Abstract
Cell-penetrating peptides (CPPs) are a class of short peptides, which have the ability of intracellular delivery. CPPs can also be used as an ideal transfection tool whereby biomacromolecules can be transferred as cargo into recipient cells. Here, a synthesized cell-penetrating peptide (R9)-mediated DNA-free transfection is presented. DNA sequences of nine tandem arginine (R9), one cysteine (cys), reporter mCherry, and histidine label were sequentially constructed into pET-45b( +) expression vector and transformed into the Escherichia coli BL21(DE3) strain. The R9-cys-mCherry fusion protein was obtained by prokaryotic expression. The Arabidopsis root tips, Chinese cabbage microspores, and 3-wk-old microspore embryos (MDEs) were used as transfected recipients. With a working concentration of R9-cys-mCherry ranging from 10 to 100 μg mL−1 and overnight incubation, R9-cys-mCherry protein can be translocated into the recipient mentioned above. The transfection efficiency of root tips reached 100%, and that of microspores and MDEs was 8.13% and 94.79%, respectively. A CPP-mediated DNA-free transfection system was built in dicots providing a technical basis for DNA-free CRISPR RNP (ribonucleoprotein) intracellular delivery in the future.
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Acknowledgements
Special thanks to the kind Senior scientist Dr. Kim Boutilier and every nice lab mate in PDS cluster, Bioscience Wageningen. The vectors in this study were kindly provided by Dr. Andriy Bilichak.
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The authors declared that they have no conflicts of interest in this work. We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted. This paper is supported by China Scholarship Council (CSC) (201608210147); Provincial doctoral research initiation funding project (2020-BS-037); fellowship of China Postdoctoral Science Foundation (2021M693846).
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Wu, H., Zhang, Z., Zhu, K. et al. Cell-penetrating peptide-mediated transfection of proteins into tissues of Arabidopsis and Chinese cabbage. In Vitro Cell.Dev.Biol.-Plant 58, 28–34 (2022). https://doi.org/10.1007/s11627-021-10221-0
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DOI: https://doi.org/10.1007/s11627-021-10221-0