Gli autori vorrebbero riconoscere il finanziamento dal Giappone Scienza e della Tecnologia Agenzia esplorativa di ricerca per la tecnologia avanzata (JST-ERATO), la New Energy and Industrial Technology Development Organization (NEDO), e la Croce-ministeriale strategico del programma per la promozione dell&#39;innovazione (SIP), il Giappone .

1. preparazione di formulazioni Peptide-Based <ol><li> Preparare soluzioni di ciascun peptide come segue: (KH) 9 -BP100 (1 mg / ml o 800 nM), Cytcox- (KH) 9 (1 mg / ml), BP100 (1 mg / ml) e (BP100) 2 K 8 (70 mM). Pesare la quantità necessaria di ciascun peptide in una provetta da 1,5 ml microcentrifuga e aggiungere acqua ultrapura autoclavato per sciogliere il peptide. Mescolare bene pipettando ripetutamente fino ad ottenere una soluzione limpida. </li><li> Amplifica e purificare il DNA plasmide (pDNA), a doppia elica del DNA (dsDNA) e l&#39;RNA a doppio filamento (dsRNA) secondo metodologie molecolari standard. In provette da 1,5 ml microcentrifuga, fare soluzioni di riserva con una concentrazione di 1 mg / ml (PDNA e dsDNA) o 400 Nm (dsRNA). </li><li> Preparare la soluzione proteica magazzino con una concentrazione di 7 pM, sciogliendo 1 mg di proteina (es., Alcool deidrogenasi, ADH) polvere in 1 ml di soluzione di carbonato di sodio (0,1 M, pH 9). Etichetta la proteina con FLe sonde luorescent come la rodamina B (RhB) secondo protocolli standard per consentire la visualizzazione microscopica della proteina consegnato nelle cellule. </li><li> Unire rispettivi componenti in una provetta da 1,5 ml microcentrifuga. <ol><li> Per le formulazioni peptide-PDNA mira il citoplasma, aggiungere 6,4 ml di (KH) 9 -BP100 (1 mg / ml) a 20 ml di pDNA (1 mg / ml) e mescolare bene pipettando. Lasciare il composto si stabilizzi per 15 minuti a 25 ° C. Aggiungere 773,6 ml di acqua ultrapura autoclavato per diluire la soluzione ad un volume finale di 800 microlitri. Utilizzare il costrutto pDNA progettata per l&#39;espressione nucleare (P35S-RLuc-NOS, tabella 1). </li><li> Per le formulazioni peptide-PDNA mira i mitocondri, aggiungere 6,6 ml di Cytcox- (KH) 9 (1 mg / ml) a 20 ml di pDNA (1 mg / ml) e mescolare bene pipettando. Lasciare il composto si stabilizzi per 15 minuti a 25 ° C e aggiungere 2,4 ml di BP100 (1 mg / ml). Lasciare il composto si stabilizzi per 15 minuti a 256; C per altri 15 min. Aggiungere 771 ml di acqua ultrapura autoclavato per diluire la soluzione ad un volume finale di 800 microlitri. Utilizzare il costrutto pDNA progettata per l&#39;espressione mitocondriale (pDONR-COX2: rluc, Tabella 1). </li><li> Per le formulazioni peptide-dsDNA, aggiungere 5,1 ml di (KH) 9 -BP100 (1 mg / ml) a 8 ml di dsDNA (1 mg / ml) e mescolare bene pipettando. Lasciare il composto si stabilizzi per 15 minuti a 25 ° C. Aggiungere 786,9 ml di acqua ultrapura autoclavato per diluire la soluzione ad un volume finale di 800 microlitri. </li><li> Per le formulazioni peptide-dsRNA, aggiungere 50 ml di (KH) 9 -BP100 (800 nm) a 50 ml di dsRNA (400 Nm) e mescolare bene pipettando. Lasciare il composto si stabilizzi per 15 minuti a 25 ° C. Aggiungere 700 ml di acqua RNase-free per diluire la soluzione ad un volume finale di 800 microlitri. </li><li> Per le formulazioni peptide-proteina, aggiungere 16 ml di (BP100) 2 K 8 (70 micron) a 16 ml di ADH (7 micron) e mescolareben pipettando. Lasciare il composto si stabilizzi per 15 minuti a 25 ° C. Aggiungere 768 ml di acqua ultrapura autoclavato per diluire la soluzione ad un volume finale di 800 microlitri. </li></ol></li><li> Lasciare le formulazioni di stabilizzarsi per 15 min a 25 ° C. </li></ol> 2. Caratterizzazione di formulazioni Peptide-Based <ol><li> Trasferire ogni soluzione (800 ml) in una vaschetta per dispersione della luce dinamica (DLS) analisi. Determinare il diametro e polidispersità indice idrodinamico di complessi formati con un Nanosizer zeta, utilizzando un 633 nm laser He-Ne a 25 ° C con un angolo di rilevamento retrodiffusione di 173 °. </li><li> misurazioni seguente formato, trasferire ciascuna soluzione (800 ml) in una cella capillare piegato per misure potenziali zeta a parametri di default di costante dielettrica, indice di rifrazione e la viscosità dell&#39;acqua a 25 ° C. </li><li> Osservare un piccolo volume di soluzione complessa, utilizzata per l&#39;analisi DLS, mediante microscopia a forza atomica (AFM). Cassetta 10 ml di soluzione complessa sulla superficie spaccati appena esposto di un foglio di mica e lasciare la mica all&#39;aria secca durante la notte in un piatto di plastica coperto petri. </li><li> Acquisire un&#39;immagine dei complessi in aria a 25 ° C utilizzando un cantilever silicio con costante elastica di 1,3 N / m in modalità 27,28 maschiatura. </li></ol> 3. L&#39;infiltrazione di foglie delle piante <ol><li> Utilizzare 3 settimane vecchi impianti terreno coltivati (Arabidopsis thaliana 24, Nicotiana benthamiana 24 o pioppo 26). Trasfezione almeno 3 foglie per servire come triplice copia per la quantificazione dell&#39;espressione genica o la consegna di proteine. </li><li> Caricare un 1 ml siringa di plastica senza ago con 100 ml di soluzione completa per la trasfezione di una foglia. Posizionare la punta della siringa sulla superficie abassiale della foglia. </li><li> Premere la punta della siringa contro la foglia leggermente spingere il pistone della siringa lentamente esercitando una contropressione con l&#39;ibarretta NDICE di una mano lattice guantata dal lato opposto. infiltrazione successo può osservare come la diffusione di una zona impregnato d&#39;acqua nella foglia. Etichettare le foglie infiltrati per facilitarne l&#39;identificazione. </li><li> Incubare la foglia transfettate per durate ottimizzati dopo infiltrazione con peptide-pDNA (12 ore), il peptide-dsDNA (12 ore), il peptide-dsRNA (9-48 ore) e peptide-proteina (6 ore) formulazioni nelle seguenti condizioni: 16 luce hr / 8 ore buio a 22 ° C per A. thaliana e pioppo, o 24 ore di luce costante a 29 ° C per N. benthamiana. </li></ol> 4. Valutazione di Transfection efficienza <ol><li> Asportare l&#39;intera foglia transfettate di impianti più piccoli (ad es., A. thaliana), o una sezione di 1 cm 2 della regione infiltrato per impianti più grandi (ad es., N. benthamiana). </li><li> Per gli esperimenti di trasfezione utilizzando Renilla luciferasi (Rluc) giornalista vettore, determinare il transfectioefficienza n quantitativamente utilizzando un kit di Rluc Assay. <ol><li> Mettere ogni foglia escissa o sezione foglia in una provetta da 1,5 ml. Aggiungere 100 ml di 1 × Rluc Assay Lysis Buffer per provetta. Allo stesso modo, preparare lisati di foglie di controllo non transfettate (triplice copia). </li><li> Macinare la foglia con un pestello omogeneizzazione e incubare il lisato risultante a 25 ° C per 6 - 10 ore. </li><li> Centrifugare il lisato a 12.470 × g in una microcentrifuga per 1 min. Trasferimento 20 ml di lisato eliminato ad un bene in una micropiastra a 96 pozzetti, e utilizzare il volume rimanente per la quantificazione della concentrazione totale di proteine ​​utilizzando un kit di test BCA Protein secondo il protocollo del produttore. </li><li> Aggiungere 100 ml di 1 × Rluc Assay Substrate (diluito con il tampone Rluc Assay) nel pozzo e mescolare lentamente pipettaggio. Posizionare la micropiastra in un lettore di micropiastre multimodale e avviare la misurazione. </li><li> Sottrarre la luminescenza di fondo (media del non-trasfettate triplice copia) dalla luminescenza di ogni campione sperimentale. Calcolare il rapporto di fotoluminescenza (unità di luce relativa, RLU) per la quantità di proteina (mg). </li></ol></li><li> Per gli esperimenti di trasfezione utilizzando verde proteina fluorescente (GFP) vettore giornalista o la proteina fluorescente (ad es., ADH-RhB), osservare la fluorescenza utilizzando un microscopio confocale a scansione laser. <ol><li> Tagliare i bordi di una foglia intera (per facilitare la rimozione di spazi aerei), mentre una foglia sezionata può essere utilizzato così com&#39;è. Rimuovere lo stantuffo di una siringa da 10 ml e posizionare ogni sezione escissa foglia o foglia nella siringa. </li><li> Sostituire il pistone e spingere delicatamente sul fondo della siringa senza schiacciare la foglia. Attingere l&#39;acqua nella siringa fino a circa mezzo pieno. </li><li> Puntare la siringa verso l&#39;alto e spingere lo stantuffo per eliminare l&#39;aria dalla siringa attraverso la punta. Coprire la punta della siringa e tirare lentamente lo stantuffo per espellere l&#39;aria dal leaf. Ripetere questa operazione più volte fino a quando la foglia appare traslucido. </li><li> Coprire la superficie di un vetrino da microscopio con nastro adesivo. Tagliare un&#39;area quadrata sul nastro abbastanza grande per accogliere il campione foglia con una lama, e sbucciare il pezzo quadrato di nastro adesivo fuori con le pinze per creare una camera di campione. Il nastro servirà come distanziale fra il vetrino e coprioggetto. </li><li> Posizionare la foglia nella camera con la superficie abaxial rivolta verso l&#39;alto e riempire la zona di camera rimanente con acqua. Sigillare la foglia all&#39;interno della camera con vetrino coprioggetti e fissare i bordi del coprioggetto con nastro adesivo. </li><li> Esaminare il campione foglia utilizzando il microscopio confocale a scansione laser sotto un obiettivo 40X o di un obiettivo ad immersione 63X acqua. GFP o RhB fluorescenza possono essere visualizzate a lunghezze d&#39;onda di eccitazione di 488 nm o 555 nm, rispettivamente. </li></ol></li></ol>

