Optimized conditions for transgenesis of the ascidian Ciona using square wave electroporation

Abstract

The protochordate Ciona has recently become an important model organism for the study of developmental gene regulation, in part because transient transgenic embryos can be produced rapidly and reliably using electroporation. Published methods are currently for the use of electroporation devices delivering exponentially decaying pulses. However, some workers have advocated the use of square wave electroporation devices for eukaryotic transgenics. This paper presents results of experiments to find optimal conditions for the use of square-wave electroporation in the ascidian Ciona. In the present analysis, a single pulse of 90 msec duration at 63-75 V/cm was found to give an optimal balance of a high proportion of cells transformed with the transgene, and a low level of abnormal development. Forty micrograms of DNA per electroporation is sufficient for effective visualization of reporter gene expression, although doses up to 100 μg provide higher proportions of transformed cells. In side-by-side comparison with exponential pulse electroporation, square wave pulses give similar penetrance of transgene expression, along with lower proportions of embryos with abnormal development at higher amounts of input DNA.

Purification of Mitochondrial Proteins HSP60 and ATP Synthase from Ascidian Eggs: Implications for Antibody Specificity

“…The ascidian myoplasm offers an accessible system to address questions concerning networks of mitochondria, such as their regulation, function, and how they are properly partitioned during cell division. Ascidians are amenable to an increasing battery of experimental approaches including micromanipulation, modification of gene expression or function, live imaging, genetics, proteomics [20], [24]–[28] and as we show here, biochemistry.…”

Multiple layers of complexity in cis‐regulatory regions of developmental genes

“…These reporter constructs are either injected into embryos to generate stable transformants (Venken and Bellen, 2007;Tasic et al, 2011;Frokjaer-Jensen et al, 2012), or electroporated (Funahashi and Nakamura, 2008;Vierra and Irvine, 2012), to observe the transient effect of the construct. In most cases, these small DNA fragments are sufficient to provide coherent functions in vivo.…”

Practical tips for imaging ascidian embryos

“…For a detailed protocol of electroporation, please refer to Christiaen et al . () and Vierra & Irvine ().…”

“…These promoters allow cell type-or lineagespecific observations. For a detailed protocol of elec- Christiaen et al (2009) and Vierra & Irvine (2012).…”