Name: direct protein delivery to mammalian cells using cell-permeable cys2-his2 zinc-finger domains
Uploaded: 2015-03-25T15:00:00
Description: Watch this Scientific Journal Video about Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains at JoVE.com

Dette arbeidet ble støttet av National Institutes of Health (DP1CA174426 til Carlos F. Barbas) og ShanghaiTech University, Shanghai, Kina (til JL). Molekylære grafikk ble generert ved hjelp PyMol.

1. Kloning <ol><li> Skaff alanine-byttet to-finger ZIF domener som har blitt under klonet inn i Dyre 28 ekspressjonsvektor system og er tilgjengelig på forespørsel (PET-2F-ZIF). 34 </li><li> PCR amplifisere EmGFP fra plasmidet Emerald-pBAD med primerne 5&#39;-Xmal-EmGFP (5&#39;-GGAAATTG CCCGGG ATGGTGAGCAAGGGCGAGGAGCTGTTCAC-3 &#39;, Xma I-sete i fet skrift) og 3&#39; SacI-EmGFP (5&#39;-CGGATCT GAGCTC TTACTTGTACAGCTCGTCCATGCCGAG-3 &#39; ; Sac jeg nettsted ...

<ol>
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Her er en trinn-for-trinn-protokoll for proteinlevering ved hjelp av celle-gjennomtrengelige sink-finger (Zif) domener presentert. ZIF domene ikke redusere aktiviteten av smeltet enzymatisk last 34; muliggjør for produksjon og rensing av proteiner i utbytter nesten identiske med de som ble observert med ikke-modifisert protein; og kan transportere proteiner og enzymer i en rekke celletyper med effektivitet som overskrider tradisjonelle cellepenetrerende peptid eller protein transduksjon domenesystemer. Samme...

