Name: genomic transformation of the picoeukaryote ostreococcus tauri
Uploaded: 2012-07-13T13:00:00
Duration: 10 min 45 s
Description: Watch this Scientific Journal Video about Genomic Transformation of the Picoeukaryote Ostreococcus tauri at JoVE.com

SynthSys et Center for Integrativ Systembiologi finansieret af BBSRC og EPSRC pris D019621. EU FP6 Network of Excellence &quot;Marine Genomics&quot; tilskud til FYB har finansieret udviklingen af ​​genetisk transformation metoder.

1. Fremstillinq af algemateriale <ol><li> Ostreococcus celler dyrkes i kunstigt havvand (ASW). Havsalt (typisk omkring 40 g per liter) opløses i deioniseret vand til en salinitet på 30 ppt, som målt ved hjælp af en salinitet meter. Berigelsesmedium 11,12, sporstoffer metalelementer og vitaminer tilsættes som beskrevet i tabel 1-3. Medierne derefter filtersteriliseret gennem et 0,22 um filter. </li><li> For vedligeholdelse, er celler af Ostreococcus stamme OTTH95 subkultiveres aseptisk i en fortynding på 1/100 i frisk ASW hver 7 dage og dyrket under konstant lys i et plantevækstregulerende inkubator forsynet med måneskinsblå filter. Lysintensiteten skal være tæt på 20 umol m-2s-1 og temperaturen holdes på 20 ° C. Cellerne ikke kræver konstant omrøring, men rystes gang hver 2. til 3. dag for at forhindre aggregering. </li><li> For hver omdannelse, er 50 ml celler kræves på en celle density 20-30 x 10 6 ml -1, som skal nås 5-7 dage efter sub-dyrkning. Omtrentlig celledensitet og axeny kan bestemmes ved anvendelse af enten flowcytometri eller et hæmocytometer og mindst en x40 forstørrelse. </li></ol> 2. Elektroporation <ol><li> Fremstilling af DNA til transformation. For hver omdannelse, 5 ug af ren, lineariserede plasmid DNA, der kræves ved en koncentration på 1 ug / ul i sterilt deioniseret vand. Til opnåelse af dette DNA, Forfatterne anbefaler anvendelse af en Qiagen Midi prep kit, selvom andre fremgangsmåder kan fungere lige så godt. Fordøje produkt med et enzym, der skærer i skelettet af den anvendte vektor, men ikke i transgen eller selektionsgen. Yderligere oprensning og koncentreres den resulterende lineære DNA ved hjælp af ethanolfældning og resuspender produktet i en passende volumen af ​​høj kvalitet sterilt deioniseret vand. </li><li> Fremstille mikrocentrifugerør indeholdende 5 ug DNA for hver transformation. En kontroversiellel uden DNA er nødvendigt for hver cellelinje, der skal transformeres. Holde disse rør på is, sammen med et 2 mm elektropora-tionskuvette for hver transformation. </li><li> Forbered 2,2 ml resuspenderingsbufferen pr transformation. Opløs Sorbitol til en 1 M opløsning i ddH2O tilsættes 0,1% pluronsyre F68 og filter-steriliseres. </li><li> Tilsættes pluronsyre F68, til en slutkoncentration på 0,1% til cellerne, og pelleten i 10 minutter ved 8000x g ved 10 ° C i et 50 ml rør med konisk bund. Umiddelbart resuspender cellerne i 1 ml resuspensionsbuffer ved at pipettere op og ned, og overføres til et mikrofugerør. Spinnes ned i 10 minutter ved 8000 xg ved 10 ° C, og arbejder hurtigt, skal du gentage dette vasketrin en gang mere. </li><li> Med en cut spids, resuspenderes hver endelige pellet i 40 ul resuspensionsbuffer. Tilsæt 40 ​​ul af de resuspenderede celler til hver rør af lineariseret DNA på is, bland forsigtigt og overføre til elektroporation kuvetten. </li><li> Sætte kuvette i electroptalen maskine. Skift indstillinger til 6 kV cm -1, 600 Ω og 25 uF. Elektroporere cellerne. </li><li> Inkubér electoporated celler i kuvetterne ved stuetemperatur i 10 minutter, og bruge tid til at forberede vævskulturkolber. Mærk dem og tilføje 30 ml frisk ASW til hver. Tag 1 ml af hver kolbe og forsigtigt tilføje den til den tilsvarende kuvette. Inkuber i 2 minutter og fjern forsigtigt ASW, der nu indeholder det dråbe af celler og forsigtigt og langsomt