Navigation Links
Transient Gene Expression Following Single and Double Exponential Decay Electroporation Pulses

James R. Wong, Ph.D. and Wei-Yu Lin, Bio-Rad Laboratories


Summary
A double pulse format for electroporation of mammalian tissue culture cells gave lower transient expression than a single exponential decay pulse. Moreover, separate administration of the two components of the double pulse revealed that transformation was due almost exclusively to the first, high voltage pulse and not to the second, long duration waveform.


Introduction
The Twin-pulse wave delivery is a novel electroporation waveform consisting of a short duration, exponential decay pulse followed by a second capacitor discharge yielding a long duration pulse. It is claimed that the first high-voltage pulse creates pores, or openings in the cell walls due to the high voltage delivered... (and) the second low-voltage pulse delivers energy to those cells shocked by the first pulse, therefore favoring entry of exogenous molecules into the cells by means of creating an active electrophoresis field.1 While the transient formation of pores in the plasma membrane following delivery of appropriate electrical fields has been documented via electron microscopy,2 the mechanism of entry and egress of materials through these pores is not known, and no evidence has been provided for the postulated Twin-pulse description.

De Chasseval and de Villartay described the use of double electroporation pulse format to introduce pairs of plasmids into human lymphoid cells.3 Their principal finding was that cotransfection of a plasmid encoding the SV40 large T antigen with a reporter plasmid bearing the SV40 origin of replication increased transient luciferase or CD2 expression several fold. The electroporation conditions were optimized for voltage, but not for capacitance, resistance, or timing between pulses. Furthermore, these researchers did not compare the results of single, exponential decay pulses to the expression produced by the double pulse procedure. De Chasseval and de Villartay did not claim that the use of the double pulse was essential for the success of these experiments.

In this report, the optimal Twin-pulse electroporation conditions from de Chasseval and de Villarty, as reported in the January, 1993 EquiBio News, were compared to single electroporation pulses generated by the Gene Pulser II system for transient gene expression in COS cells.


Results and Discussion
The results in Table 1 and Figure 1 show the reporter gene activity obtained for optimal electroporation conditions of the two instruments. Maximum transient expression was obtained with both the Gene Pulser II system and the EasyJect Plus with a single pulse of 210V and 450 F capacitance. The third treatment duplicates the optimum Twin-pulse conditions reported by EquiBio for mammalian cells a 500 V, 25 F pulse with a 99 ohm resistor in parallel, a 7 second inter-pulse delay, and then a 74 V, 2,100 F pulse with a 99 ohm resistor in parallel.

It is clearly apparent that a single, optimized exponential decay pulse is more effective than this Twin-pulse form, generating about twice the level of transient gene expression. Moreover, when the two parts of the double pulse were delivered to cell samples separately in treatments 4 and 5, ca. 70% of the Twinpulse expression was obtained when the first, high voltage component was administered alone. Most of the gene transfer occurring during a double pulse is therefore taking place during the brief initial, pore-formation phase, with very little enhancement in expression contributed by the second pulse element, which is purported to be the DNA movement phase. While the Twin-pulse mechanism seems plausible in theory, in practice its benefits were not observed, and published reports demonstrating its efficacy compared to single pulses could not be found.

Comparison of luciferase and β-galactosidase expression resulting from each of the electroporation treatments in Table 1 also shows that the two reporter enzymes varied in a parallel fashion. This indicates that these measurements of enzyme activity are an accurate representation of the plasmid uptake and gene expression in these cell samples.

Waveform optimization experiments using single pulses produced by the Gene Pulser II system were performed with either low voltage and high capacitance or high voltage and low capacitance. The results (not shown) indicate that the pulse parameters used in the first experiment are essentially the optimum combinations of voltage and capacitance, i.e. pulse energy.


Materials and Methods
Cells and Electroporation Conditions
COS-M7 cells were grown in DMEM containing 10% fetal calf serum so that cultures were ca. 70% confluent on the day of electroporation. Cells were trypsinized, collected by centrifugation, washed twice in PBS, and resuspended in DMEM without serum at a density of 1.5 x 106 cells per 200 l aliquot, the amount used for one electroporation. DNA in a total mass of 0.33 g as a mixture of equal amounts of two plasmids was placed in a 0.2 cm gap electroporation cuvette, then the cell suspension was added. Exponential decay electroporation pulses were administered as specified in Table 1 by a Gene Pulser II system with Capacitance Extender II or an EasyJect Plus electroporator (EquiBio) according to the manufacturers instructions. The cells were then cultured in 4 ml DMEM containing 10% serum in hexwell plates in a humidified 37 C, 5% CO2 environment.

