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A novel electroporation system for efficient molecular delivery into Chlamydomonas reinhardtii with a 3-dimensional microelectrode.

ABSTRACT: Electroporation is one of the most widely used transfection methods because of its high efficiency and convenience among the various transfection methods. Previous micro-electroporation systems have some drawbacks such as limitations in height and design, time-consuming and an expensive fabrication process due to technical constraints. This study fabricates a three dimensional microelectrode using the 3D printing technique. The interdigitated microstructure consisting of poly lactic acid was injected by a 3D printer and coated with silver and aluminum with a series of dip-coatings. With the same strength of electric field (V cm(-1)), a higher efficiency for molecular delivery and a higher cellular viability are achieved with the microelectrode than with a standard cuvette. In addition, this study investigates chemicophysical changes such as Joule heating and dissolved metal during electroporation and showed the micro-electroporation system had less chemicophysical changes. It was concluded that the proposed micro-electroporation system will contribute to genetic engineering as a promising delivery tool, and this combination of 3D printing and electroporation has many potential applications for diverse designs or systems.

SUBMITTER: Kang S

PROVIDER: S-EPMC4629139 | biostudies-literature | 2015 Nov

REPOSITORIES: biostudies-literature

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A novel electroporation system for efficient molecular delivery into Chlamydomonas reinhardtii with a 3-dimensional microelectrode.

Kang Seongsu S Kim Kwon-Ho KH Kim Yeu-Chun YC

Scientific reports 20151102

Electroporation is one of the most widely used transfection methods because of its high efficiency and convenience among the various transfection methods. Previous micro-electroporation systems have some drawbacks such as limitations in height and design, time-consuming and an expensive fabrication process due to technical constraints. This study fabricates a three dimensional microelectrode using the 3D printing technique. The interdigitated microstructure consisting of poly lactic acid was inj ...[more]

PMID: 26522846

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Project description:BACKGROUND:Chlamydomonas reinhardtii is an unicellular green alga used for functional genomics studies and heterologous protein expression. A major hindrance in these studies is the low level and instability of expression of nuclear transgenes, due to their rearrangement and/or silencing over time. RESULTS:We constructed dedicated vectors for Agrobacterium-mediated transformation carrying, within the T-DNA borders, the Paromomycin (Paro) selectable marker and an expression cassette containing the Luciferase (Luc) reporter gene. These vectors and newly developed co-cultivation methods were used to compare the efficiency, stability and insertion sites of Agrobacterium- versus electroporation-mediated transformation. The influence of different transformation methods, of the cell wall, of the virulence of different Agrobacterium strains, and of transgene orientation with respect to T-DNA borders were assessed. False positive transformants were more frequent in Agrobacterium-mediated transformation compared to electroporation, compensating for the slightly lower proportion of silenced transformants observed in Agrobacterium-mediated transformation than in electroporation. The proportion of silenced transformants remained stable after 20 cycles of subculture in selective medium. Next generation sequencing confirmed the nuclear insertion points, which occurred in exons or untraslated regions (UTRs) for 10 out of 10 Agrobacterium-mediated and 9 out of 13 of electroporation-mediated insertions. Electroporation also resulted in higher numbers of insertions at multiple loci. CONCLUSIONS:Due to its labor-intensive nature, Agrobacterium transformation of Chlamydomonas does not present significant advantages over electroporation, with the possible exception of its use in insertional mutagenesis, due to the higher proportion of within-gene, single-locus insertions. Our data indirectly support the hypothesis that rearrangement of transforming DNA occurs in the Chlamydomonas cell, rather than in the extracellular space as previously proposed.

Dataset Information

A novel electroporation system for efficient molecular delivery into Chlamydomonas reinhardtii with a 3-dimensional microelectrode.

Publications

A novel electroporation system for efficient molecular delivery into Chlamydomonas reinhardtii with a 3-dimensional microelectrode.

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