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	<li>Altman, A., Hasegawa, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Plant biotechnology and agriculture: Prospects for the 21st century. , (2011).</li><li>Horn, M. E., Woodard, S. L., Howard, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+molecular+farming:+systems+and+products.">Plant molecular farming: systems and products.</a> Plant Cell Rep. 22, 711-720 (2004).</li><li>Gelvin, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agrobacterium-mediated+plant+transformation:+the+biology+behind+the+&amp;#34;gene-jockeying&amp;#34;+tool.">Agrobacterium-mediated plant transformation: the biology behind the &amp;#34;gene-jockeying&amp;#34; tool.</a> Microbiol. Mol. Biol. Rev. 67, 16-37 (2003).</li><li>Klein, T. M., Wolf, E. D., Wu, R., Sanford, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-velocity+microprojectiles+for+delivering+nucleic+acids+into+living+cells.">High-velocity microprojectiles for delivering nucleic acids into living cells.</a> Nature. 327, 70-73 (1987).</li><li>Fromm, M., Taylor, L. P., Walbot, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+genes+transferred+into+monocot+and+dicot+plant+cells+by+electroporation.">Expression of genes transferred into monocot and dicot plant cells by electroporation.</a> Proc. Natl. Acad. Sci. U. S. A. 82, 5824-5828 (1985).</li><li>Krens, F. A., Molendijk, L., Wullems, G. J., Schilperoort, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+transformation+of+plant+protoplasts+with+Ti-plasmid+DNA.">In vitro transformation of plant protoplasts with Ti-plasmid DNA.</a> Nature. 296, 72-74 (1982).</li><li>Birch, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+transformation:+problems+and+strategies+for+practical+application.">Plant transformation: problems and strategies for practical application.</a> Annu. Rev. Plant Physiol. Plant Mol. Biol. 48, 297-326 (1997).</li><li>Newell, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+transformation+technology.+Developments+and+applications.">Plant transformation technology. Developments and applications.</a> Mol. Biotechnol. 16, 53-65 (2000).</li><li>Nielsen, K. M., Bones, A. M., Smalla, K., van Elsas, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Horizontal+gene+transfer+from+transgenic+plants+to+terrestrial+bacteria--a+rare+event?.">Horizontal gene transfer from transgenic plants to terrestrial bacteria--a rare event?.</a> FEMS Microbiol. Rev. 22, 79-103 (1998).</li><li>Chuah, J. A., Kaplan, D., Numata, K., Cai, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+25,+Engineering+peptide-based+carriers+for+drug+and+gene+delivery.">Chapter 25, Engineering peptide-based carriers for drug and gene delivery.</a> Engineering in Translational Medicine. , 667-689 (2014).</li><li>Nitta, S., Numata, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biopolymer-based+nanoparticles+for+drug/gene+delivery+and+tissue+engineering.">Biopolymer-based nanoparticles for drug/gene delivery and tissue engineering.</a> Int. J. Mol. Sci. 14, 1629-1654 (2013).</li><li>Numata, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poly(amino+acid)s/polypeptides+as+potential+functional+and+structural+materials.">Poly(amino acid)s/polypeptides as potential functional and structural materials.</a> Polym. J. 47, 537-545 (2015).</li><li>Numata, K., Kaplan, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silk-based+delivery+systems+of+bioactive+molecules.">Silk-based delivery systems of bioactive molecules.</a> Adv. Drug Deliv. Rev. 62, 1497-1508 (2010).</li><li>Numata, K., Subramanian, B., Currie, H. A., Kaplan, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioengineered+silk+protein-based+gene+delivery+systems.">Bioengineered silk protein-based gene delivery systems.</a> Biomaterials. 30, 5775-5784 (2009).</li><li>Chugh, A., Eudes, F., Shim, Y. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-penetrating+peptides:+nanocarrier+for+macromolecule+delivery+in+living+cells.">Cell-penetrating peptides: nanocarrier for macromolecule delivery in living cells.</a> IUBMB Life. 62, 183-193 (2010).</li><li>Eggenberger, K., Mink, C., Wadhwani, P., Ulrich, A. S., Nick, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+the+peptide+Bp100+as+a+cell-penetrating+tool+for+the+chemical+engineering+of+actin+filaments+within+living+plant+cells.">Using the peptide Bp100 as a cell-penetrating tool for the chemical engineering of actin filaments within living plant cells.</a> ChemBioChem. 12, 132-137 (2011).</li><li>Numata, K., Kaplan, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silk-based+gene+carriers+with+cell+membrane+destabilizing+peptides.">Silk-based gene carriers with cell membrane destabilizing peptides.</a> Biomacromolecules. 11, 3189-3195 (2010).</li><li>Numata, K., Hamasaki, J., Subramanian, B., Kaplan, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+delivery+mediated+by+recombinant+silk+proteins+containing+cationic+and+cell+binding+motifs.">Gene delivery mediated by recombinant silk proteins containing cationic and cell binding motifs.</a> J. Control. Release. 146, 136-143 (2010).</li><li>Numata, K., Mieszawska-Czajkowska, A. J., Kvenvold, L. A., Kaplan, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silk-based+nanocomplexes+with+tumor-homing+peptides+for+tumor-specific+gene+delivery.">Silk-based nanocomplexes with tumor-homing peptides for tumor-specific gene delivery.</a> Macromol. Biosci. 12, 75-82 (2012).</li><li>Numata, K., Reagan, M. R., Goldstein, R. H., Rosenblatt, M., Kaplan, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spider+silk-based+gene+carriers+for+tumor+cell-specific+delivery.">Spider silk-based gene carriers for tumor cell-specific delivery.</a> Bioconjugate Chem. 22, 1605-1610 (2011).</li><li>Chuah, J. A., Yoshizumi, T., Kodama, Y., Numata, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+introduction+into+the+mitochondria+of+Arabidopsis+thaliana+via+peptide-based+carriers.">Gene introduction into the mitochondria of Arabidopsis thaliana via peptide-based carriers.</a> Sci. Rep. 5, 7751 (2015).</li><li>Numata, K., Yoshizumi, T., Kodama, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+transformation+method.">Plant transformation method.</a> Patent. , (2015).</li><li>Ng, K. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+delivery+of+proteins+in+intact+plants+via+fusion+peptides.">Intracellular delivery of proteins in intact plants via fusion peptides.</a> PLoS ONE. 11, e0154081 (2016).</li><li>Lakshmanan, M., Kodama, Y., Yoshizumi, T., Sudesh, K., Numata, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+and+efficient+gene+delivery+into+plant+cells+using+designed+peptide+carriers.">Rapid and efficient gene delivery into plant cells using designed peptide carriers.</a> Biomacromolecules. 14, 10-16 (2012).</li><li>Lakshmanan, M., Yoshizumi, T., Sudesh, K., Kodama, Y., Numata, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Double-stranded+DNA+introduction+into+intact+plants+using+peptide-DNA+complexes.">Double-stranded DNA introduction into intact plants using peptide-DNA complexes.</a> Plant Biotechnol. 32, 39-45 (2015).</li><li>Numata, K., Ohtani, M., Yoshizumi, T., Demura, T., Kodama, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+gene+silencing+in+plants+via+synthetic+dsRNA+and+carrier+peptide.">Local gene silencing in plants via synthetic dsRNA and carrier peptide.</a> Plant Biotechnol. J. 12, 1027-1034 (2014).</li><li>Numata, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzymatic+degradation+processes+of+poly[(R)-3-hydroxybutyric+acid]+and+poly[(R)-3-hydroxybutyric+acid-co-(R)-3-hydroxyvaleric+acid]+single+crystals+revealed+by+atomic+force+microscopy:+effects+of+molecular+weight+and+second-monomer+composition+on+erosion+rates.">Enzymatic degradation processes of poly[(R)-3-hydroxybutyric acid] and poly[(R)-3-hydroxybutyric acid-co-(R)-3-hydroxyvaleric acid] single crystals revealed by atomic force microscopy: effects of molecular weight and second-monomer composition on erosion rates.</a> Biomacromolecules. 6, 2008-2016 (2005).</li><li>Numata, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adsorption+of+biopolyester+depolymerase+on+silicon+wafer+and+poly[(R)-3-hydroxybutyric+acid]+single+crystal+revealed+by+real-time+AFM.">Adsorption of biopolyester depolymerase on silicon wafer and poly[(R)-3-hydroxybutyric acid] single crystal revealed by real-time AFM.</a> Macromol. Biosci. 6, 41-50 (2006).</li><li>Kawai, S., Nishizawa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+procedure+for+DNA+transfection+with+polycation+and+dimethyl+sulfoxide.">New procedure for DNA transfection with polycation and dimethyl sulfoxide.</a> Mol. Cell. Biol. 4, 1172-1174 (1984).</li><li>Liu, F., Song, Y., Liu, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrodynamics-based+transfection+in+animals+by+systemic+administration+of+plasmid+DNA.">Hydrodynamics-based transfection in animals by systemic administration of plasmid DNA.</a> Gene Ther. 6, 1258-1266 (1999).</li><li>Luo, D., Saltzman, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancement+of+transfection+by+physical+concentration+of+DNA+at+the+cell+surface.">Enhancement of transfection by physical concentration of DNA at the cell surface.</a> Nat. Biotechnol. 18, 893-895 (2000).</li><li>Boynton, J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chloroplast+transformation+in+Chlamydomonas+with+high+velocity+microprojectiles.">Chloroplast transformation in Chlamydomonas with high velocity microprojectiles.</a> Science. 240, 1534-1538 (1988).</li><li>Sikdar, R. S., Serino, G., Chaudhuri, S., Maliga, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plastid+transformation.">Plastid transformation.</a> Arabidopsis thaliana Plant Cell Rep. 18, 20-24 (1998).</li><li>Svab, Z., Hajdukiewicz, P., Maliga, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+transformation+of+plastids+in+higher+plants.">Stable transformation of plastids in higher plants.</a> Proc. Natl. Acad. Sci. U. S. A. 87, 8526-8530 (1990).</li><li>Liu, Y. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complementation+of+plant+mutants+with+large+genomic+DNA+fragments+by+a+transformation-competent+artificial+chromosome+vector+accelerates+positional+cloning.">Complementation of plant mutants with large genomic DNA fragments by a transformation-competent artificial chromosome vector accelerates positional cloning.</a> Proc. Natl. Acad. Sci. U. S. A. 96, 6535-6540 (1999).</li><li>Park, H. S., Lee, B. M., Salas, G. M., Srivatanakul, M., Smith, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shorter+T-DNA+or+additional+virulence+genes+improve+Agrobactrium-mediated+transformation.">Shorter T-DNA or additional virulence genes improve Agrobactrium-mediated transformation.</a> Theor. Appl. Genet. 101, 1015-1020 (2000).</li><li>Frary, A., Hamilton, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficiency+and+stability+of+high+molecular+weight+DNA+transformation:+an+analysis+in+tomato.">Efficiency and stability of high molecular weight DNA transformation: an analysis in tomato.</a> Transgenic Res. 10, 121-132 (2001).</li><li>Que, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maize+transformation+technology+development+for+commercial+event+generation.">Maize transformation technology development for commercial event generation.</a> Front. Plant Sci. 5, 379 (2014).</li><li>Miranda, A., Janssen, G., Hodges, L., Peralta, E. G., Ream, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agrobacterium+tumefaciens+transfers+extremely+long+T-DNAs+by+a+unidirectional+mechanism.">Agrobacterium tumefaciens transfers extremely long T-DNAs by a unidirectional mechanism.</a> J. Bacteriol. 174, 2288-2297 (1992).</li><li>Loeb, T. A., Spring, L. M., Steck, T. R., Reynolds, T. L., Bajaj, Y. P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+wheat+(Triticum+spp.).">Transgenic wheat (Triticum spp.).</a> Transgenic Crops I. , 14-36 (2000).</li></ol>

