Hurtig og effektiv ekspression af flere proteiner i aviær embryoner ved hjælp af mRNA-elektroporation
Summary
Vi rapporterer Messenger RNA (mRNA) elektroporation som en metode, der tillader hurtig og effektiv ekspression af flere proteiner i vagtler embryo modelsystemet. Denne metode kan anvendes til fluorescently label celler og registrere deres in vivo bevægelser ved time-lapse mikroskopi kort efter elektroporation.
Abstract
Vi rapporterer, at mRNA elektroporation tillader fluorescerende proteiner til at mærke celler i levende vagtler embryoner hurtigere og bredt end DNA-elektroporation. Den høje transfektering effektivitet tillader mindst 4 særskilte mRNAs at blive Co-transfected med ~ 87% effektivitet. De fleste af de elektro porterede mRNAs nedbrydes i løbet af de første 2 h post-elektro poration, hvilket tillader tidsfølsomme eksperimenter, der skal udføres i det udviklende embryon. Endelig beskriver vi, hvordan man dynamisk billede levende embryoner elektroporeret med mRNAs at indkode forskellige subcellulære målrettede fluorescerende proteiner.
Introduction
Elektro poration er en fysisk transfektering metode, der bruger en elektrisk puls til at skabe forbigående porer i plasma membranen, så stoffer som nukleinsyre eller kemikalier til at passere ind i cytosol. Elektro poration anvendes i vid udstrækning til at levere DNA til bakterier, gær, planter og pattedyrsceller1,2,3. Det bruges rutinemæssigt til at introducere genetiske nyttelast i målceller og væv inden for det udviklende aviær embryo for at studere den genetiske kontrol med udvikling eller mærke migrerende populationer af celler4,5,6, 7. Der findes dog også flere eksperimentelle begrænsninger med DNA-elektroporation8. For eksempel introducerer DNA-elektro portering ofte meget varierende antal udtryks vektorer pr. celle og efterfølgende mRNAs og proteiner, som de koder. Denne variation kan føre til en betydelig celle celle heterogenitet, der komplicerer både billedanalyse og data tolkning9,10. Desuden, proteiner fra DNA-elektroporation kun begynde at udtrykke ~ 3 h post-elektro poration og ikke når den maksimale effektivitet i celle nummer og fluorescens intensitet indtil 12 h, sandsynligvis på grund af den tid, der kræves for at overføre til kernen og komplet både transskription og oversættelse in vivo11.
I modsætning hertil har mRNA transfektering været anvendt effektivt i en række forskellige modelsystemer, herunder xenopus laevis oocytter ved mikroinjektion12,13, omprogrammering af humane stamceller ved mRNA lipofectamintransfection14 og elektroporerende genstridige neurale stamceller i voksne mus15. Vi testede mRNA electroporations evne til effektivt at mærke celler under tidlig aviær embryonal udvikling ved hjælp af in vitro syntetiserede Mrna’er, der indkoder distinkte fluorescerende proteiner (FPs). Til vores studier brugte vi pCS2 + Vector, en multifunktions ekspressions vektor, der almindeligvis anvendes til at udtrykke proteiner i xenopus og Zebra embryoner. SP6-og T7 RNA-polymerasepromotorerne i pCS2 + tillader syntesen af mRNA og protein fra ethvert klonet gen, når det anvendes i et in vitro-transskription/oversættelsessystem.
Her viser vi, at mRNA elektroporation giver mulighed for hurtig og effektiv ekspression af fluorescerende proteiner (fps) i gastrulerende vagtler embryoner. Vi designede og genererede mange af de udtryks vektorer, der anvendes i disse studier. For eksempel under klappede vi LifeAct-eGFP gene16 i pCS2 + Vector17 for at udtrykke fra CMV promoter og SP6 Promoter. Det indsatte gen ligger nedstrøms for SP6-promotoren og opstrøms for SV40 poly (A) hale18. I embryoner Co-elektroporeret med mRNA og DNA, FPs kodet fra in vitro transkriberet mRNAs blev først detekteret inden for 20 min af elektro poration, hvorimod FPs fra DNA-udtryks vektorer blev detekteret først efter 3 h. multiple mRNAs kodning for nuklear, Golgi, og membran proteiner kan elektroporeres ind i et embryon samtidigt, hvilket resulterer i hurtig og effektiv ekspression af flere proteiner i individuelle celler. Endelig, ved hjælp af en in vivo fluorescens opsving efter photobleaching (FRAP) assay, viser vi, at et flertal af de elektro porterede mRNAs forfald inden for 2 h. Således, hurtig indledende protein produktion kombineret med begrænset ny protein oversættelse gør mRNA elektro poration en værdifuld teknik, når tidsmæssig kontrol af udtryk er nødvendig.
Protocol
Representative Results
Discussion
I denne protokol, vi gav trin for trin instruktioner om, hvordan man præcist mikroinjicere og elektroporat mRNA i cellerne i gastrulerende vagtler embryoner. Vi påviste, at in vitro syntetiseret mRNA elektroporation giver hurtig og effektiv ekspression af fluorescerende proteiner (FPs) i gastrulerende vagtler embryoner (figur 2 og 3). Fluorescens fra H2B-Citrine protein oversat fra elektro porteret mRNAs kunne påvises ved Konfokal mikroskopi inden for ~ 20 min og steg i F…
Declarações
The authors have nothing to disclose.