pipetteres direkte i ASW i dyrkningskolbe. </li><li> Celler typisk forblive i en dråbe. Pas på ikke at ryste eller forstyrre de aggregerede cellerne i dette øjeblik. Tillade cellerne at genvinde i inkubatoren i 1-2 timer, og derefter genopslæmme ved omrystning af kolberne. På dette tidspunkt, bør cellerne resuspenderes frit og ingen klumper skal være synlig. Lad kulturerne til at inddrive natten i inkubatoren. </li></ol> 3. Inddragelse af celler på plader i halvfast medium <ol><li&gt; Den næste dag autoklaveres en opløsning af 2,1% agarose med lavt smeltepunkt i ddH2O i en flaske indeholdende en rørepind. Holde smeltet ved 65-90 ° C i et større bægerglas indeholdende vand på en opvarmning magnetomrører. For hver omdannelse fremstilling 8 petriskåle (55 mm diameter) og 8 x 15 ml indeholdt hvert 9 ml ASW plus det ønskede valg. Hvis du bruger nourseothricin eller G418, skal du bruge 2 mg / ml. </li><li> Indsamle de transformerede celler fra inkubatoren. Arbejder i en steril flowhood, tilsæt 1 ml kogende LMP-agarose til 9 ml i et af rørene. Glasset lukkes, og blandes ved at vende. Derpå tilsættes 0,5 ml frisk transformerede celler hurtigt blandes og hæld i pladen. Gentag denne proces med de resterende 7 rørene og derefter gå videre til den næste transformation kolben. </li><li> Lade pladerne åbne i stinkskab for en time, for agarose at indstille. Luk derefter pladerne og overføre dem til store firkantede petriskåle, som afholder 4 plader hver. Forsegl kvadratiske plader with parafilm. Bemærk, at pladerne ikke bliver sat helt, og som følge heraf er gelen meget skrøbelige. Man skal passe på ikke at bryde gelen ved håndtering af pladerne. Placer alle kvadratiske plader i inkubatoren. </li></ol> 4. Udvælgelse af transformerede kolonier <ol><li> Kolonier skal vises efter 10 til 21 dage om omdannelse plader, men ikke på mock transformerede plader. Hvis du vil vælge kolonier bruge en 200 gl pipette med cut-off tips. Du skal blot vælge frie kolonier og suge ud den grønne koloni. Pas på ikke at inkludere alle celler fra nærliggende kolonier. </li><li> Overføre cellerne til 2 ml flydende medium indeholdende selektion i en 24 brønde plade. Ved anvendelse nourseothricin, anvende 1,75 mg / ml. Blandes ved pipettering op og ned. Vælg 24-50 kolonier pr transformation. Forsegle pladerne med parafilm og overførsel til inkubatoren. </li><li> Efter en uge, overføres 100 ul af hver brønd til 2 ml frisk ASW med selektion i et 24 brønde plade og vokse til en additional 7 dage. Overlevende cellelinier kan anvendes til yderligere undersøgelser. Stabil integration i genomet og insertion nummer skal analyseres under anvendelse af PCR og Southern Blot. Genomisk DNA kan opnås for disse formål ved hjælp af en ned-skaleret standardmetode til plante-DNA-ekstraktion. </li></ol> 5. Repræsentative resultater Cellerne delt 1/100 nåede en densitet på 25 millioner celler pr efter dyrkning i 7 dage. Elektroporering af celler med de passende indstillinger resulterede i en tidskonstant på mellem 10 og 14 millisekunder. Når celler overføres til mediet i dyrkningskolber, bør en dråbe af celler dannes. Da let rystet efter en time, bør cellerne let opblandingen. Optagelse af 1 ml transformerede celler i 0,2% LMP agar bør resultere i en ensartet, men kun halvfast gel. Kolonier skal vises efter 10 til 21 dage. Når transformerende 5 ug lineariseret DNA under anvendelse af protokollen ovenfor, 50-100 kolonier pr transformationPladen blev typisk forventes, versus ingen af ​​de negative kontrolprøver plader. ~ 80% af opsamlet kolonier blev positivt selekteret ved antibiotisk resistens i flydende medium, og blev anvendt i efterfølgende studier. <table border="1"><tbody><tr><td></td><td> Slutkoncentration </td><td> Stamopløsning </td><td> Volumen til 1 liter </td></tr><tr><td> NaNO 3 </td><td> 8,83 x 10 -4 M </td><td> 75 g / L dH2O </td><td> 1 ml </td></tr><tr><td> NH4Cl </td><td> 3,63 x 10 -5 M </td><td> 2,68 g / L dH2O </td><td> 1 ml </td></tr><tr><td> β-glycerophosphat </td><td> 1 x 10 -5 M </td><td> 2,16 g / L dH2O </td><td> 1 ml </td></tr><tr><td> H2 Seo 3 </td><td> 1 x 10-8M </td><td> 1,29 mg / L dH2O </td><td> 1 ml </td></tr><tr><td> Tris-base (pH 7,2) </td><td> 1 x 10 -3 M </td><td> 121,1 g / L dH2O </td><td> 1 ml </td></tr><tr><td> K spormetal-opløsning </td><td></td><td> Tabel 2 </td><td> 1 ml </td></tr><tr><td> f / 2 vitaminopløsning </td><td></td><td> Tabel 3 </td><td> 0,5 ml </td></tr></tbody></table> Tabel 1. Medium bestanddele for vækst af O. Tauri 11, 12. udgør et samlet volumen på 1 liter af kunstigt havvand med en salinitet på 30 ppt, og der tilsættes ovennævnte komponenter fra stamopløsninger som angivet. Filter-sterilisere mediet og brug inden for en uge. Lagre af alle forbindelser undtagen vitamin opløsningen kan samles til at tilføje 6 ml af denne opløsning til hver liter medium. <table border="1"><tbody><tr><td></td><td> Slutkoncentration </td><td> Stamopløsning </td><td> Beløb til 1 liter </td></tr><tr><td> Na2 </sub&gt; EDTA • 2H 2 O </td><td> 1 x 10 -4 M </td><td></td><td> 41,6 g </td></tr><tr><td> FeCl3 • 6H H2O </td><td> 1 x 10 -5 M </td><td></td><td> 3,15 g </td></tr><tr><td> Na 2 Moo 4 • 2H 2 O </td><td> 1 x 10-8M </td><td> 6,3 g / L dH2O </td><td> 1 ml </td></tr><tr><td> ZnSO 4 • 7H 2 O </td><td> 1 x 10-9M </td><td> 22,0 g / L dH2O </td><td> 1 ml </td></tr><tr><td> CoCl 2 • 6H 2 O </td><td> 1 x 10-9M </td><td> 10,0 g / L dH2O </td><td> 1 ml </td></tr><tr><td> MnCl 2 • 4H 2 O </td><td> 1 x 10-8M </td><td> 180,0 g / L dH2O </td><td> 1 ml </td></tr><tr><td> CuSO 4 • 5H 2 O </td><td> 1 x 10-8M </td><td> 9,8 g / L dH <sub&gt; H2O </td><td> 1 ml </td></tr></tbody></table> Tabel 2 nedenfor. Sporstofanalyse løsning 11,12. Fyld op til i alt 1 liter, i dH2O, og varme for at opløse. Alikvot af opløsningen og fryse til opbevaring. Slutkoncentrationer angivet vedrører den endelige medium, ikke spormetal-opløsning. <table border="1"><tbody><tr><td></td><td> Slutkoncentration </td><td> Stamopløsning </td><td> Beløb til 1 liter </td></tr><tr><td> Vitamin B 12 </td><td> 1 x 10 -10 M </td><td> 1 g / L dH2O </td><td> 1 ml </td></tr><tr><td> Biotin </td><td> 1 x 10-9M </td><td> 0,1 g / L dH2O </td><td> 10 ml </td></tr><tr><td> Thiamin • HCl </td><td> 1 x 10-7M </td><td></td><td> 200 mg </td></tr></tbody></table> Tabel 3. f / 2vitaminopløsning 11. fyldes op til i alt 1 liter i dH2O, filter-sterilisere alikvot og fryse. Slutkoncentrationer angivet vedrører den endelige medium, der ikke vitamin opløsningen. <img alt="Figur 1" src="/files/ftp_upload/4074/4074fig1.jpg" /> Figur 1. Grafisk overblik over proceduren for transformation. Skematisk fremstilling af den procedure, der genetisk transformere Ostreococcus Tauri af elektroporation. <img alt="Figur 2" src="/files/ftp_upload/4074/4074fig2.jpg" /> Figur 2. Kulturvækst. Celler subkultiveres aseptisk i en fortynding på 1/100 i frisk ASW hver 7 dage og dyrket under konstant lys i et plantevækstregulerende inkubator forsynet med måneskinsblå filter. Lysintensiteten skal være tæt på 20 umol m-2s-1 og temperaturen holdes på 20 ° C Cellerne ikke kræver konstant omrøring, men erHaken gang hver 2. til 3. dag for at forhindre aggregering. <img alt="Figur 3" src="/files/ftp_upload/4074/4074fig3.jpg" /> Figur 3. Colony prisdannelse på 0,2% LMP agarose. Overførsel 4 plader til en stor firkant petriskål, og tætning med parafilm (øverst til venstre). Når kolonier er dannet, skal du blot vælge gratis (ideelt set konisk formede) kolonier (øverst til højre) og suge dem ud af pladen med en P200 pipette. Der bør ikke være nogen kolonier på den negative kontrol (nederst til højre).