Plasmids and Reporter Enzyme Assays
The expression plasmids pCH1104 and pSVZALAΔ5'5, which contain the SV40 early promoter, were used to produce E. coli β-galactosidase and firefly luciferase respectively. These reporter enzyme activities were assayed in cell extracts prepared 24 hours after electroporation. Adherent cells were washed twice with cold PBS and lysed in 300 l luciferase lysis buffer. Extracts were centrifuged briefly to remove cell debris. Luciferase was quantitated using luciferin (Analytical Luminescence Laboratory, San Diego) employed according to the manufacturers protocol, collecting the light produced by 20l of extract for 10 sec with an MGM Optocomp I luminometer. Galactosidase was determined in the same cell extracts using the chemiluminescent Galacto-Light assay (Tropix, Cambridge, Massachusetts) in the same luminometer.


Conclusion
A single, capacitor discharge electroporation pulse gave better transformation results than a Twin-pulse regime. A single pulse treatment is also much easier to optimize, since there are only two electrical parameters to set.


References
1. EasyJect Electroporator Instruction Manual, Stratagene, La Jolla, California (1994).

2. Chang, D. C. and Reese, T. S. Changes in membrane structure induced by electroporation as revealed by rapid-freezing electron microscopy, Biophys. J., 58, 1-12 (1990).

3. De Chasseval, R. and de Villartay, J.-P. 1992. High level transient gene expression in human lymphoid cells by SV40 large T antigen boost. Nucleic Acids Res., 20, 245-250 (1992).

4. Hall, C. V., Jacob, P. E., Ringold, G. M. and Lee, F. Expression and regulation of E. coli lacZ gene fusions in mammalian cells. J. Mol. Appli. Genet., 2, 101-109 (1983).

5. DeWet, J. R., Wood, K. V., DeLuca, M., Helinski, D. R. and Subramani, S. Firefly luciferase gene: structure and expression in mammalian cells. Mol. Cell. Biol., 7, 725-737 (1987).


Twin-pulse and EasyJect are trademarks of EquiBio Company.


back to top
'"/>

Source:


Page: All 1 2 3 4 5

Related biology technology :