Svært effektive og allsidige protein levering strategier er kritisk for mange grunnleggende forskning og terapeutiske anvendelser. Den direkte levering av rensede proteiner i celler representerer en av de sikreste og enkleste metoder for å oppnå dette. 1,2 motsetning strategier som er avhengige av genekspresjon fra nukleinsyrer, proteiner utgjør 3-5 levering ingen risiko for insertional mutagenese, er uavhengig av cellulær transkripsjon / oversettelse maskiner og åpner for en umiddelbar effekt. Men forundrer mangelen på enkle og generaliseres metoder for endowing celle-trengende aktivitet på proteiner rutinemessig sine direkte inntreden i cellene. Nåværende metoder for å lette intracellulær proteinlevering er basert på bruk av naturlig forekommende 6-8 eller utformet cellepenetrerende peptider, 9-12 kompressor transduksjon domener, 13,14 nanopartikler 15 og liposomer, 16-viruslignende partikler 17,18 </sup&gt; og polymere mikro materialer. 19 Dessverre er mange av disse tilnærmingene er hemmet av lave cellulært opptak priser, 20,21 dårlig stabilitet, 22 utilsiktet celle-type spesifisitet, 23 lav endosomal rømningsegenskaper 24 og toksisitet. 25 I tillegg er mange protein transduksjon teknologier redusere bioaktiviteten av de leverte proteiner. 14 Vårt laboratorium tidligere vist at sink-finger-nuklease (ZFN) proteiner - kimære restriksjonsendonukleaser som består av en programmerbar Cys to -Hans 2 sink-finger-DNA-bindende protein, og spalting domenet til FoKI restriksjonsendonuklease 26-28 - er iboende celle- 29 Denne overraskende celle-trengende aktivitet ble vist seg å være en iboende egenskap ved spesialdesignede sink-finger domene, et DNA-bindende plattform som har dukket opp som et kraftig verktøy for målrettet genom en gjennomtrengelig.gineering, 30-32 og anses å være et resultat av den konstellasjon av seks positivt ladede rester på proteinoverflaten. Faktisk har flere DNA-bindende proteiner, inkludert c-Jun og N-DEK blitt vist å ha en iboende evne til å krysse cellemembraner. 33 Mer nylig vårt laboratorium utvidet på disse resultater og viste at cellepenetrerende aktivitet av sink- finger (ZIF) domener kan utnyttes for intracellulær protein levering. Genetisk fusjon av enten en- eller to-finger ZIF domener til spesifikke protein last førte til opptak effektivitet som oversteg mange konvensjonelle celletrengende peptid leveringssystemer. 34 Mest spesielt, gjorde ZIF-mediert levering ikke kompromiss aktiviteten av smeltet enzymatisk last og tilrettelagt høye nivåer av cytosolisk levering. Sammen er disse funnene viser potensialet av ZIF domene for tilrettelegging for effektiv og lettvinte levering av proteiner, og potensielt mer varierte typer av makromolekyler, inn i cellene. Her er en detaljert steg-for-steg-protokoll på hvordan man implementerer ZIF-teknologi for protein levering i pattedyrceller presentert. Vi har tidligere konstruert en pakke med en-, to-, tre-, fire-, fem- og seks-finger-domener Zif som mangler evnen til å binde DNA, på grunn av substitusjon av hver av de α-heliks DNA-bindingsrester, men er i stand til å levere proteiner i celler 34 (figur 1). Produksjon og transduksjon av Emerald GFP (EmGFP) i HeLa celler ved hjelp av en to-finger ZIF domenet er beskrevet. Denne protokollen er utvidbar til nesten hvilken som helst protein i stand til ekspresjon av oppløselig i Escherichia coli, og nesten en hvilken som helst pattedyrcelletype. Forventede resultater er gitt og strategier for å maksimere ytelsen til dette systemet blir også diskutert.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>XmaI</td>
			<td>New England Biolabs</td>
			<td>R0180L</td>
			<td></td>
		</tr>
		<tr>
			<td>SacI</td>
			<td>New England Biolabs</td>
			<td>R0156L</td>
			<td></td>
		</tr>
		<tr>
			<td>Expand High Fidelity PCR system</td>
			<td>Roche</td>
			<td>11759078001</td>
			<td></td>
		</tr>
		<tr>
			<td>dNTPs</td>
			<td>New England Biolabs</td>
			<td>N0446S</td>
			<td></td>
		</tr>
		<tr>
			<td>4-20% Tris-Glycine Mini protein gels, 1.5 mm, 10 wells</td>
			<td>Life Technologies</td>
			<td>EC6028BOX</td>
			<td></td>
		</tr>
		<tr>
			<td>2x Laemmli Sample Buffer</td>
			<td>BioRad</td>
			<td>161-0737</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 DNA Ligase</td>
			<td>Life Technologies</td>
			<td>15224-017</td>
			<td></td>
		</tr>
		<tr>
			<td>BL21 (DE3) Competent E. coli</td>
			<td>New England Biolabs</td>
			<td>C2527I</td>
			<td></td>
		</tr>
		<tr>
			<td>IPTG</td>
			<td>Thermo Scientific</td>
			<td>R0391</td>
			<td></td>
		</tr>
		<tr>
			<td>Zinc Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>208086-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Kanamycin Sulfate</td>
			<td>Fisher Scientific</td>
			<td>BP906-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G8270-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris Base</td>
			<td>Fisher Scientific</td>
			<td>BP152-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>S9888-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>Fisher Scientific</td>
			<td>PR-V3151&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>PMSF</td>
			<td>Thermo Scientific</td>
			<td>36978</td>
			<td></td>
		</tr>
		<tr>
			<td>Ni-NTA Agarose Resin</td>
			<td>QIAGEN</td>
			<td>30210</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G5516-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Imidazole</td>
			<td>Sigma-Aldrich</td>
			<td>I5513-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Amicon Ultra-15 Centrifugal Filter Units</td>
			<td>EMO Millipore</td>
			<td>UFC900324</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Life Technologies</td>
			<td>11966-025</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Life Technologies</td>
			<td>10437-028</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic&#160;</td>
			<td>Life Technologies</td>
			<td>15240-062</td>
			<td></td>
		</tr>
		<tr>
			<td>24-Well Flat Bottom Plate</td>
			<td>Sigma-Aldrich</td>
			<td>CLS3527-100EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-Lysine</td>
			<td>Sigma-Aldrich</td>
			<td>P7280</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS, No Calcium, No Magnesium</td>
			<td>Life Technologies</td>
			<td>21600010</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparan Sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>H4777</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Life Technologies</td>
			<td>25300054</td>
			<td></td>
		</tr>
		<tr>
			<td>Hela cells</td>
			<td>ATCC</td>
			<td>CCL-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Nano Drop ND-1000 spectrophotometer&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAquick PCR Purification Kit</td>
			<td>QIAGEN</td>
			<td>28104</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAquick Gel Extraction Kit</td>
			<td>QIAGEN</td>
			<td>28704</td>
			<td></td>
		</tr>
	</tbody>
</table>

direct protein delivery to mammalian cells using cell-permeable cys2-his2 zinc-finger domains

