Localized Electroporation: A Method for Targeting Expression of Genes in Avian Embryos

Atkins, Ross; Wang, Diana; Burke, Robert D.

doi:10.2144/00281st04

Cited by 28 publications

(12 citation statements)

References 2 publications

(2 reference statements)

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“…The first successful electroporation of chick tissue was performed on retinal explants in vitro (Pu and Young, 1990). Muramatsu et al (1996Muramatsu et al ( , 1997 succeeded in electroporating chick embryos in ovo, and Momose et al (1999) optimized the technique to specifically target tissues of interest in ovo by the use of microelectrodes (reviewed in Atkins et al, 2000). Since then, many functional studies based on electroporation-mediated gene transfer in chick have been published.…”

Section: Introductionmentioning

confidence: 99%

In ovo electroporation of avian somites

Scaal

Gros

Lesbros

et al. 2004

Developmental Dynamics

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Section: Introductionmentioning

confidence: 99%

In ovo electroporation of avian somites

Scaal

Gros

Lesbros

et al. 2004

Developmental Dynamics

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“…Recently, several approaches have been used to electroporate smaller numbers of cells successfully. These include the use of small electrode arrays to electroporate cells in just one region of a Petri dish [13] or single, large-tipped electrodes to electroporate only a small population of cells in single embryos [1]. A very recent investigation has succeeded in inserting genes in single cells [5].…”

Section: Introductionmentioning

confidence: 99%

Single-cell electroporation

Rae

Levis

2001

Pflügers Arch - Eur J Physiol

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Using modified patch-clamp methodology, we demonstrated that it is possible to insert genes or other compounds routinely into single cells by electroporation. When the cell is indented by a small-tipped microelectrode, a voltage of 10 V or less in the pipette is divided by the pipette resistance and the series resistance of the cleft between the pipette tip and the cell surface. The voltage at the cell membrane can be high enough to cause localized dielectric breakdown of the membrane and create pores that allow compounds in the pipette to enter the cell. Rectangular pulses from 20 micros to more than 300 ms are effective, as are frequencies from DC to 5 kHz. The most significant parameter was the total time for which the voltage was applied. Pipette voltages of 2-10 V were required, with larger genes requiring larger voltages. With optimal parameters, transfection rates in excess of 80% were also possible routinely. This approach offers an effective alternative to intracellular pressure injection and iontophoresis for placing genes, drugs, and other compounds in cells. Because of the small size of the electrode tips, substances can be inserted in cells from almost any location on their surfaces. In addition, the small tips electroporated only a limited area and so did little cell damage.

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“…Electroporation (EP) recently has been used for the introduction of genes into the chick embryo (Momose et al, 1999;Itasaki et al, 1999;Yasuda et al, 2000;Atkins et al, 2000;DeLise and Tuan, 2000;Swartz et al, 2001a,b). In EP, current creates micropores in the cell membrane and drives the charged DNA into cells.…”

Section: Introductionmentioning

confidence: 99%

Efficient ectopic gene expression targeting chick mesoderm

Oberg

Pira

Revelli

et al. 2002

Developmental Dynamics

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The chick model has been instrumental in illuminating genes that regulate early vertebrate development and pattern formation. Targeted ectopic gene expression is critical to dissect further the complicated gene interactions that are involved. In an effort to develop a consistent method to ectopically introduce and focally express genes in chick mesoderm, we evaluated and optimized several gene delivery methods, including implantation of 293 cells laden with viral vectors, direct adenoviral injection, and electroporation (EP). We targeted the mesoderm of chick wing buds between stages 19 and 21 (Hamburger and Hamilton stages) and used ␤-galactosidase and green fluorescent protein (GFP) to document gene transfer. Expression constructs using the cytomegalovirus (CMV) promoter, the ␤-actin promoter, and vectors with an internal ribosomal entry sequence linked to GFP (IRES-GFP) were also compared. After gene transfer, we monitored expression for up to 3 days. The functionality of ectopic expression was demonstrated with constructs containing the coding sequences for Shh, a secreted signaling protein, or Hoxb-8, a transcription factor, both of which can induce digit duplication when ectopically expressed in anterior limb mesoderm. We identified several factors that enhance mesodermal gene transfer. First, the use of a vector with the ␤-actin promoter coupled to the 69% fragment of the bovine papilloma virus yielded superior mesodermal expression both by markers and functional results when compared with several CMV-driven vectors. Second, we found the use of mineral oil to be an important adjuvant for EP and direct viral injection to localize and contain vector within the mesoderm at the injection site. Lastly, although ectopic expression could be achieved with all three methods, we favored EP confined to the mesoderm with insulated microelectrodes (confined microelectroporation-CMEP), because vector construction is rapid, the method is efficient, and results were consistent and reproducible.

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Localized Electroporation: A Method for Targeting Expression of Genes in Avian Embryos

Cited by 28 publications

References 2 publications

In ovo electroporation of avian somites

In ovo electroporation of avian somites

Single-cell electroporation

Efficient ectopic gene expression targeting chick mesoderm

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