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Passaggi critici all&#39;interno del protocollo che sono stati identificati empiricamente sono discussi. Con l&#39;infiltrazione della siringa, le formulazioni a base di peptidi vengono introdotti gli spazi aerei all&#39;interno del foglio della pianta attraverso gli stomi. Per garantire l&#39;assimilazione massima della soluzione, il processo di infiltrazione deve essere effettuata quando le piante sono in condizioni favorevoli alla stomi ie., Dotate di acqua sufficiente e durante il periodo di luce. Per quanto riguarda la preparazione di complessi trasfezione, per formulazioni che coinvolgono una combinazione di due peptidi (consegna DNA mitocondriale targeting), la sequenza per aggiunta di ciascun componente è cruciale e non dovrebbe essere invertito. Ci sono molte modifiche plausibili alla procedura. Un&#39;altra opzione per l&#39;infiltrazione di cellule vegetali è l&#39;uso di infiltrazione sotto vuoto, che è in grado di introdurre la soluzione complesso in piante intere e / o tessuti parziali inclusimeristemi apicali. Per questa procedura alternativa, piantine vengono immersi nella soluzione di trasfezione, e in base alla pressione generata dal vuoto, i complessi peptide-carico vengono forzati attraverso gli stomi e nelle cellule vegetali (Yoshizumi, T., dati non pubblicati). Espressione genica transitoria, sulla base di studi utilizzando cellule animali, ha dimostrato di aumentare con più elevate concentrazioni di DNA 29-31. È importante notare che il rapporto di peptide di DNA influisce sulle proprietà biofisiche (dimensioni, carica superficiale) di complessi (figura 4), che influenza l&#39;efficienza di trasfezione; di conseguenza, il rapporto ottimale deve essere mantenuta anche con una maggiore concentrazione di DNA. Uno dei principali vantaggi nell&#39;uso di peptidi come agente di consegna gene / proteina è che la sua sequenza è suscettibile di sintonizzazione ad espletare una funzione desiderata. Ad esempio, il dominio mitocondriale targeting del peptide carrier descritto in questo Study possono essere sostituiti con sequenze cloroplastici o perossisomiali targeting per la localizzazione di questi organelli. Mentre trasformazione cloroplasto è possibile in diverse piante usando biolistics 32-34, né l&#39;Agrobacterium né il metodo biolistic potrebbe introdurre geni nei mitocondri o altri organelli oltre il nucleo (Tabella 2). Non ci sono limitazioni host-gamma rigidi per trasfezione peptide-based, a differenza del metodo basata su Agrobacterium (Tabella 2). Finora, formulazioni per la trasfezione di A. thaliana, N. benthamiana e pioppo sono stati ottimizzati (tabella 1), ma il metodo può essere applicato anche per la Nicotiana tabacum, pomodoro cultivar Micro-Tom, il riso (sulla base di esperimenti preliminari), e altre piante mono e dicotiledoni. Finora, non vi è alcun limite noto dimensioni transgene quando peptidi sono utilizzati unvettori s trasfezione. Al contrario, grandi molecole di DNA sono stati trovati per ridurre l&#39;efficienza di trasformazione di Agrobacterium metodi mediata 35,36, ed un limite superiore per dimensione per transgeni di circa 200 kb è stato segnalato 37-39. Utilizzando biolistics, invece, grandi frammenti di DNA possono essere tranciate durante la preparazione o la consegna in piante 38. Sebbene nessun limite superiore è stato determinato per la trasformazione biolistica finora, vincoli fisici sono stati trovati per limitare le dimensioni di DNA che possono essere trasferiti a molto meno di 150 kb 40. I principali vantaggi di utilizzare il sistema di peptide-based per il trasferimento di geni rispetto agli approcci esistenti che impiegano sia Agrobacterium o bombardamento microproiettili, di cui sopra, sono riassunti nella tabella 2. Esistono alcune limitazioni per questo metodo così com&#39;è. In primo luogo, mirato consegna di pDNA di organelli specifici, come l&#39;mitochondria, è stato dimostrato possibile da una semplice combinazione di sequenze cellule penetrante e organello transito DNA-binding sebbene l&#39;efficienza era basso. espressione del transgene potrebbe essere rilevato, mediante microscopia confocale, solo in una piccola popolazione di mitocondri all&#39;interno delle cellule. Quindi, sono necessarie ulteriori modifiche: (i) migliorare la traslocazione di più complessi attraverso la membrana cellulare / organelli, e (ii) migliorare la dissociazione e trasferimento di pDNA dal peptide carrier nel organello di destinazione per l&#39;espressione. In secondo luogo, con questo sistema di consegna DNA, l&#39;espressione transiente dei geni reporter esogeni stata ottenuta con successo nel compartimento citosolico e mitocondriale di cellule. incorporazione stabile e l&#39;espressione dei geni introdotti nel genoma centrale nucleare / organelli non sono stati ancora stabilito, tuttavia, per l&#39;assenza di opportune strategie di selezione. Pur riconoscendo che ci sono aree di miglioramento o ulteriori dviluppo, la strategia peptide derivato da qui descritto rimane una tecnica semplice e versatile che ha aperto la strada per la consegna dei vari carichi in diversi tipi di piante.

