Branched polyethylenimine (PEI) might be considered the closest commercially available analog to the polymeric carrier developed here. PEI has been widely used for DNA delivery in the mammalian cells (52-55). The mechanisms of action of the PEI, as well as our developed polymer, might be the following: (i) condensing DNA, (ii) providing protection against potential nuclease degradation, and (iii) promoting association with the plasma membrane of a target cell to enhance entry into the cell. Following an uptake, DNA should be capable of escape from the endosome and be released into the cytoplasm to be capable of reaching the nucleus. We found that PEI exhibited significantly lower transformation efficiencies with yeast cells, compared with our oligoelectrolyte polymeric nanoscale carriers. Transformation of Saccharomyces cerevisiae yeast
Saccharomyces cerevisiae yeast is a unicellular eukaryotic organism most intensively used and studied in molecular and cell biology; in addition, it is widely used in biotechnology (6, 7, 16, 56). Although current transformation methodologies are enough sufficient for S. cerevisiae, we were interested to see if our oligoeletrolyte polymeric carriers could further enhance transformation efficiency.
Comparison of transformation efficiency using a circular episomal plasmid DNA (pYEp352 of 5.2 kb (containing URA3 gene as a selectable marker), Institute of Cell Biology, National Academy of Sciences of Ukraine, Lviv, Ukraine) and yeast Saccharomyces cerevisiae BY4742 MATα his3Δ leu2Δ lysΔ ura3Δ cells were performed. Ura+ transformants of S. cerevisiae was selected on a solid minimal modified Burkholder medium without uracil with transformants selected based on uracil independence.
Our polymeric carrier based transformation approach yielded 17,100 colonies of yeast transformants per 1 µg of DNA, while the standard electroporation protocol resulted in 1000 colonies, and chemical LiAc-based transformation yielded 8300 colonies (Figure 6).
Thus, there is no significant increase in the transformation efficiency when using our carrier with S. cerevisiae cells whose surface is well permeable to plasmid DNA. However, a distinct advantage in the transformation efficiency could be demonstrated when using yeast species such as Hansenula polymorpha and Pichia pastoris which have proven less emmenable to transformation in previous studies. These findings strongly suggest that the developed polymeric carrier possesses chemical characteristics necessary for efficient DNA delivery. We can hypothesize that such characteristics could include the hydrophobic core chain required for the improved penetration through the plasma membrane or possibly the positively charged polymer branches involved in binding DNA molecule, although this remains to be further investigated.
It is important to note that the polymeric carrier is highly efficiency for both transient and stable transformation when using either linearized or circular plasmid DNA. Moreover, there is no need for additional pretreatment steps to preparing competent cells or the requirement of special equipment for conducting the transformation. In addition, the developed method of yeast transformation is more convenient and rapid in use, and the proposed DNA carrier exhibits low toxicity and is not mutagenic (Supplementary Figures S3 and S4). This is unlike the widely used chemical-based LiAc method, in which costly geneticine antibiotic or other reagents are required for positive selection, and, thus, cells have to be incubated for long periods of time after transformation. Such prolonged cell incubation without selection makes experiments time consuming and decreases significantly the reproducibility of the results. A detailed comparison between the various transformation approaches can be found in Table 2.
In conclusion, we have developed a novel method for efficient delivery of DNA into yeast cells using polymeric nanocomposites. We clearly demonstrated this method is more efficient and reproducible than other currently used methods for genetic transformation such as LiAc and electroporation. The developed method yielded a higher number of genetic transformants for the yeast species H. polymorpha and P. pastoris which are known to have a very low efficiency using current methods. Thus, our polymeric carrier represents the first nanoscale carrier for DNA delivery into the yeast cells, an approach that previously had only been applied for DNA delivery into the mammalian cells.
This work was partly supported by the grants from WUBMRC (Ukraine-USA), CRDF (USA), and F-46 project of the National Academy of Sciences of Ukraine, as well as by the project funded by the Ministry of Education and Science of Ukraine. The authors thank V. Nazarko, PhD, A. Polupanov, Y. Ryabec, PhD, I. Bohovych, and Y. Pynyaha, PhD (all from the Institute of Cell Biology, NAS of Ukraine) for their great help and fruitful discussions.
Authors declare no competing interests.
Address correspondence to Rostyslav Stoika, Department of Regulation of Cell Proliferation and Apoptosis, Institute of Cell Biology, NAS of Ukraine, Drahomanov St. 14/16, Lviv, Ukraine. E-mail: [email protected]
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