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Cellulær stat og kultur axeny er af afgørende betydning for proceduren for transformation. Selvom celler normalt holdes i cyklusser på 12 timers lys og blev 12 timer mørke, transformationseffektivitet i celler dyrket under disse betingelser viser sig at være utilstrækkelige. I løbet af de gentagne foderpelleteringsforhold / resuspension trin, bør celler altid opblandingen nemt. Hvis celler aggregat eller en slim-lignende stof er til stede, er det bedre at starte proceduren fra begyndelsen igen. Typisk kan ~ 50 kolonier forventes på hver transformation plade, versus ingen på den negative kontrol pladen. I tilfælde af lav transformationseffektivitet, bør dyrkningsbetingelser varieres for at sikre cellerne er i den optimale tilstand. Variabler, der påvirker transformationseffektivitet inkludere omgivende temperatur i laboratoriet (bør være tæt på 20 ° C) og håndtering af hastighed (Procedure fra pelletering cellerne [2,3] til nyttiggørelse [2,7] bør tage ~ 1 time). Det er også vigtigt, at alle opløsninger enre lavet frisk før hver transformation. Optages i plader, er koncentrationen af LMP-agarose afgørende. Ostreococcus celler vil ikke vokse i høje agarose koncentrationer og vil diffundere i lave koncentrationer. Tre transformationsvektorer for Ostreococcus er blevet offentliggjort tidligere 6, og flere vektorer forventes at blive genereret og offentliggjort inden længe. Den eksisterende vektor Pot-Luc 6 tillader anvendelsen af en promotor valg i kombination med et C-terminalt ildflueluciferase tag tillader hurtigere transgene linjevalg plus medgørlige ekspressionsmønstre, hvorimod Potox vektoren 6 bærer en stærk inducerbar promotor 13 tages fra Ostreococcus høj affinitet Phosphate Transporter (HAPT)-genet for overekspression af genet af interesse. Den Potox-Luc vektoren stammer fra Potox, der bærer en yderligere luciferase markør, der kan anvendes til indirekte selektion af overekspression linier <sop&gt; 14. Succesfulde vælgemærkerne på disse vektorer er nourseothricin og G148. Imidlertid Ostreococcus celler er meget resistente over for mange antibiotika, og koncentrationerne af forbindelserne, der er nødvendige for selektion af transgene Ostreococcus celler er højere end for konventionelle modelsystemer (2 mg / ml). Selv om processen synes ineffektiv, det store antal celler og plasmid-DNA er nødvendig for denne fremgangsmåde udgør nogen væsentlig hindring. En større begrænsning er, at hver genereret linje, der er resistent over for den selekterbare markør skal analyseres individuelt til at kontrollere, at hele konstruktionen er integreret i DNA. Vi har observeret eksempler, i hvilke vellykket ekspression af den selekterbare markør ikke svarer til indsættelse af selve genet af interesse; dvs partielle integrationer kan forekomme. Åbenbart tilfældig insertion i genomet betyder også, at der ikke er nogen styring af indsættelsesstedet anvendelse af denne metode, og fremgangsmåder based på homolog rekombination er i øjeblikket ved at blive udviklet. Men den metode, der er beskrevet her, er, at vores bedste viden, der i øjeblikket er den eneste karakteriseret metode til at generere genetisk modificerede Ostreococcus celler. Som Ostreococcus Tauri er først for nylig blevet brugt som en eksperimentel model organisme, de fleste metoder arbejder med disse celler vil sandsynligvis udvikle sig og blive forbedret af stigende forskningsmæssig involverede samfund. Denne protokol har til formål at hjælpe folk indlede arbejdet med Ostreococcus, men på ingen måde hævder at beskrive alt, hvad der er at vide om genomisk transformation af denne microalga. Forfatterne tillid til, at læserne vil være i stand til at tilpasse denne protokol til deres egne behov, som små forskelle i inkubatorer (lysniveauer eller kvalitet) og andre parametre vil eksistere, og sandsynligvis skal optimeres i hvert enkelt laboratorium for at få de sunde kulturer er nødvendige for denne protokol. Efter at have læst de teknikker, der er beskrevet her,vektorer kan konstrueres til at generere luminescerende, fluorescerende eller andre mærkede fusionsproteiner linier under kontrol af en række promotorer. Denne spændende roman eksperimentel platform vil være nyttigt at undersøge, hvordan komplicerede biokemiske problemer løses i en eukaryot af reduceret kompleksitet, hvor farmakologiske tilgange er meget lettere 3.