Acknowledgements
Vi takker David Huss for nyttige indblik i dette arbejde. Dette arbejde blev delvist støttet af Rose Hills Foundation Summer Research Fellowship (2016-2018) og USC Provosts undergrad Research Fellowship til M.T., Saban Research Institute Intramural Training pre-doktor Award til M.D., og University of Southern California bachelor forskning Associates program Award til R.L.
Materials
| BamHI-HF | New England Biolabs | R3136L | |
| BglII | New England Biolabs | R0144S | |
| BsrG1-HF | New England Biolabs | R3575S | |
| NotI-HF | New England Biolabs | R3189L | |
| SalI-HF | New England Biolabs | R3138L | |
| Phenol:Chloroform:Isoamyl Alcohol | Thermo Fisher | 15593031 | |
| SP6 mMessage Machine in vitro transcription kit | Thermo Fisher | AM1340 | |
| Fast Green FCF | Sigma Aldrich | F7252 | |
| Triton X-100 | Sigma Aldrich | 93443 | 4-(1,1,3,3-Tetramethylbutyl)phenyl-polyethylene glycol, t-Octylphenoxypolyethoxyethanol, Polyethylene glycol tert-octylphenyl ether |
| DAPI | Sigma Aldrich | D9542 | 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride, 4′,6-Diamidino-2-phenylindole dihydrochloride, DAPI dihydrochloride |
| Whatman No.1 filter paper | Sigma Aldrich | WHA1001125 | |
| glycerol | Sigma Aldrich | G9012 | |
| Urea | Sigma Aldrich | 51457 | |
| pmTurquoise2-Golgi | Addgene | 36205 | pmTurquoise2-Golgi was a gift from Dorus Gadella (Addgene plasmid # 36205 ; http://n2t.net/addgene:36205 ; RRID:Addgene_36205) |
| pmEGFP-N1-LifeAct | Nat. Methods 2008;5:605-7. PubMed ID: 18536722 | ||
| pCS2.Lifeact-mGFP | Addgene | This paper | |
| pCS.H2B-citrine | Addgene | 53752 | pCS-H2B-citrine was a gift from Sean Megason (Addgene plasmid # 53752 ; http://n2t.net/addgene:53752 ; RRID:Addgene_53752) |
| pCS.memb-mCherry | Addgene | #53750 | pCS-memb-mCherry was a gift from Sean Megason (Addgene plasmid # 53750 ; http://n2t.net/addgene:53750 ; RRID:Addgene_53750) |
| Zeiss LSM-780 inverted microscope | Carl Zeiss Microscopy GmbH | The LSM-780 is a confocal and multi-photon microscope that offers the sensitivity required for vital imaging work. Equipped with a motorized stage, an autofocus device, and a full stage-top blackout incubator, the 780 is an excellent microscope for high-end live cell/embryo imaging. The high-sensitivity 32-channel Quasar detector allows for spectral imaging, linear unmixing, and high color count (>4) image acquisition. Excitation can be performed with 6 lines single photon lasers (405, 458, 488, 514, 564 and 633 nm), Chameleon (Coherent) 2-photon laser (range from 690nm to 1000nm), and run with ZEN 2011 SP7 (Black) system software. | |
| CUY-21 EDIT in vivo electroporator | Bex Co., Ltd. | ||
| Platinum flat square electrode, side length 5 mm | Bex Co., Ltd. | LF701P5E | |
| Olympus MVX10 FL Stereo Microscope | Olympus LifeScience | ||
| XM10 Monochrome camera | Olympus LifeScience | ||
| Phosphate-Buffered Saline (PBS) for HCR (10×, pH 7.4) | To prepare 1 L of a 10× stock solution, combine 80 g of NaCl (Sigma-Aldrich S3014), 2 g of KCl (Sigma-Aldrich P9541), 11.4 g of Na2HPO4 (anhydrous; Sigma-Aldrich S3264), and 2.7 g of KH2PO4 (anhydrous; Sigma-Aldrich P9791). Adjust the pH to 7.4 with HCl, and bring the final volume to 1 L with ultrapure H2O. Avoid using CaCl2 and MgCl2 in PBS for HCR. It is important that the PBS for HCR is prepared as an RNase-free solution (e.g., via diethylpyrocarbonate [DEPC] treatment). | ||
| 1.37 M NaCl | |||
| 27 mM KCl | |||
| 80 mM Na2HPO4 20 mM KH2PO4 | |||
| PBS/Triton | Add 1 mL of Triton X-100 (Sigma Aldrich 93443) and 100 mL of 10× PBS to 890 mL of ultrapure distilled H2O. Filter the solution through a 0.2-μm filter and store it at 4 ̊C until use. | ||
| 1× phosphate-buffered saline (PBS) (DEPC-treated; pH 7.4) | |||
| 0.1% Triton X-100 |
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