Plant genetic engineering is conventionally used for transferring beneficial traits to plants. In recent years, this technology has been applied to convert plants into bio-factories for the production of pharmaceutically important and commercially valuable proteins, many of which cannot be chemically synthesized and are very costly to produce using animal or microbial systems1. By the introduction of new genes, the plant's own metabolism can be manipulated for the production of various biopharmaceuticals like antibodies, metabolic enzymes, hormones, antigens or vaccine2.
Established gene transfer technologies for plants are the Agrobacterium-mediated delivery3, bombardment with DNA-coated microprojectiles (biolistics)4, and electroporation5 or polyethylene glycol6 treatment of protoplasts. Techniques requiring protoplasts are generally avoided because they are time-consuming, cumbersome and yield inconsistent results7. As a result, virtually all plant modification work at present utilizes either Agrobacterium or microprojectiles for gene transfer. The Agrobacterium method is more extensively used but not applicable to many economically important plant species. Meanwhile, microprojectile bombardment is more versatile due to a broad range of susceptible plants but requires specialized equipment and often causes severe tissue damage. Furthermore, these methods involve either a random (biolistics) or complex (Agrobacterium) delivery mechanism and have variable transformation rates8. Hence, a novel plant transformation technology that is simple yet effective, and applicable to different plant types is required.
Currently, plants are subjected to genetic modification primarily by the delivery of exogenous DNA encoding a desired trait, rather than by direct delivery of a target protein. The higher stability of DNA over proteins is a prime advantage; nevertheless, potential problems associated with DNA delivery include the random insertion of exogenous DNA into the plant genome and unintended transmission of antibiotic resistance genes to pathogenic bacteria via horizontal gene transfer9. For genome-editing purposes, the ability to edit plant genomes without introducing foreign DNA into cells may circumvent regulatory concerns related to genetically modified plants. Thus, an alternative DNA-free strategy for the modification of plants by direct delivery of protein will be able to cater to these needs.
Here we introduce a peptide-based system, originally developed for human gene therapy10-14, for the targeted delivery of exogenous genes or proteins in intact plants. Peptides are able to protect DNA from nuclease degradation and can mediate gene transfer across cell as well as organellar membranes15-17. They also have diverse and tunable properties besides being non-cytotoxic18-20. More importantly, with the use of peptides, genes can be precisely targeted to intracellular organelles such as the mitochondria21 or plastids (chloroplasts)22 for expression-a task not achievable by biolistic or Agrobacterium-mediated transformation. Depending on the cargo type, this new plant modification technology can be exploited to deliver proteins23 and either express (plasmid21,24 or double-stranded DNA25) or down-regulate (double-stranded RNA26) specific genes within the plant, throughout its cytosolic space24-26 or within a specific organellar compartment21. The designed carrier peptides consist of a cationic domain in the form of either polylysine (K8) or polylysine-co-histidine (KH)9 for binding and/or condensation of negatively-charged cargoes, which is conjugated to cell penetrating (BP100 peptide) or mitochondria transit (Cytcox peptide) sequences.