<table border="1"><tbody><tr><td colspan="4"> Medium bestanddele </td></tr><tr><td> Kemisk </td><td colspan="2" > Katalog nummer </td><td> Leverandør </td></tr><tr><td> Instant Ocean Marine Salt </td><td colspan="3"> fra <a href="http://amazon.co.uk" target="_blank">amazon.co.uk</a> eller <a href="http://rocketaquatics.co.uk" target="_blank">rocketaquatics.co.uk</a> </td></tr><tr><td> NaNO 3 </td><td colspan="2"> S5506-250G </td><td> Sigma </td></tr><tr><td> NH4Cl </td><td colspan="2"> A9434-500G </td><td> Sigma </td></tr><tr><td> Glycerol-2-phosphat-dinatriumsalt-hydrat </td><td colspan="2"> G9422-100G </td><td> Sigma </td></tr><tr><td> H2 Seo 3 </td><td colspan="2"> 84.920-250G </td><td> Sigma </td></tr><tr><td> Ultrarent tris </td><td colspan="2"> 15504-020 </td><td> Invitrogen </td></tr><tr><td> Na 2 EDTA • 2H 2 O </td><td colspan="2"> BPE120-500G </td><td> Fisher Scientific </td></tr><tr><td> FeCl3 • 6H H2O </td><td colspan="2"> 157740 </td><td> Sigma </td></tr><tr><td> Na 2 Moo 4 • 2H 2 O </td><td colspan="2"> 31.439-100G-R </td><td> Sigma </td></tr><tr><td> ZnSO 4 • 7H 2 O </td><td colspan="2"> Z0251 </td><td> Sigma </td></tr><tr><td> CoCl 2 • 6H 2 O </td><td colspan="2"> 31277 </td><td> Sigma </td></tr><tr><td> MnCl 2 • 4H 2 O </td><td colspan="2"> 203.734-5G </td><td> Sigma </td></tr><tr><td> CuSO 4 • 5H 2 O </td><td colspan="2"> C8027 </td><td> Sigma </td></tr><tr><td> Vitamin B 12 (cyanocobalamin) </td><td colspan="2"> V2876 </td><td> Sigma </td></tr><tr><td> Biotin </td><td colspan="2"> 47868 </td><td> Sigma </td></tr><tr><td> Thiamin • HCl </td><td colspan="2"> T4625 </td><td> Sigma </td></tr><tr><td> Ampicillin </td><td colspan="2"> A9518-25G </td><td> Sigma </td></tr><tr><td> Neomycin </td><td colspan="2"> N6386 </td><td> Sigma </td></tr><tr><td> Kanamycin </td><td colspan="2"> K4000 </td><td> Sigma </td></tr><tr><td colspan="4"> Forbrugsstoffer til dyrkning </td></tr><tr><td colspan="2"> Item </td><td> Katalog nummer </td><td> Leverandør </td></tr><tr><td colspan="2"> Vævskulturkolbe T25 Suspensionskulturer ventileres med støbekanalen </td><td> 83.1810.502 </td><td> Starstedt </td></tr><tr><td colspan="2"> Vævskulturkolbe T75 Suspensionskulturer ventileres med støbekanalen </td><td> 83.1813.502 </td><td> Starstedt </td></tr><tr><td colspan="2"> Vævskulturkolbe T175 Suspensionskulturer ventileres med støbekanalen </td><td> 83.1812.502 </td><td> Starstedt </td></tr><tr><td colspan="2"> TPP flasketoppen filtre, 1L, komplette </td><td> 99950T </td><td> Helena Bioscience </td></tr><tr><td colspan="2"> TPP flasketoppen filtre, 500 ml, </td><td> 99500T </td><td> Helena Bioscience </td></tr><tr><td colspan="2"> TPP flasketoppen filtre, 250 ml, </td><td> 99250T </td><td> Helena Bioscience </td></tr><tr><td colspan="2"> TPP flasketoppen filtre, 150 ml, </td><td> 99150T </td><td> Helena Bioscience </td></tr><tr><td colspan="2"> Saltindhold Meter for Marine Aquarium </td><td> DSG-10 </td><td> Daeyoon </td></tr></tbody></table><table border="1"><tbody><tr><td colspan="3" > Kemikalier til transformation </td></tr><tr><td> Kemisk </td><td> Katalognummer </td><td> Leverandør </td></tr><tr><td> D-sorbitol </td><td> S6021-1KG </td><td> Sigma </td></tr><tr><td> Pluronic F-68-opløsning </td><td> P5556-100ML </td><td> Sigma </td></tr><tr><td> ClonNat </td><td> 51000 </td><td> Werner </td></tr><tr><td> Agarose med lavt smeltepunkt </td><td> 16520-050 </td><td> Invitrogen </td></tr><tr><td colspan="3"> Forbrugsstoffer til transformation </td></tr><tr><td> Item </td><td> Katalog nummer </td><td> Leverandør </td></tr><tr><td> Vævskulturkolbe T25 Suspensionskulturer ventileres med støbekanalen </td><td> 83.1810.502 </td><td> Starstedt </td></tr><tr><td> Vævskulturkolbe T75 Suspensionskulturer ventileres med støbekanalen </td><td> 83.1813.502 </td><td> Starstedt </td></tr><tr><td> Vævskulturkolbe T175 Suspension Cultures Frigjort med støbekanalen </td><td> 83.1812.502 </td><td> Starstedt </td></tr><tr><td> Greiner centrifugerør 50ml </td><td> 227261 </td><td> GBO </td></tr><tr><td> Gene Pulser / MicroPulser Kuvetter, 0,2 cm gap, 50 </td><td> 165-2086 </td><td> Bio-Rad </td></tr><tr><td> Petriskål runde PS 3 Vents H14.2 Φ55 </td><td> 391-0895 </td><td> VWR International </td></tr><tr><td> Petriskål firkant med fire porte polystyren steril ved bestråling klart 120mm </td><td> FB51788 </td><td> Fisher Scientific </td></tr><tr><td> Moonlight Blåt lys filter </td><td> 183 </td><td> Lee Belysning </td></tr></tbody></table>