Due to their modularity and ability to be reprogrammed to recognize a wide range of DNA sequences, Cys2-His2 zinc-finger DNA-binding domains have emerged as useful tools for targeted genome engineering. Like many other DNA-binding proteins, zinc-fingers also possess the innate ability to cross cell membranes. We recently demonstrated that this intrinsic cell-permeability could be leveraged for intracellular protein delivery. Genetic fusion of zinc-finger motifs leads to efficient transport of protein and enzyme cargo into a broad range of mammalian cell types. Unlike other protein transduction technologies, delivery via zinc-finger domains does not inhibit enzyme activity and leads to high levels of cytosolic delivery. Here a detailed step-by-step protocol is presented for the implementation of zinc-finger technology for protein delivery into mammalian cells. Key steps for achieving high levels of intracellular zinc-finger-mediated delivery are highlighted and strategies for maximizing the performance of this system are discussed.

To-finger ZIF-EmGFP fusjonsproteiner kan uttrykkes i E. coli med&gt; 95% homogenitet og høyt utbytte (&gt; 25 mg / ml) (figur 2). Generelt, en- og to-finger Zif-fusjonsproteiner som kan produseres i mengder som nesten er identiske med dem for villtype-modifisert protein. Men i noen sammenhenger, fem- og seks-finger ZIF fusjonsproteiner er ikke i stand til å bli produsert i rentene høye nok for nedstrøms applikasjoner. Direkte påføring av to fingre Zif-EmGFP pr...

Watch this Scientific Journal Video about Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains at JoVE.com

Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains

Sink finger-domener er iboende celle-gjennomtrengelige og i stand til å mediere proteinlevering til et bredt utvalg av pattedyrcelletyper. Her er en detaljert steg-for-steg-protokollen for å implementere sink-finger-teknologien for intracellulær protein levering presentert.

Direkte Protein Levering til pattedyrceller Bruke Cell-gjennomtrengelig Cys<sub&gt; 2</sub&gt; -Hans<sub&gt; 2</sub&gt; Sink-finger domener

Due to their modularity and ability to be reprogrammed to recognize a wide range of DNA sequences, Cys2-His2 zinc-finger DNA-binding domains have emerged ...

Research

JoVE Journal

Biology

Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains

Long-term Imaging Mammalian Cells using Wide-Field Microscopy

Trypsinizing and Subculturing Mammalian Cells

As cells reach confluency, they must be subcultured or passaged. Failure to subculture confluent cells results in reduced mitotic index and eventually in ...

Engineering Cell-permeable Protein

The protein transduction technique enables the direct delivery of biologically active material into mammalian cells [for review see 1,2]. For this one can ...

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

Genome Editing with CompoZr Custom Zinc Finger Nucleases (ZFNs)

Genome Editing with CompoZr Custom Zinc Finger Nucleases ZFNs

Genome Redigerer med CompoZr Custom sink finger nukleaser (ZFNs)

Genome editing is a powerful technique that can be used to elucidate gene function and the genetic basis of disease. Traditional gene editing methods such ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

siRNA Screening for å identifisere Ubiquitin og Ubiquitin-lignende system regulatorer av biologiske mekanismer i dyrkede pattedyrceller

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...

Identification of Protein Interaction Partners in Mammalian Cells Using SILAC-immunoprecipitation Quantitative Proteomics

Identifisering av protein interaksjon Partnere i pattedyrceller Bruke SILAC-immunoprecipitation Kvantitative Proteomikk

Quantitative proteomics combined with immuno-affinity purification, SILAC immunoprecipitation, represent a powerful means for the discovery of novel ...

Zinc-finger Nuclease Enhanced Gene Targeting in Human Embryonic Stem Cells

Sink-finger Nuclease Forbedret Gene Targeting i humane embryonale stamceller

One major limitation with current human embryonic stem cell (ESC) differentiation protocols is the generation of heterogeneous cell ...

Examination of Proteins Bound to Nascent DNA in Mammalian Cells Using  BrdU-ChIP-Slot-Western Technique

Undersøkelse av Proteiner Bundet til begynnende DNA i pattedyrceller Bruke BrdU-chip-Slot-vestlig Technique

Histone deacetylases 1 and 2 (HDAC1,2) localize to the sites of DNA replication. In the previous study, using a selective inhibitor and a genetic ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Et grafisk brukergrensesnitt for programvareassistert sporing av proteinkonsentrasjon i dynamiske cellulære protrusjoner

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...