<table border="0" cellpadding="0" cellspacing="0" width="2358">
	<tbody>
		<tr height="25">
			<td height="25" style="height:25px;width:453px;">(KH)9-BP100 peptide</td>
			<td style="width:355px;">Custom-synthesized by RIKEN Brain Science Institute</td>
			<td style="width:315px;">N/A</td>
			<td style="width:1235px;">Sequence: KHKHKHKHKHKHKHKH- 
			KHKKLFKKILKYL</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:453px;">(BP100)2-K8 peptide</td>
			<td>Custom-synthesized by RIKEN Brain Science Institute</td>
			<td style="width:315px;">N/A</td>
			<td>Sequence: KKLFKKILKYLKKLFKKIL- 
			KYLKKKKKKKK</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BP100 peptide</td>
			<td>Custom-synthesized by RIKEN Brain Science Institute</td>
			<td style="width:315px;">N/A</td>
			<td>Sequence: KKLFKKILKYL</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Cytcox-(KH)9 peptide</td>
			<td>Custom-synthesized by RIKEN Brain Science Institute</td>
			<td style="width:315px;">N/A</td>
			<td>Sequence: MLSLRQSIRFFKKHKHKH- 
			KHKHKHKHKHKH</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">P35S-GFP(S65T)-TNOS and P35S-RLuc-TNOS</td>
			<td style="width:355px;">N/A</td>
			<td style="width:315px;">N/A</td>
			<td style="width:1235px;">Encodes green fluorescent protein and Renilla luciferase genes, respectively, under the control of CaMV 35S constitutive promoter (Ref: Lakshmanan et al. Biomacromolecules 2013, 14, 10)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pDONR-cox2:gfp and pDONR-cox2:rluc</td>
			<td style="width:355px;">N/A</td>
			<td style="width:315px;">N/A</td>
			<td style="width:1235px;">Encodes green fluorescent protein and Renilla luciferase genes, respectively, under the control of cox2 mitochondrial-specific promoter (Ref: Chuah et al. Sci Rep 2015, 5, 7751)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">dsDNA (PCR-amplified from pBI221-P35S-Rluc-TNOS)</td>
			<td style="width:355px;">N/A</td>
			<td style="width:315px;">N/A</td>
			<td style="width:1235px;">Encodes green fluorescent protein and Renilla luciferase genes, respectively, under the control of CaMV 35S constitutive promoter (Ref: Lakshmanan et al. Plant Biotechnol 2015, 32, 39)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GFP5 siRNA</td>
			<td style="width:355px;">N/A</td>
			<td style="width:315px;">N/A</td>
			<td style="width:1235px;">For RNA interference-mediated silencing of green fluorescent protein (Ref: Numata et al. Plant Biotechnol J 2014, 12, 1027)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CHS siRNA</td>
			<td style="width:355px;">N/A</td>
			<td style="width:315px;">N/A</td>
			<td style="width:1235px;">For RNA interference-mediated silencing of chalcone synthase (Ref: Numata et al. Plant Biotechnol J 2014, 12, 1027)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">1 mL and 10 mL Plastic Syringes</td>
			<td>TERUMO Corporation</td>
			<td>SS-01T, SS-10ESZ</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">1.5 mL Microcentrifuge Tube</td>
			<td>AS ONE Corporation</td>
			<td>151212</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">96-Well Flat-Bottom Plate</td>
			<td>Asahi Glass Co., Ltd.</td>
			<td>3860-096</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Adhesive Tape</td>
			<td>Sekisui Chemical Co., Ltd.</td>
			<td>No. 835</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Alcohol Dehydrogenase</td>
			<td style="width:355px;">Sigma-Aldrich Co., LLC.</td>
			<td style="width:315px;">A-7011</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Atomic Force Microscope</td>
			<td>Seiko Instruments Inc.</td>
			<td>SPI3800, SPA 300HV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Atomic Force Microscope</td>
			<td>Hitachi High-Tech Science Corporation</td>
			<td>AFM5300E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">BCA Protein Assay Kit</td>
			<td>Thermo Fisher Scientific Inc.</td>
			<td>23227</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Cantilever</td>
			<td>Hitachi High-Tech Science Corporation</td>
			<td>K-A102001593</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Confocal Laser Scanning Microscope</td>
			<td>Carl Zeiss</td>
			<td>LSM700</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Cork Borer</td>
			<td style="width:355px;">Sigma-Aldrich Co., LLC.</td>
			<td>Z165220</td>
			<td>For excision of leaves into 1-cm diameter disks</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Coverslip</td>
			<td style="width:355px;">Matsunami Glass Ind., Ltd.</td>
			<td style="width:315px;">C02261</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Cuvette</td>
			<td style="width:355px;">Sarstedt</td>
			<td style="width:315px;">759116</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Folded Capillary Cell</td>
			<td style="width:355px;">Malvern Instruments, Ltd.</td>
			<td style="width:315px;">DTS1070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Forceps</td>
			<td style="width:355px;">Shimizu Akira Inc.</td>
			<td style="width:315px;">Stainless pincet 150</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Homogenization Pestle</td>
			<td>Ieda Trading Corp.</td>
			<td style="width:315px;">9993</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Mica</td>
			<td>Nisshin EM Co., Ltd.</td>
			<td style="width:315px;">LC23Z</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Microcentrifuge</td>
			<td>Beckman Coulter</td>
			<td style="width:315px;">BKA46472</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microplate Reader</td>
			<td>Molecular Devices Corporation</td>
			<td style="width:315px;">Spectra MAX M3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Microscope Slide</td>
			<td style="width:355px;">Matsunami Glass Ind., Ltd.</td>
			<td>S011120</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Pipette</td>
			<td style="width:355px;">Eppendorf Research&#174; plus</td>
			<td>3120000909</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Pipette Tips</td>
			<td>AS ONE Corporation</td>
			<td>2-5138-01, 2-5138-02, 2-5138-03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Plastic Petri Dish</td>
			<td>AS ONE Corporation</td>
			<td>1-7484-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Renilla Luciferase Assay Kit</td>
			<td>Promega corporation</td>
			<td>E2810</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Rhodamine B Isothiocyanate</td>
			<td style="width:355px;">Sigma-Aldrich Co., LLC.</td>
			<td style="width:315px;">283924</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">RNase-Free Water</td>
			<td style="width:355px;">Qiagen</td>
			<td style="width:315px;">129112</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Sodium Carbonate</td>
			<td>Wako Pure Chemical Industries, Ltd.</td>
			<td>199-01585</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Surgical Blade and Handle</td>
			<td>FEATHER Safety Razor Co., Ltd.</td>
			<td>Stainless steel No. 14 (blade), No. 3L (handle)</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Syringe Tip Cap</td>
			<td>Musashi Engineering Inc.</td>
			<td>NC-3E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Weighing Balance</td>
			<td>Sartorius</td>
			<td>CPA225D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Zeta Potentiometer</td>
			<td>Malvern Instruments, Ltd.</td>
			<td>Zetasizer Nano-ZS</td>
			<td style="width:1235px;"></td>
		</tr>
	</tbody>
</table>

peptide-derived method to transport genes and proteins across cellular and organellar barriers in plants

The capacity to introduce exogenous proteins and express (or down-regulate) specific genes in plants provides a powerful tool for fundamental research as well as new applications in the field of plant biotechnology. Viable methods that currently exist for protein or gene transfer into plant cells, namely Agrobacterium and microprojectile bombardment, have disadvantages of low transformation frequency, limited host range, or a high cost of equipment and microcarriers. The following protocol outlines a simple and versatile method, which employs rationally-designed peptides as delivery agents for a variety of nucleic acid- and protein-based cargoes into plants. Peptides are selected as tools for development of the system due to their biodegradability, reduced size, diverse and tunable properties as well as the ability to gain intracellular/organellar access. The preparation, characterization and application of optimized formulations for each type of the wide range of delivered cargoes (plasmid DNA, double-stranded DNA or RNA, and protein) are described. Critical steps within the protocol, possible modifications and existing limitations of the method are also discussed.