1. An Epitope Tagging Vector for Gene Expression in Mammalian Cells
2. Antibodies for Studying NMDA Receptor Protein Expression and Synapse-Specific Immunolabeling
3. High-Level Expression of Peanut Allergens Affected by Rare Codon Usage
4. Versatile Vectors for Ponasterone A- Inducible Control of Gene Expression in Mammalian Cells
5. Innovative Tissue Array Technology for High-Throughput Screening of Gene Expression
6. New Mammalian Expression Vectors Employ Stable, High-Level Fluorescence Humanized Renilla GFP Reporter
7. Functional Cloning Using ViraPort Retroviral cDNA Expression Libraries
8. High-Level Protein Expression, One-Column Purification, and FLAG Epitope Tagging in E. coli
9. A New Lambda Vector for Mammalian Expression
10. Expression and Purification of Recombinant Proteins That Have Native Amino Acid Sequence
11. Codon Bias-Adjusted BL21 Derivatives for Protein Expression
Post Your Comments:
(Date:7/29/2015)... July 29, 2015  HealthSouth Corporation ... largest providers of post-acute healthcare services, offering both ... results of operations for the second quarter ended ... was characterized by strong volume growth in both ... EBITDA," said Jay Grinney, President and Chief Executive ...
(Date:7/29/2015)... ... July 29, 2015 , ... ... announced its GHS Label Guide . To align chemical container labeling with ... guide includes explanations of label components, an example of an accurate label, and ...
(Date:7/29/2015)...  Indivior PLC (LON: INDV) today announced that the ... accepted and received Priority Review by the U.S. Food ... overdose. This naloxone nasal spray comes as a pre-filled ... into the nasal mucosa. 1 The device has ... be better equipped to help an opioid overdose victim. ...
(Date:7/29/2015)... CA (PRWEB) , ... July 29, 2015 , ... Finding ... the morning is quite normal. Although there are a considerable number of terms in ... understood that this post-sleep material is a mixture of mucus, blood cells, skin cells ...
Breaking Biology Technology:HealthSouth Reports Results for Second Quarter 2015 2HealthSouth Reports Results for Second Quarter 2015 3HealthSouth Reports Results for Second Quarter 2015 4HealthSouth Reports Results for Second Quarter 2015 5HealthSouth Reports Results for Second Quarter 2015 6HealthSouth Reports Results for Second Quarter 2015 7HealthSouth Reports Results for Second Quarter 2015 8HealthSouth Reports Results for Second Quarter 2015 9HealthSouth Reports Results for Second Quarter 2015 10HealthSouth Reports Results for Second Quarter 2015 11HealthSouth Reports Results for Second Quarter 2015 12HealthSouth Reports Results for Second Quarter 2015 13HealthSouth Reports Results for Second Quarter 2015 14HealthSouth Reports Results for Second Quarter 2015 15HealthSouth Reports Results for Second Quarter 2015 16HealthSouth Reports Results for Second Quarter 2015 17HealthSouth Reports Results for Second Quarter 2015 18HealthSouth Reports Results for Second Quarter 2015 19HealthSouth Reports Results for Second Quarter 2015 20HealthSouth Reports Results for Second Quarter 2015 21HealthSouth Reports Results for Second Quarter 2015 22HealthSouth Reports Results for Second Quarter 2015 23HealthSouth Reports Results for Second Quarter 2015 24HealthSouth Reports Results for Second Quarter 2015 25HealthSouth Reports Results for Second Quarter 2015 26HealthSouth Reports Results for Second Quarter 2015 27HealthSouth Reports Results for Second Quarter 2015 28HealthSouth Reports Results for Second Quarter 2015 29HealthSouth Reports Results for Second Quarter 2015 30HealthSouth Reports Results for Second Quarter 2015 31HealthSouth Reports Results for Second Quarter 2015 32HealthSouth Reports Results for Second Quarter 2015 33HealthSouth Reports Results for Second Quarter 2015 34HealthSouth Reports Results for Second Quarter 2015 35HealthSouth Reports Results for Second Quarter 2015 36
... senior vice,president and co-director of the Vaccine and ... today was named as one of 65 new,members ... Election to the IOM is considered one of ... medicine and recognizes individuals who have,demonstrated outstanding professional ...
... October 13 Mentice, a leading,supplier of solutions ... Larsson as Vice President of Global Marketing., ... up and coordinate,Mentice,s marketing activities globally from a ... over 15 years of experience of global,sales, marketing ...
... Mass., Oct. 13 Proteon Therapeutics is,presenting ... of the potential of PRT-201 to enhance ... arterial disease (PAD). The,presentation, entitled, "Local Catheter-Directed ... and Restenosis," is being made at the ...
Cached Biology Technology:Mentice Appoints Bjorn Larsson as Vice President of Global Marketing 2Proteon Therapeutics to Present New Data on PRT-201 at Transcatheter Cardiovascular Therapeutics (TCT) Annual Symposium in Washington, D.C., October 13, 2008 2
(Date:7/2/2015)... 2015 Research and Markets( ... "Next Generation Biometrics Market by Application, Technology, Function & ... their offering. The next generation biometrics market ... a CAGR of 17.9% between 2015 and 2020 ... the market. Safran SA ( France ...
(Date:6/29/2015)... , June 24, 2015 ... addition of the "Latin America Biomedical Sensors Market ... their offering. The Latin America Biomedical Sensors ... a CAGR of 2.04% over the period 2014-2020 ... are adaptable to the genetic formulation of each individual. ...
(Date:6/25/2015)... 2015  TAKE Solutions Ltd., a global business technology ... States Patent and Trademark Office (USPTO) for its "Method ... Solutions, Clinical Accelerators to reduce the time taken to ... standardization without the accelerators), thus reducing time to market. ... At the heart of the patented Clinical Data Standardization ...
Breaking Biology News(10 mins):Next Generation Biometrics Market 2015 - Global Forecast to 2020 with Safran, NEC, 3M Cogent, Fujitsu & Suprema Dominating the $24 Billion Market 2Next Generation Biometrics Market 2015 - Global Forecast to 2020 with Safran, NEC, 3M Cogent, Fujitsu & Suprema Dominating the $24 Billion Market 3Latin America Biomedical Sensors Market Report 2015-2020 2TAKE Solutions Awarded Patent By USPTO 2
... Barry T. Katzen, M.D., FSIR, an interventional radiologist ... & Vascular Institute in Miami, Fla., was awarded ... II MD Award for Physician Excellence. This prestigious ... or patient care in vascular disease. "For ...
... unlikely the United States will meet some specific biofuel ... unless innovative technologies are developed or policies change, says ... Council, which adds that the standard may be an ... Achieving this standard would likely increase federal budget outlays ...
... Cell-penetrating peptides, such as the HIV TAT peptide, ... mechanisms, from direct entry to endocytosis, a process by ... these cell-penetrating peptides, or CPPs, can facilitate the cellular ... to nano-sized particles and large fragments of DNA. Because ...
Cached Biology News:Society of Interventional Radiology member receives award for career contributions 2Society of Interventional Radiology member receives award for career contributions 3Certain biofuel mandates unlikely to be met by 2022; unless new technologies, policies developed 2Certain biofuel mandates unlikely to be met by 2022; unless new technologies, policies developed 3Certain biofuel mandates unlikely to be met by 2022; unless new technologies, policies developed 4UCLA study shows cell-penetrating peptides for drug delivery act like a Swiss Army Knife 2UCLA study shows cell-penetrating peptides for drug delivery act like a Swiss Army Knife 3
Human peripheral blood PBMCs, For immunohistochemistry (IHC) Cell Chip with Human Peripheral Blood PBMC and T Cells...
Umbilical cord blood CD14+ Monocytes, For immunohistochemistry (IHC) Cell Chip with Human Umbilical Cord Blood Monocytes, B Cells and NK Cells...
Human peripheral blood CD8+ T cells, For immunohistochemistry (IHC) Cell Chip with Human Peripheral Blood PBMC and T Cells...
Human peripheral blood CD56+ NK cells, For immunohistochemistry (IHC) Cell Chip with Human Peripheral Blood Monocytes, B Cells, NK Cells and Stem Cells...
Biology Products: