A Simple and Non-Invasive Method for Nuclear Transformation of Intact-walled Chlamydomonas reinhardtii

Title
A Simple and Non-Invasive Method for Nuclear Transformation of Intact-walled Chlamydomonas reinhardtii
Authors
허윤석
Issue Date
2014-07
Publisher
PLOS ONE
Series/Report no.
PLOS ONE ; Vol9 no.7 Article e101018 Startpage 1 Endpage 9
Abstract
Genetic engineering in microalgae is gaining attraction but nuclear transformation methods available so far are either inefficient or require special equipment. In this study, we employ positively charged nanoparticles, 3-aminopropyl- functionalized magnesium phyllosilicate (aminoclay, approximate unit cell composition of [H2N(CH2)3]8Si8Mg6O12(OH)4), for nuclear transformation into eukaryotic microalgae. TEM and EDX analysis of the process of transformation reveals that aminoclay coats negatively-charged DNA biomolecules and forms a self-assembled hybrid nanostructure. Subsequently, when this nanostructure is mixed with microalgal cells and plated onto selective agar plates with high friction force, cell wall is disrupted facilitating delivery of plasmid DNA into the cell and ultimately to the nucleus. This method is not only simple, inexpensive, and non-toxic to cells but also provides efficient transformation (5.036102 transformants/mg DNA), second only to electroporation which needs advanced instrumentation. We present optimized parameters for efficient transformation including pre-treatment, friction force, concentration of foreign DNA/aminoclay, and plasticity of agar plates. It is also confirmed the successful integration and stable expression of foreign gene in Chlamydomonas reinhardtii through molecular methods.
URI
https://doi.org/10.1371/journal.pone.0101018
http://dspace.inha.ac.kr/handle/10505/53462
ISSN
1932-6203
Appears in Collections:
College of Engineering(공과대학) > Biotechnology (생명공학) > Journal Papers, Reports(생명공학 논문, 보고서)
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