Egfp

Brain-derived neurotrophic factor GFP

EGFP, luciferase ß-galactosidase Tbx5, GFP ß-galactosidase Luciferase

Luciferase, ß-galactosidase

ß-galactosidase, CAT ß-galactosidase, neuronal e-NOS Alkaline phosphatase CAT, luciferase Luciferase, ß-galactosidase bcl-xs, CAT

Luciferase GFP

Human growth hormone Luciferase, EGFP

Table 5 Electroporation of Embryos

Transgene

Species

Endpoint

Ref.

Truncated Pax6 fused to Drosophila engrailed repressor domain, GFP Winged-helix transcription factor

FoxD3, fusion protein with EGFP Sonic Hedgehog GFP

Interfering RNA, GFP, p-

galactosidase Slug, GFP

EGFP, N-cadherin-EGFP fusion construct, DsRED Pax-5, EGFP, p-galactosidase Pdx-1, ngn3, p-galactosidase

Krox-20, p-galactosidase p-galactosidase dsRNA against Otx2, Foxa2, p-

galactosidase R-cadherin, cadherin-6, GFP p-galactosidase, EGFP EphA4/ephrin-A5, EGFP EGFP

Pax6, EGFP

Tlx, fusion genes Tlx-DNA-binding domain-engrailed repressor domain, TlxVP16 activation domain Six3 and deletion mutants, Grg5, luciferase, CAT

Chick

Chick Chick Mouse

Chick

Chick

Chick

Chick

Chick Mouse

Mouse

Chick

Mouse

Mouse

Chick

Chick

Imaging, antibody staining of Pax6, Islet1/2,

Nkx2.2, Wnt7b, Hu protein In situ hybridization, fluorescence microscopy, immunohistochemistry Whole mount in situ hybridization, histochemistry Microscopy

Fluorescence microscopy, histochemistry

In situ hybridization, Immunohistochemistry, fluorescence microscopy Confocal and 2-photon microscopy

Histochemistry, in situ hybridization Histochemistry, in situ hybridization, immunohistochemistry Histochemistry, in situ hybridization, immunohistochemistry PCR

Histochemistry, in situ hybridization, immunocytochemistry, histology Fluorescence microscopy, Immunohistochemistry Histochemistry, fluorescence microscopy Immunocytochemistry, confocal imaging Fluorescence microscopy, histology Fluorescence microscopy, immunocytochemistry Histochemistry, in situ hybridization

In situ hybridization, immunohistochemistry, CAT activity

embedded in a tissue can vary from case to case, or tissue to tissue, a predetermined voltage may not necessarily produce a predetermined current (98). As an example, Fig. 3 depicts a direct comparison of the 2 methods in the pig muscle. Notice that a predetermined voltage pulse causes an increase in the current flowing through a porcine muscle tissue during the duration of the pulse with the loss of a perfect square wave; in contrast, a constant current source actually maintains a constant current through a porcine muscle tissue. Thus, in many cases, the experiments do not provide a means to delineate the exact amount of current to which the cells are exposed. For this reason, conventional electroporators may generate amounts of heat in tissues that can easily kill cells (99,100). For example, a typical electronic 50-mS pulse with an average current of 5 Amperes across a typical load impedance of 25 ohms can theoretically raise the temperature in tissue 7.5°C, which is enough to kill cells. The physics of tissue injury caused by electrical shock is reviewed by Lee et al. (101). In contrast, the power dissipation decreases in a constant current system and prevents heating of a tissue, which may reduce tissue damage and contribute to the overall success of the procedure. Thus, there is a need to overcome the technological problems associated with constant voltage electroporation by providing a means to control effectively the amount of electricity delivered to the cells. This can be accomplished by precisely controlling the ionic flux that impinges on the cell membrane conduits. A constant current electrokinetic device has been tested in our laboratory, in collaboration with Robert H. Carpenter, DVM, MS. The paired-needle electrodes that use a predetermined voltage pulse across opposing electrode pairs creates a centralized pattern during an electroporation event in an area where congruent and intersecting overlap points develop. This area can be visualized as an asterisk pattern, as shown in Fig. 3C. However, asymmetrically arranged needle electrodes without opposing pairs will produce a decentralized pattern during an electroporation event in an area in which there are no congruent electroporation overlap points. One example of such symmetry is shown in Fig. 3D, with a decentralized pattern area of electroporation that resembles a pentagon. In this case, plasmids would be stereotactically

dayO day 5

Figure 3 Constant voltage electroporation with paired needles vs. constant current electroporation using a pentagonal electrode array. (A) A predetermined voltage pulse causes an increase in the current flowing through a porcine muscle tissue during the duration of the pulse. In contrast, (B) a constant current source actually maintains a constant current through a porcine muscle tissue. (C) Maximum intensity electric field after constant voltage electroporation using a paired needle array. (D) Diffuse electric field after constant current electroporation using a pentagonal needle array. (E) Comparison of gene expression between the 2 delivery methods using similar electric field intensities.

dayO day 5

Figure 3 Constant voltage electroporation with paired needles vs. constant current electroporation using a pentagonal electrode array. (A) A predetermined voltage pulse causes an increase in the current flowing through a porcine muscle tissue during the duration of the pulse. In contrast, (B) a constant current source actually maintains a constant current through a porcine muscle tissue. (C) Maximum intensity electric field after constant voltage electroporation using a paired needle array. (D) Diffuse electric field after constant current electroporation using a pentagonal needle array. (E) Comparison of gene expression between the 2 delivery methods using similar electric field intensities.

delivered in-between the internal electrodes, followed by the constant current electric field pulses. A comparison of in vivo results obtained with a classical electroporator and a constant current device with asymmetrically arranged electrodes in adult pigs is depicted in Fig. 3E.

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