genomic transformation of the picoeukaryote ostreococcus tauri

Almindelige problemer hindrer hurtige fremskridt i Plant Sciences omfatter celle, væv og hele organismen kompleksitet, og især det høje niveau af genomisk redundans påvirker simple genetik i højere planter. Den hidtil ukendte modelorganisme Ostreococcus Tauri er den mindste fritlevende eukaryot kendt til dato, og har en stærkt reduceret genomstørrelse og cellulær kompleksitet 1,2, manifesteret ved tilstedeværelsen af kun et af de fleste organeller (mitokondrier chloroplast, Golgi stabel) pr celle, og et genom, der kun indeholder ~~~HEAD=NNS 8000 gener. Endvidere, at kombinationen af ​​unicellularity og let kultur tilvejebringer en platform egnet til kemiske biologi fremgangsmåder. For nylig er Ostreococcus held blevet anvendt til at undersøge de grundlæggende mekanismer bag cirkadiske tidtagning 3-6. Resultater fra denne model organisme har påvirket ikke kun Plant Science, men også pattedyr biologi 7. Dette eksempel belyser, hvordan hurtige eksperimenter påsimple eukaryote fra den grønne linie kan accelerere forskning i mere komplekse organismer ved at generere testbare hypoteser ved hjælp af metoder, det er teknisk muligt kun på denne baggrund af reduceret kompleksitet. Kendskab til et genom og mulighed for at ændre gener er vigtige redskaber i enhver model arter. Genomisk 1, transkriptom 8, og proteom 9 oplysninger om denne art er frit tilgængelige, mens de tidligere rapporterede metoder 6,10 til genetisk omdanne Ostreococcus er kendt for at få laboratorier verden over. I denne artikel, at de eksperimentelle fremgangsmåder genetisk transformation denne hidtil ukendte model organisme med en overekspression konstruktion ved hjælp af elektroporering som beskrevet i detaljer, samt fremgangsmåden til optagelse af transformerede celler i lav procentdel agarose for at tillade selektion af transformerede linier stammer fra en enkelt transformeret celle. Efter den vellykkede anvendelse af OstreocoCCUS til circadian forskning, kan stigende interesse i Ostreococcus kan forventes fra forskellige forskningsområder inden for og uden plante videnskaber, herunder bioteknologiske områder. Forskere fra en bred vifte af biologiske og medicinske videnskaber, der arbejder på bevarede biokemiske veje kan overveje at videreføre forskning i Ostreococcus, fri fra genomiske og organismal kompleksiteten af større model arter.

Watch this Scientific Journal Video about Genomic Transformation of the Picoeukaryote Ostreococcus tauri at JoVE.com

Genomic Transformation of the Picoeukaryote Ostreococcus tauri

Denne artikel beskriver genetisk transformation af unicellulær marine alger<em&gt; Ostreococcus Tauri</em&gt; Ved hjælp af elektroporation. Dette eukaryot organisme er en effektiv model platform for højerestående planter, possesing stærkt reduceret genomisk og cellulær kompleksitet og er let kan både cellekultur og kemisk biologi.

Genomisk Transformation af Picoeukaryote<em&gt; Ostreococcus Tauri</em

Common problems hindering rapid progress in Plant Sciences include cellular, tissue and whole organism complexity, and notably the high level of genomic ...

