Transient Expression of Recombinant Small Interfering RNA to mRNA of \[\delta-Isoform\] Human Protein Kinase C in Lactuca Sativa Biomass

Anton Peterson, Victor Dosenko, Victoria Bidiuk, Maryna Korshevniuk, Vita Linovytska, Mykola Kuchuk

Abstract


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

\[\delta-isoform\]

 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).


Keywords


Plant expression system; Transient expression; Lactuca sativa; Recombinant small interfering RNA; Delta isoform of human protein kinase C

References


T. Novokhatska et al., “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, no. 1, pp. 78–84, 2014.

T. Novokhatska et al., “Correction of vascular hypercontractility in spontaneously hypertensive rats using shRNAs-induced delta protein kinase C gene silencing”, Eur. J. Pharmacol., vol. 718, no. 1-3, pp. 401–407, 2013. doi: 10.1016/j.ejphar.2013.08.003

A. Auerbach et al., “Uptake of dietary milk miRNAs by adult humans: a validation study”, F1000Res., vol. 721, no. 5, pp. 1–14, 2016. doi: 10.12688/f1000research.8548.1

K.W. Witwer et al., “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 Biology, vol. 10, no. 7, pp. 1080–1086, 2013. doi: 10.4161/rna.25246

K.W. Witwer and K.D. Hirschi, “Transfer and functional consequences of dietary microRNAs in vertebrates: Concepts in search of corroboration”, BioEssays, vol. 36, no. 4, pp. 394–406, 2014. doi: 10.1002/bies.201300150

L. Zhang et al., “Exogenous plant MIR168a specifically targets mammalian LDLRAP1: evidence of cross-kingdom regulation by microRNA”, Cell Res., vol. 22, no. 1, pp. 107–126, 2012. doi: 10.1038/cr.2011.158

S. Mlotshwa et al., “A novel chemopreventive strategy based on therapeutic microRNAs produced in plants”, Cell Res., vol. 25, no. 4, pp. 521–524, 2015. doi: 10.1038/cr.2015.25

K.-C. Kwon and H. Daniell, “Oral delivery of protein drugs bioencapsulated in plant cells”, Molecular Therapy, vol. 24, no. 8, pp. 1342–1350, 2016. doi: 10.1038/mt.2016.115

I.M. Gerasymenko et al., “Establishment of transgenic lettuce plants producing potentially anti-hypertensive shRNA”, Cytol. Genet., vol. 51, no. 1, pp. 1–7, 2017. doi: 10.3103/s0095452717010054

S. Marillonnet et al., “Systemic Agrobacterium tumefaciens–mediated transfection of viral replicons for efficient transient expression in plants”, Nature Biotechnol., vol. 23, no. 6, pp. 718–723, 2005. doi: 10.1038/nbt1094

O. Voinnet et al., “An enhanced transient expression system in plants based on suppression of gene silencing by the p19 protein of tomato bushy stunt virus”, Plant J., vol. 83, no. 4, pp. 949–956, 2003. doi: 10.1046/j.1365-313X.2003.01676.x

Agrobacterium Protocols, vol. 1, Methods in Molecular Biology, Kan Wang, Ed. Springer Science & Business Media, 2006.


GOST Style Citations






DOI: https://doi.org/10.20535/1810-0546.2017.3.95355

Refbacks

  • There are currently no refbacks.




Copyright (c) 2017 NTUU KPI