Una serie di carichi di acidi nucleici e proteine ​​sono state introdotte con successo in diversi impianti che utilizzano i peptidi studiati come vettori di consegna. Interazioni elettrostatiche tra peptidi cationici e carichi caricati negativamente portato alla formazione di complessi trasfezione che può essere infiltrato direttamente in pianta foglie con una siringa senza ago (Figura 1). Formulazioni ottimizzate (empiricamente determinati in questi studi 21,23-26) per la trasfezione di cellule vegetali sono elencati nella Tabella 1, in cui è rappresentata ciascuna tipologia della vasta gamma di carichi trasportati (pDNA, dsDNA, dsRNA e proteine). I diametri medi di tutte le formulazioni basati su peptidi erano nella gamma approssimativa di 150 - 300 nm. Sulla base di analisi di DLS, tutte le formulazioni visualizzati polidispersità relativamente bassa dimensione, indicando che gli aggregati del peptide-carico formate hanno una distribuzione uniforme dimensioni. Le morfologie dicomplessi tra peptide e pDNA (Figura 2A) o proteine (Figura 2B), in mica, sono state ripreso mediante AFM. Omogenei complessi globulari sono stati osservati per entrambe le combinazioni peptide-PDNA e peptide-proteina, in accordo con i dati derivati ​​dalla misurazione DLS. In termini di potenziale zeta di complessi (Tabella 1), pDNA- e complessi dsDNA-derivati avuto oneri netti di superficie negativa mentre complessi dsRNA basati avevano una carica superficiale quasi neutro. complessi peptide-proteina, d&#39;altra parte, sono state caricate positivamente. Le efficienze di peptide-pDNA e formulazioni del peptide-dsDNA nel mediare la trasfezione di A. thaliana o N. benthamiana come sistemi modello di piante sono stati valutati quantitativamente e qualitativamente. Il saggio di espressione genica RLuc è stato impiegato per la quantificazione dei livelli di espressione genica (Tabella 1), quindi per questo esperimento pDNA odsDNA codificante il gene RLuc deve essere utilizzato per complessazione con i rispettivi peptidi carrier. Utilizzando la (KH) 9 -BP100 / formulazione pDNA, consegna nucleare mirata ed espressione di pDNA può essere realizzato, dopo un periodo di incubazione di 12 ore, con una RLU / Valore mg stimato di circa 1 × 10 5. Per la consegna mitocondriale-mirata ed espressione di pDNA, una combinazione di peptidi, Cytcox- (KH) 9 e BP100, è necessario per la formazione del complesso. Con lo stesso periodo di incubazione ottimizzata di 12 ore, tuttavia, un livello molto più basso di trasfezione (circa 1 × 10 3 RLU / mg) è stato raggiunto. Nel frattempo, è stato richiesto simile periodo di incubazione (12 ore) e il livello di espressione genica (circa 1 × 10 3 RLU / mg) / registrata per i complessi dsDNA-based, anche formulata utilizzando il (KH) 9 -BP100 peptide. valutazioni qualitative di espressione genica sono stati eseguiti da osservazione microscopica diretta di foglie trattati con complessi preparEd usando pDNA o dsDNA codificante il gene reporter GFP. In cellule trasfettate con i complessi non mirati peptide-PDNA, diffondere fluorescenza verde in corrispondenza con l&#39;espressione della GFP è stato chiaramente osservato e ha trovato di localizzare nel citoplasma (Figura 3A). chiare differenze nel modello di localizzazione di fluorescenza GFP sono stati evidenti in cellule infiltrate con complessi peptide-PDNA mitocondriali mirati. Qui, puntiformi fluorescenza verde che colocalize con la macchia mitocondriale era visibile, confermando la specificità dell&#39;espressione genica esclusivamente nel compartimento mitocondriale di cellule (Figura 3B). Nel caso di formulazioni peptide-proteina, coniugazione del carico proteine ​​(ADH) per un fluoroforo (FR) permetterà la visualizzazione della proteina erogata nel compartimento intracellulare. Nel giro di un breve periodo di incubazione di 6 ore, è stato trovato per essere distribuito in tutto ADH-RhB proteine ​​(blu)citoplasma e vacuolo di cellule infiltrate (Figura 3C). Nel frattempo, rapida ed efficace down-regolazione dell&#39;espressione genica potrebbe essere realizzato in diversi impianti che utilizzano formulazioni peptide-dsRNA. Nel primo esperimento, foglia A. thaliana è stato infiltrato con peptide-dsRNA Complessi di mettere a tacere il gene calcone sintasi (CHS) responsabile di antociani (pigmento rosso) biosintesi in condizioni di siccità. La differenza nella comparsa di A. thaliana lascia in condizioni normali (Figura 3D, a) e le condizioni di siccità (Figura 3D, b) ha fornito un facile mezzo per valutare CHS silenziamento utilizzando la formulazione peptide-dsRNA ottimizzata (freccia 1 indica la regione infiltrato). Nel secondo esperimento, complessi peptide-dsRNA sono stati infiltrati nelle foglie di transgenico A. thaliana che esprimono la proteina fluorescente gialla (YFP). Un apparente riduzione della espressione YFP potrebbe essere visto nelle cellule epidermiche 9 ore post-trasfezione (Figura 3E, F). L&#39;efficacia della formulazione nell&#39;espressione genica down-regolazione in un sistema diverso impianto (pioppo, 12 hr dopo la trasfezione) è stato inoltre verificato (figura 3G, H). <img alt="Figura 1" src="/files/ftp_upload/54972/54972fig1.jpg" /> Figura 1: Formulazioni Peptide-based per la consegna di acidi nucleici e proteine in carichi piante viventi. I peptidi carrier progettati sono costituiti da policationi coniugati con penetrante cellulare o sequenze di transito organellari. Policationi consentono vincolanti e / o condensa di carichi carica negativa così come fuga dal vano endosomal seguente internalizzazione nelle cellule. Consegna delle merci nelle cellule e, successivamente, a organelli specifici è mediata da sequenze di cellule penetrante e sequenze di transito organellari, rispettivamente. I vari carichi che potrebbero essere successfully erogato nella pianta includono acidi nucleici come pDNA, dsDNA e dsRNA, nonché proteine ​​modello come albumina di siero bovino (BSA), alcol deidrogenasi (ADH) e citrino (una variante della proteina fluorescente gialla). Molecole bioattive sono in grado di formare complessi trasfezione con coniugati peptide tramite interazioni elettrostatiche, che vengono introdotti in foglie delle piante da siringa infiltrazione. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54972/54972fig1large.jpg" target="_blank">Clicca qui per vedere una versione più grande di questa figura.</a> <img alt="figura 2" src="/files/ftp_upload/54972/54972fig2.jpg" /> Figura 2: morfologie del Formulazioni Peptide-base. (A) AFM ampiezza immagine di (KH) 9 -BP100 formulazione / pDNA a N / P 0.5. (B) Altezza immagine AFM di (BP100) 2 K 8 / formulazione ADH a molare ratio 10. riprodotto con permesso da fonti pubblicate 23,24. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54972/54972fig2large.jpg" target="_blank">Clicca qui per vedere una versione più grande di questa figura.</a> <img alt="Figura 3" src="/files/ftp_upload/54972/54972fig3.jpg" /> Figura 3: Valutazione della microscopica Transfection efficienze usando formulazioni Peptide-based ottimizzati. (A) espressione citosolico GFP (green), chiaramente distinto da autofluorescenza cloroplasto (rosso), è stato osservato nelle cellule del mesofillo spugnoso di A. thaliana foglie infiltrato con (KH) 9 formulazione -BP100 / pDNA (N / P 0,5; 12 ore ). (B) l&#39;espressione della GFP (verde) è stato rilevato nei mitocondri (rosso) nelle cellule epidermiche di foglia di A. thaliana infiltrati con Cytcox- (KH) 9 / BP100 / formulazione pDNA (N / P 0,5 per ogni peptidee; 12 ore). immagini ingrandite di mitocondri con espressione GFP sono mostrati nel pannello estrema destra. (C) Consegna di ADH-RhB (blu) nelle cellule del mesofillo spugnoso di A. thaliana foglie mediata da (BP100) 2 K 8 in un rapporto molare di peptidi / proteine 10, visualizzate 6 ore post-infiltrazione. (D) foglie A. thaliana prima (a) e dopo (b) il trattamento con (KH) 9 -BP100 / formulazione dsRNA (rapporto molare 2; 48 ore), che ha portato alla soppressione della via di biosintesi delle antocianine. Una formulazione analoga contenente GFP5 dsRNA stato infiltrato nella foglia come controllo negativo (c). Frecce 1 e 3 indicano l&#39;area infiltrati mentre le frecce 2 e 4 indicano la zona non infiltrata all&#39;interno della foglia. (E) A. thaliana lascia esprimere la proteina fluorescente gialla (YFP) e (F) la fluorescenza YFP diminuita dopo infiltrazione con<html> (KH) 9 -BP100 / formulazione dsRNA (rapporto molare 2; 9 ore). (G) foglie di pioppo transgenici che esprimono la proteina fluorescente gialla (YFP) e (H) la diminuzione YFP fluorescenza dopo infiltrazione con (KH) 9 formulazione -BP100 / dsRNA (rapporto molare 2; 12 ore). Riprodotto con il permesso di fonti pubblicate 21,23,24,26. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54972/54972fig3large.jpg" target="_blank">Clicca qui per vedere una versione più grande di questa figura.</a> <img alt="Figura 4" src="/files/ftp_upload/54972/54972fig4.jpg" /> Figura 4: Variazione Peptide-to-DNA (N / P) Rapporto e l&#39;effetto sulle proprietà biofisiche di complessi. Con l&#39;aumento del peptide al rapporto di DNA, le formulazioni a base di peptidi diminuiscono in termini di dimensioni, mentre il loro valore di transizione zeta-potenziale da negativo a positivo./files/ftp_upload/54972/54972fig4large.jpg &quot;target =&quot; _ blank &quot;&gt; Clicca qui per vedere una versione più grande di questa figura. <img alt="Tabella 1" src="/files/ftp_upload/54972/54972table1.jpg" /> Tabella 1: caratterizzazione e valutazione di diverse formulazioni del peptide-based. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54972/54972table1large.jpg" target="_blank">Clicca qui per vedere una versione più grande di questa tabella.</a> <img alt="Tabella 2" src="/files/ftp_upload/54972/54972table2.jpg" /> Tabella 2: un confronto di peptide-based e altre tecnologie esistenti DNA di consegna per le Piante intatto. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54972/54972table2large.jpg" target="_blank">Clicca qui per vedere una versione più grande di questa tabella.</a>

Watch this Scientific Journal Video about Peptide-derived Method to Transport Genes and Proteins Across Cellular and Organellar Barriers in Plants at JoVE.com

Peptide-derived Method to Transport Genes and Proteins Across Cellular and Organellar Barriers in Plants

Existing methods for the modification of plants have limited applicability. The novel peptide-derived technology proposed here promises both simplicity and efficiency in the introduction of exogenous protein or genes into the desired intracellular compartments of intact plants.

Peptide di derivazione Metodo di Trasporto geni e proteine ​​Attraverso cellulari e barriere organelli in Piante

The capacity to introduce exogenous proteins and express (or down-regulate) specific genes in plants provides a powerful tool for fundamental research as ...

peptide-derived-method-to-transport-genes-proteins-across-cellular

Universidad San Sebastian

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Genetics

10.5K Views.  RIKEN Center for Sustainable Resource Science. Existing methods for the modification of plants have limited applicability. The novel peptide-derived technology proposed here promises both simplicity and efficiency in the introduction of exogenous protein or genes into the desired intracellular compartments of intact plants.

Video: Peptide-derived Method to Transport Genes and Proteins Across Cellular and Organellar Barriers in Plants

Preparation of rAAV9 to Overexpress or Knockdown Genes in Mouse Hearts

Controlling the expression or activity of specific genes through the myocardial delivery of genetic materials in murine models permits the investigation of gene functions. Their therapeutic potential in the heart can also be determined. There are limited approaches for in vivo molecular intervention in the mouse heart. Recombinant adeno-associated virus (rAAV)-based genome engineering has been utilized as an essential tool for in vivo cardiac gene manipulation. The specific advantages of this technology include high efficiency, high specificity, low genomic integration rate, minimal immunogenicity, and minimal pathogenicity. Here, a detailed procedure to construct, package, and purify the rAAV9 vectors is described. Subcutaneous injection of rAAV9 into neonatal pups results in robust expression or efficient knockdown of the gene(s) of interest in the mouse heart, but not in the liver and other tissues. Using the cardiac-specific TnnT2 promoter, high expression of GFP gene in the heart was obtained. Additionally, target mRNA was inhibited in the heart when a rAAV9-U6-shRNA was utilized. Working knowledge of rAAV9 technology may be useful for cardiovascular investigations.

