Highly efficient transformation of the model zygnematophycean alga Closterium peracerosum-strigosum-littorale complex by square-pulse electroporation.

The zygnematophycean algae occupy an important phylogenetic position as the closest living relatives of land plants. Reverse genetics is quite useful for dissecting the functions of genes. However, this strategy requires genetic transformation, and there are only a few reports of successful transformation in zygnematophycean algae. Here, we established a simple and highly efficient transformation technique for the unicellular zygnematophycean alga Closterium peracerosum-strigosum-littorale complex using a square electric pulse-generating electroporator without the need for cell wall removal. Using this method, the transformation efficiency increased > 100-fold compared with our previous study using particle bombardment. We also succeeded in performing CRISPR/Cas9-based gene knockout using this new method. Our method requires only small amounts of labor, time and incubator space. Moreover, our technique could also be utilized to transform other charophycean algae with available genome information by optimizing the electric pulse conditions.

In vivo silencing of a molecular target by short interfering RNA electroporation: tumor vascularization correlates to delivery efficiency.

Screening for a molecular target for cancer therapy requires multiple steps, of which an important one is evaluation of the knockdown effect of the target molecule on pregrown xenograft tumors. However, methods currently used for local administration of knockdown reagents, such as short interfering RNA (siRNA), are not satisfactory as to simplicity and efficiency. We established an electroporation method involving a constant voltage and "plate and fork" type electrodes and used it for in vivo delivery of siRNA. The delivery efficiency correlated to the electric current. The electric current correlated to the microvascular density and vascular endothelial growth factor (VEGF) expression and exhibited a threshold that guaranteed efficient delivery. Consequently, we showed that the vascularization and VEGF expression in tumors determined the efficiency of delivery of siRNA by electroporation. VEGF was chosen as a model target. VEGF siRNA electroporation suppressed the growth of tumors exhibiting high VEGF expression to less than 10% of the control level, but it had no effect on low VEGF-expressing tumors. Notably, a long interval (20 days) of electroporation was enough to obtain a satisfactory effect. Systemically injected siRNA could also be delivered into tumors by this method. Our data will provide the technical basis for in vivo electroporation, and this simple and efficient siRNA delivery method is applicable to in vivo comprehensive screening for a molecular target.

Optimized universal protocol for electroporation of both coagulase-positive and -negative Staphylococci.

Electroporation is a common technique necessary for genomic manipulation of Staphylococci. However, because this technique has too low efficiency to be applied to some Staphylococcal species and strains, especially to coagulase-negative Staphylococcus (CNS) isolates, basic researches on these clinically important Staphylococci are limited. Here we report on the optimization of electroporation parameters and conditions as well as on the generation of a universal protocol that can be efficiently applicable to both CNS and Coagulase-positive Staphylococci (CPS). This protocol could generate transformants of clinical Staphylococcus epidermidis isolate, with an efficiency of up to 1400 CFU/µg of plasmid DNA. Transformants of 12 other clinically important Staphylococcal species, including CNS and CPS, were also generated with this protocol. To our knowledge, this is the first report on successful electroporation in nine these Staphylococcal species.

Efficient transformation of Staphylococcus aureus using multi-pulse electroporation.

A new multi-pulse electroporation system was evaluated to transform Staphylococcus aureus. Compared to the conventional electroporation system, it yielded high transformation efficiency to obtain more than 3.9×105S. aureus RN4220 transformed cells/1µg plasmid DNA using a single electroporation by manipulating the poring pulse and transfer pulse.