular cross­talk in tumor metastasis. IOS Press, Netherlands. Review

Brownell, H.L. and Raptis, L.. 1998. Electroporation of nucleotides.
Assessment of Ras activity. 32P­labelling of cellular components, J.C. Celis (ed.), Cell Biology: A laboratory handbook. Academic Press Inc., Volume 4:65-74. Review

Raptis, L., H.L. Brownell, K.L. Firth, S. Shoelson, and S. Peraldi. 1998.
In situ electroporation for the study of signal transduction, J.C. Celis (ed.), Cell Biology: A laboratory handbook. Academic Press Inc., Volume 4:75-87. Review

Firth, K.L., H.L. Brownell, and L. Raptis. 1997.
An improved procedure for electroporation of peptides into adherent cells in situ. Biotechniques, 23:644-645.
Changes in the geometry of the slide were introduced, to offer electroporated and control cells growing on the same coated surface.

Brownell, H.L., J.F. Whitfield, and L. Raptis. 1997.
Elimination of intercellular junctional communication requires lower Rasleu61 levels than stimulation of anchorage­independent proliferation. Cancer Detect. Prev. 21:289­294.
Electroporation on a partly conductive slide was used to demonstrate that low levels of activated Rasleu61 can interrupt gap junctional permeability.

Brownell, H.L., J.F. Whitfield, and Raptis, L. 1996.
Cellular Ras partly mediates gap junction closure by the polyoma virus middle Tumor antigen. Cancer Letters, 103:99-106.
Electroporation on a partly conductive slide was used to demonstrate that Ras downregulation prevents the polyoma virus mT antigen oncogene from closing gap junctions.

Raptis, L., Liu, S.K.W., Firth, K.L., Stiles C.D. and Alberta, J.A. 1995.
Electroporation of peptides into adherent mammalian cells in situ. Biotechniques, 18:104-114.
A peptide was electroporated on a partly conductive slide and detected with an antibody.

Raptis, L., K.L. Firth, H.L. Brownell, A. Todd, W.C. Simon, B.M. Bennett, and M. Zannis­Hadjopoulos. 1995.
Electroporation of adherent cells in situ for the introduction of nonpermeant molecules, J.A. Nickoloff (ed.), Methods in Molecular Biology. Protocols for electroporation and electrofusion of Plant and Animal Cells. The Humana Press Inc., Totowa, NJ., Chapter 7:93-113.
Review.

Raptis, L., H.L. Brownell, Liu, S.K.W., K.L. Firth, and L.W. MacKenzie. 1995.
Applications of electroporation of adherent cells in situ, on a partly conductive slide. Molecular Biotechnology, 4:129-138. Review.

Simon, W.C., Raptis, L., Pang, S.C. and Bennett, B.M. 1992.
Comparison of liposome fusion and electroporation for the intracellular delivery of nonpermeant molecules to cultured, adherent cells. Journal of Pharmacological and Toxicological Methods, 29:29-35. This is an application of the technique above.

Raptis, L. and Firth, K. L. 1990.
Electroporation of adherent cells in situ. DNA and Cell Biology 9:615­621.
This is the initial publication on electroporation of adherent cells in situ, without detatching them from the solid support where they grow. The current was delivered with an aluminum electrode to introduce antibodies that were detected with fluorescent secondary antibodies.