In this manuscript, a method to prepare recombinant adeno-associated virus 9 (rAAV9) vectors to manipulate gene expression in the mouse heart is described.

Preparazione di rAAV9 per iperespressione o Knockdown geni in Cuori mouse

Controlling the expression or activity of specific genes through the myocardial delivery of genetic materials in murine models permits the investigation ...

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Genetica

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most mutagenesis-based screens, researchers have relied on the use of toxic chemicals, carcinogens, or irradiation, which requires designated equipment, safety setup, and/or disposal of hazardous materials. We have developed a simple approach to induce heritable mutations in C. elegans using germline-expressed histone-miniSOG, a light-inducible potent generator of reactive oxygen species. This mutagenesis method is free of toxic chemicals and requires minimal laboratory safety and waste management. The induced DNA modifications include single-nucleotide changes and small deletions, and complement those caused by classical chemical mutagenesis. This methodology can also be used to induce integration of extrachromosomal transgenes. Here, we provide the details of the LED setup and protocols for standard mutagenesis and transgene integration.

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Genetically-encoded histone-miniSOG induces genome-wide heritable mutations in a blue&#160;light-dependent manner. This mutagenesis method is simple, fast, free of toxic chemicals, and well-suited for forward genetic screening and transgene integration.

Optogenetic caso Mutagenesi Utilizzando istone-miniSOG in<em&gt; C. elegans</em

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most ...

Analysis of Cap-binding Proteins in Human Cells Exposed to Physiological Oxygen Conditions

Translational control is a focal point of gene regulation, especially during periods of cellular stress. Cap-dependent translation via the eIF4F complex is by far the most common pathway to initiate protein synthesis in eukaryotic cells, but stress-specific variations of this complex are now emerging. Purifying cap-binding proteins with an affinity resin composed of Agarose-linked m7GTP (a 5' mRNA cap analog) is a useful tool to identify factors involved in the regulation of translation initiation. Hypoxia (low oxygen) is a cellular stress encountered during fetal development and tumor progression, and is highly dependent on translation regulation. Furthermore, it was recently reported that human adult organs have a lower oxygen content (physioxia 1-9% oxygen) that is closer to hypoxia than the ambient air where cells are routinely cultured. With the ongoing characterization of a hypoxic eIF4F complex (eIF4FH), there is increasing interest in understanding oxygen-dependent translation initiation through the 5' mRNA cap. We have recently developed a human cell culture method to analyze cap-binding proteins that are regulated by oxygen availability. This protocol emphasizes that cell culture and lysis be performed in a hypoxia workstation to eliminate exposure to oxygen. Cells must be incubated for at least 24 hr for the liquid media to equilibrate with the atmosphere within the workstation. To avoid this limitation, pre-conditioned media (de-oxygenated) can be added to cells if shorter time points are required. Certain cap-binding proteins require interactions with a second base or can hydrolyze the m7GTP, therefore some cap interactors may be missed in the purification process. Agarose-linked to enzymatically resistant cap analogs may be substituted in this protocol. This method allows the user to identify novel oxygen-regulated translation factors involved in cap-dependent translation.

Here, we present human cell culture protocols to analyze translation initiation factors that bind the 5' cap of mRNA during physiological oxygen conditions. This method utilizes an Agarose-linked m7GTP cap analog and is suitable to investigate cap-binding factors and their interacting partners.

Analisi delle proteine ​​di legame della cap in cellule umane esposte a condizioni fisiologiche di ossigeno

Translational control is a focal point of gene regulation, especially during periods of cellular stress. Cap-dependent translation via the eIF4F complex ...

Optimization and Comparative Analysis of Plant Organellar DNA Enrichment Methods Suitable for Next-generation Sequencing

Plant organellar genomes contain large, repetitive elements that may undergo pairing or recombination to form complex structures and/or sub-genomic fragments. Organellar genomes also exist in admixtures within a given cell or tissue type (heteroplasmy), and an abundance of subtypes may change throughout development or when under stress (sub-stoichiometric shifting). Next-generation sequencing (NGS) technologies are required to obtain deeper understanding of organellar genome structure and function. Traditional sequencing studies use several methods to obtain organellar DNA: (1) If a large amount of starting tissue is used, it is homogenized and subjected to differential centrifugation and/or gradient purification. (2) If a smaller amount of tissue is used (i.e., if seeds, material, or space is limited), the same process is performed as in (1), followed by whole-genome amplification to obtain sufficient DNA. (3) Bioinformatics analysis can be used to sequence the total genomic DNA and to parse out organellar reads. All these methods have inherent challenges and tradeoffs. In (1), it may be difficult to obtain such a large amount of starting tissue; in (2), whole-genome amplification could introduce a sequencing bias; and in (3), homology between nuclear and organellar genomes could interfere with assembly and analysis. In plants with large nuclear genomes, it is advantageous to enrich for organellar DNA to reduce sequencing costs and sequence complexity for bioinformatics analyses. Here, we compare a traditional differential centrifugation method with a fourth method, an adapted CpG-methyl pulldown approach, to separate the total genomic DNA into nuclear and organellar fractions. Both methods yield sufficient DNA for NGS, DNA that is highly enriched for organellar sequences, albeit at different ratios in mitochondria and chloroplasts. We present the optimization of these methods for wheat leaf tissue and discuss major advantages and disadvantages of each approach in the context of sample input, protocol ease, and downstream application.

The comparison and optimization of two plant organellar DNA enrichment methods are presented: traditional differential centrifugation and fractionation of the total gDNA based on methylation status. We assess the resulting DNA quantity and quality, demonstrate performance in short-read next-generation sequencing, and discuss the potential for use in long-read single-molecule sequencing.

Ottimizzazione e analisi comparativa dei metodi di arricchimento del DNA organologico vegetale adatti per la sequenziazione di nuova generazione

Plant organellar genomes contain large, repetitive elements that may undergo pairing or recombination to form complex structures and/or sub-genomic ...

Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage

DNA damage induces specific signaling and repair responses in the cell, which is critical for protection of genome integrity. Laser microirradiation became a valuable experimental tool to investigate the DNA damage response (DDR) in vivo. It allows real-time high-resolution single-cell analysis of macromolecular dynamics in response to laser-induced damage confined to a submicrometer region in the cell nucleus. However, various laser conditions have been used without appreciation of differences in the types of damage induced. As a result, the nature of the damage is often not well characterized or controlled, causing apparent inconsistencies in the recruitment or modification profiles. We demonstrated that different irradiation conditions (i.e., different wavelengths as well as different input powers (irradiances) of a femtosecond (fs) near-infrared (NIR) laser) induced distinct DDR and repair protein assemblies. This reflects the type of DNA damage produced. This protocol describes how titration of laser input power allows induction of different amounts and complexities of DNA damage, which can easily be monitored by detection of base and crosslinking damages, differential poly (ADP-ribose) (PAR) signaling, and pathway-specific repair factor assemblies at damage sites. Once the damage conditions are determined, it is possible to investigate the effects of different damage complexity and differential damage signaling as well as depletion of upstream factor(s) on any factor of interest.

Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage

The goal of this protocol is to describe how to use laser microirradiation to induce different types of DNA damage, including relatively simple strand breaks and complex damage, to study DNA damage signaling and repair factor assembly at damage sites in vivo.

Laser Microirradiation per lo Studio In Vivo delle risposte cellulari al danno al DNA semplici e complesse

DNA damage induces specific signaling and repair responses in the cell, which is critical for protection of genome integrity. Laser microirradiation ...

Simultaneous Video-EEG-ECG Monitoring to Identify Neurocardiac Dysfunction in Mouse Models of Epilepsy

In epilepsy, seizures can evoke cardiac rhythm disturbances such as heart rate changes, conduction blocks, asystoles, and arrhythmias, which can potentially increase risk of sudden unexpected death in epilepsy (SUDEP). Electroencephalography (EEG) and electrocardiography (ECG) are widely used clinical diagnostic tools to monitor for abnormal brain and cardiac rhythms in patients. Here, a technique to simultaneously record video, EEG, and ECG in mice to measure behavior, brain, and cardiac activities, respectively, is described. The technique described herein utilizes a tethered (i.e., wired) recording configuration in which the implanted electrode on the head of the mouse is hard-wired to the recording equipment. Compared to wireless telemetry recording systems, the tethered arrangement possesses several technical advantages such as a greater possible number of channels for recording EEG or other biopotentials; lower electrode costs; and greater frequency bandwidth (i.e., sampling rate) of recordings. The basics of this technique can also be easily modified to accommodate recording other biosignals, such as electromyography (EMG) or plethysmography for assessment of muscle and respiratory activity, respectively. In addition to describing how to perform the EEG-ECG recordings, we also detail methods to quantify the resulting data for seizures, EEG spectral power, cardiac function, and heart rate variability, which we demonstrate in an example experiment using a mouse with epilepsy due to Kcna1 gene deletion. Video-EEG-ECG monitoring in mouse models of epilepsy or other neurological disease provides a powerful tool to identify dysfunction at the level of the brain, heart, or brain-heart interactions.

