Transient Expression of Recombinant Small Interfering RNA to mRNA of \[\delta-Isoform\] Human Protein Kinase C in Lactuca Sativa Biomass
Keywords:Plant expression system, Transient expression, Lactuca sativa, Recombinant small interfering RNA, Delta isoform of human protein kinase C
Background. The perspective of research of small interfering RNA (siRNA) application in medical practice was proved by its efficiency of chemically synthesized siRNA in the experiment in vitro. The high cost of RNA production through chemical synthesis determines the importance of searching the biotechnological methods of obtaining these compounds. A promising area of biopharmaceuticals creation is a combination of active pharmaceutical substances with innovative tools of delivery, such as bio-encapsulation in plant cells.
Objective.The aim of the paper is testing of capabilities and efficiency of transient expression of the gene encoding the small interfering RNA to mRNA
of human protein kinase C \[(anti-PKC\delta)\] in edible raw plant biomass of lettuce Lactuca sativa.
Methods. Obtaining the biomass which accumulates anti-PKCd small interfering RNA molecules was carried out by the method that was developed by our research group for the expression of genes encoding recombinant proteins. Evaluation of small interfering RNA accumulation was performed by a reverse transcription method with the subsequent Real-Time Polymerase Chain Reaction (PCR).
Results. The level of accumulation of target product is 22 fmol/g of lyophilized plant biomass.
Conclusions. The method of transient expression allows obtaining the lettuce Lactuca sativa biomass, which contains small interfering RNA recombinant anti-PKCd (detected by a reverse transcription method with the subsequent Real-Time PCR).
Novokhatska T, Klymenko K, Dosenko V, Soloviev A. Fact or fancy: Does the high level of PKC-delta gene expression contribute to potassium channels malfunction at arterial hypertension and diabetes? Pharmacology and Drug Toxicology. 2014;1:78-84.
Novokhatska T, Tishkin S, Dosenko V, Boldyriev A, Ivanova I, Strielkov I, et al. Correction of vascular hypercontractility in spontaneously hypertensive rats using shRNAs-induced delta protein kinase C gene silencing. Eur J Pharmacol. 2013 Oct;718(1-3):401-7. DOI 10.1016/j.ejphar.2013.08.003
Auerbach A, Vyas G, Li A, Halushka M, Witwer K. Uptake of dietary milk miRNAs by adult humans: a validation study. F1000Res. 2016 Apr 22;5:721. DOI 10.12688/f1000research.8548.1
Witwer KW, McAlexander MA, Queen SE, Adams RJ. Real-time quantitative PCR and droplet digital PCR for plant miRNAs in mammalian blood provide little evidence for general uptake of dietary miRNAs: Limited evidence for general uptake of dietary plant xenomi Rs. RNA Biol. 2013 Jul;10(7):1080-6. DOI 10.4161/rna.25246
Witwer KW, Hirschi KD. Transfer and functional consequences of dietary microRNAs in vertebrates: Concepts in search of corroboration. Bioessays. 2014 Apr;36(4):394-406. DOI 10.1002/bies.201300150
Zhang L, Hou D, Chen X, Li D, Zhu L, Zhang Y, et al. Exogenous plant MIR168a specifically targets mammalian LDLRAP1: evidence of cross-kingdom regulation by microRNA. Cell Res. 2012 Jan;22(1):107-26. DOI 10.1038/cr.2011.158
Mlotshwa S, Pruss GJ, MacArthur JL, Endres MW, Davis C, Hofseth LJ, et al. A novel chemopreventive strategy based on therapeutic microRNAs produced in plants. Cell Res. 2015 Apr;25(4):521-4. DOI 10.1038/cr.2015.25
Kwon KC, Daniell H. Oral delivery of protein drugs bioencapsulated in plant cells. Mol Ther. 2016 Aug;24(8):1342-50. DOI 10.1038/mt.2016.115
Gerasymenko IM, Kleschevnikov VV, Kedlian VR, Sakhno LO, Arbuzova IA, Sheludko YV, et al. Establishment of transgenic lettuce plants producing potentially anti-hypertensive shRNA. Cytol Genet. 2017;51(1):1-7. DOI 10.3103/s0095 452717010054
Marillonnet S, Thoeringer C, Kandzia R, Klimyuk V, Gleba Y. Systemic Agrobacterium tumefaciens-mediated transfection of viral replicons for efficient transient expression in plants. Nature Biotechnol. 2005 May;23(6):718-23. doi: 10.1038/nbt1094
Voinnet O, Rivas S, Mestre P, Baulcombe D. An enhanced transient expression system in plants based on suppression of gene silencing by the p19 protein of tomato bushy stunt virus. Plant J. 2003 Mar;33(5):949-56. doi: 10.1046/j.1365-313X.2003.01676.x
Methods in molecular biology. In: Kan Wang, editor. Agrobacterium Protocols. New York: Springer-Verlag; 2016. 484 p.
How to Cite
Copyright (c) 2017 Igor Sikorsky Kyiv Polytechnic Institute
This work is licensed under a Creative Commons Attribution 4.0 International License.
The ownership of copyright remains with the Authors.
Authors may use their own material in other publications provided that the Journal is acknowledged as the original place of publication and National Technical University of Ukraine “Igor Sikorsky Kyiv Polytechnic Institute” as the Publisher.
Authors are reminded that it is their responsibility to comply with copyright laws. It is essential to ensure that no part of the text or illustrations have appeared or are due to appear in other publications, without prior permission from the copyright holder.IBB articles are published under Creative Commons licence:
- Authors retain copyright and grant the journal right of first publication with the work simultaneously licensed under CC BY 4.0 that allows others to share the work with an acknowledgement of the work's authorship and initial publication in this journal.
- Authors are able to enter into separate, additional contractual arrangements for the non-exclusive distribution of the journal's published version of the work (e.g., post it to an institutional repository or publish it in a book), with an acknowledgement of its initial publication in this journal.
- Authors are permitted and encouraged to post their work online (e.g., in institutional repositories or on their website) prior to and during the submission process, as it can lead to productive exchanges, as well as earlier and greater citation of published work.