Here, we present a protocol to record brain and heart bio signals in mice using simultaneous video, electroencephalography (EEG), and electrocardiography (ECG). We also describe methods to analyze the resulting EEG-ECG recordings for seizures, EEG spectral power, cardiac function, and heart rate variability.

Video-EEG-ECG simultaneo monitoraggio per identificare la disfunzione Neurocardiac in modelli murini di epilessia

In epilepsy, seizures can evoke cardiac rhythm disturbances such as heart rate changes, conduction blocks, asystoles, and arrhythmias, which can ...

An In Vitro Protocol for Evaluating MicroRNA Levels, Functions, and Associated Target Genes in Tumor Cells

MicroRNAs (miRNAs) are small regulatory RNAs which are recognized to modulate numerous intracellular signaling pathways in several diseases including cancers. These small regulatory RNAs mainly interact with the 3&#8217; untranslated regions (3&#8217; UTR) of their target messenger RNAs (mRNAs) ultimately resulting in the inhibition of decoding processes of mRNAs and the augmentation of target mRNA degradations. Based on the expression levels and intracellular functions, miRNAs are able to serve as regulatory factors of oncogenic and tumor-suppressive mRNAs. Identification of bona fide target genes of a miRNA among hundreds or even thousands of computationally predicted targets is a crucial step to discern the roles and basic molecular mechanisms of a miRNA of interest. Various miRNA target prediction programs are available to search possible miRNA-mRNA interactions. However, the most challenging question is how to validate direct target genes of a miRNA of interest. This protocol describes a reproducible strategy of key methods on how to identify miRNA targets related to the function of a miRNA. This protocol presents a practical guide on step-by-step procedures to uncover miRNA levels, functions, and related target mRNAs using the probe-based real-time polymerase chain reaction (PCR), sulforhodamine B (SRB) assay following a miRNA mimic transfection, dose-response curve generation, and luciferase assay along with the cloning of 3&#8217; UTR of a gene, which is necessary for proper understanding of the roles of individual miRNAs.

This protocol uses a probe-based real-time polymerase chain reaction (PCR), a sulforhodamine B (SRB) assay, 3&#8217; untranslated regions (3&#8217; UTR) cloning, and a luciferase assay to verify the target genes of a miRNA of interest and to understand the functions of miRNAs.

Un protocollo in vitro per la valutazione dei livelli, delle funzioni e dei geni target associati nelle cellule tumorali

MicroRNAs (miRNAs) are small regulatory RNAs which are recognized to modulate numerous intracellular signaling pathways in several diseases including ...

Exploring Sequence Space to Identify Binding Sites for Regulatory RNA-Binding Proteins

Gene regulation plays an important role in all cells. Transcriptional, post-transcriptional (or RNA processing), translational, and post-translational steps are used to regulate specific genes. Sequence-specific nucleic acid-binding proteins target specific sequences to control spatial or temporal gene expression. The binding sites in nucleic acids are typically characterized by mutational analysis. However, numerous proteins of interest have no known binding site for such characterization. Here we describe an approach to identify previously unknown binding sites for RNA-binding proteins. It involves iterative selection and amplification of sequences starting with a randomized sequence pool. Following several rounds of these steps-transcription, binding, and amplification-the enriched sequences are sequenced to identify a preferred binding site(s). Success of this approach is monitored using in vitro binding assays. Subsequently, in vitro and in vivo functional assays can be used to assess the biological relevance of the selected sequences. This approach allows identification and characterization of a previously unknown binding site(s) for any RNA-binding protein for which an assay to separate protein-bound and unbound RNAs exists.

Sequence specificity is critical for gene regulation. Regulatory proteins that recognize specific sequences are important for gene regulation. Defining functional binding sites for such proteins is a challenging biological problem. An iterative approach for identification of a binding site for an RNA-binding protein is described here and is applicable to all RNA-binding proteins.

Esplorare lo spazio di sequenza per identificare i siti di legame per le proteine di legame dell'RNA regolamentare

Gene regulation plays an important role in all cells. Transcriptional, post-transcriptional (or RNA processing), translational, and post-translational ...

In Vivo Forward Genetic Screen to Identify Novel Neuroprotective Genes in Drosophila melanogaster

There is much to understand about the onset and progression of neurodegenerative diseases, including the underlying genes responsible. Forward genetic screening using chemical mutagens is a useful strategy for mapping mutant phenotypes to genes among Drosophila and other model organisms that share conserved cellular pathways with humans. If the mutated gene of interest is not lethal in early developmental stages of flies, a climbing assay can be conducted to screen for phenotypic indicators of decreased brain functioning, such as low climbing rates. Subsequently, secondary histological analysis of brain tissue can be performed in order to verify the neuroprotective function of the gene by scoring neurodegeneration phenotypes. Gene mapping strategies include meiotic and deficiency mapping that rely on these same assays can be followed by DNA sequencing to identify possible nucleotide changes in the gene of interest.

In Vivo Forward Genetic Screen to Identify Novel Neuroprotective Genes in Drosophila melanogaster

We present a protocol using a forward genetic approach to screen for mutants exhibiting neurodegeneration in Drosophila melanogaster. It incorporates a climbing assay, histology analysis, gene mapping and DNA sequencing to ultimately identify novel genes related to the process of neuroprotection.

In Vivo Avanti Schermo Genetico per Identificare Nuovi Geni Neuroprotettivi in Drosophila melanogaster

There is much to understand about the onset and progression of neurodegenerative diseases, including the underlying genes responsible. Forward genetic ...

A Bioinformatics Pipeline to Accurately and Efficiently Analyze the MicroRNA Transcriptomes in Plants

MicroRNAs (miRNAs) are 20- to 24-nucleotide (nt) endogenous small RNAs (sRNAs) extensively existing in plants and animals that play potent roles in regulating gene expression at the post-transcriptional level. Sequencing sRNA libraries by Next Generation Sequencing (NGS) methods has been widely employed to identify and analyze miRNA transcriptomes in the last decade, resulting in a rapid increase of miRNA discovery. However, two major challenges arise in plant miRNA annotation due to increasing depth of sequenced sRNA libraries as well as the size and complexity of plant genomes. First, many other types of sRNAs, in particular, short interfering RNAs (siRNAs) from sRNA libraries, are erroneously annotated as miRNAs by many computational tools. Second, it becomes an extremely time-consuming process for analyzing miRNA transcriptomes in plant species with large and complex genomes. To overcome these challenges, we recently upgraded miRDeep-P (a popular tool for miRNA transcriptome analyses) to miRDeep-P2 (miRDP2 for short) by employing a new filtering strategy, overhauling the scoring algorithm and incorporating newly updated plant miRNA annotation criteria. We tested miRDP2 against sequenced sRNA populations in five representative plants with increasing genomic complexity, including Arabidopsis, rice, tomato, maize and wheat. The results indicate that miRDP2 processed these tasks with very high efficiency. In addition, miRDP2 outperformed other prediction tools regarding sensitivity and accuracy. Taken together, our results demonstrate miRDP2 as a fast and accurate tool for analyzing plant miRNA transcriptomes, therefore a useful tool in helping the community better annotate miRNAs in plants.

A bioinformatics pipeline, namely miRDeep-P2 (miRDP2 for short), with updated plant miRNA criteria and an overhauled algorithm, could accurately and efficiently analyze microRNA transcriptomes in plants, especially for species with complex and large genomes.

Una pipeline di bioinformatica per analizzare in modo accurato ed efficiente i trascrittomi di microRNA nelle piante

MicroRNAs (miRNAs) are 20- to 24-nucleotide (nt) endogenous small RNAs (sRNAs) extensively existing in plants and animals that play potent roles in ...

Peptide di derivazione Metodo di Trasporto geni e proteine ​​Attraverso cellulari e barriere organelli in Piante

Peptide di derivazione Metodo di Trasporto geni e proteine Attraverso cellulari e barriere organelli in Piante