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Research

JoVE Journal

Biology

Genomic Transformation of the Picoeukaryote Ostreococcus tauri

Isolation of Genomic DNA from Mouse Tails

Large Insert Environmental Genomic Library Production

The vast majority of microbes in nature currently remain inaccessible to traditional cultivation methods. Over the past decade, culture-independent environmental genomic (i.e. metagenomic) approaches have emerged, enabling researchers to bridge this cultivation gap by capturing the genetic content of indigenous microbial communities directly from the environment. To this end, genomic DNA libraries are constructed using standard albeit artful laboratory cloning techniques. Here we describe the construction of a large insert environmental genomic fosmid library with DNA derived from the vertical depth continuum of a seasonally hypoxic fjord. This protocol is directly linked to a series of connected protocols including coastal marine water sampling [1], large volume filtration of microbial biomass [2] and a DNA extraction and purification protocol [3]. At the outset, high quality genomic DNA is end-repaired with the creation of 5 -phosphorylated blunt ends. End-repaired DNA is subjected to pulsed-field gel electrophoresis (PFGE) for size selection and gel extraction is performed to recover DNA fragments between 30 and 60 thousand base pairs (Kb) in length. Size selected DNA is purified away from the PFGE gel matrix and ligated to the phosphatase-treated blunt-end fosmid CopyControl vector pCC1 (EPICENTRE <a href="http://www.epibio.com/item.asp?ID=385">http://www.epibio.com/item.asp?ID=385</a>). Linear concatemers of pCC1 and insert DNA are subsequently headfull packaged into phage particles by lambda terminase, with subsequent infection of phage-resistant E. coli cells. Successfully transduced clones are recovered on LB agar plates under antibiotic selection and archived in 384-well plate format using an automated colony picking robot (Qpix2, GENETIX). The current protocol draws from various sources including the CopyControl Fosmid Library Production Kit from EPICENTRE and the published works of multiple research groups [4-7]. Each step is presented with best practice in mind. Whenever possible we highlight subtleties in execution to improve overall quality and efficiency of library production. The whole process of fosmid library production and automated colony picking takes at least 7-10 days as there are many incubation steps included. However, there are several stopping points possible which are mentioned within the protocol.

Construction of a fosmid library with environmental genomic DNA isolated from the vertical depth continuum of a seasonally hypoxic fjord is described. The resulting clone library is picked into 384-well plates and archived for downstream sequencing and functional screening by the application of an automated colony picking system.

The vast majority of microbes in nature currently remain inaccessible to traditional cultivation methods. Over the past decade, culture-independent ...

Generation of RNA/DNA Hybrids in Genomic DNA by Transformation using RNA-containing Oligonucleotides

Synthetic short nucleic acid polymers, oligonucleotides (oligos), are the most functional and widespread tools of molecular biology. Oligos can be produced to contain any desired DNA or RNA sequence and can be prepared to include a wide variety of base and sugar modifications. Moreover, oligos can be designed to mimic specific nucleic acid alterations and thus, can serve as important tools to investigate effects of DNA damage and mechanisms of repair. We found that Thermo Scientific Dharmacon RNA-containing oligos with a length between 50 and 80 nucleotides can be particularly suitable to study, in vivo, functions and consequences of chromosomal RNA/DNA hybrids and of ribonucleotides embedded into DNA. RNA/DNA hybrids can readily form during DNA replication, repair and transcription, however, very little is known about the stability of RNA/DNA hybrids in cells and to which extent these hybrids can affect the genetic integrity of cells. RNA-containing oligos, therefore, represent a perfect vector to introduce ribonucleotides into chromosomal DNA and generate RNA/DNA hybrids of chosen length and base composition. Here we present the protocol for the incorporation of ribonucleotides into the genome of the eukaryotic model system yeast /Saccharomyces cerevisiae/. Yet, our lab has utilized Thermo Scientific Dharmacon RNA-containing oligos to generate RNA/DNA hybrids at the chromosomal level in different cell systems, from bacteria to human cells.

This work shows how to form an RNA/DNA hybrid at the chromosomal level and reveal transfer of genetic information from RNA to genomic DNA in yeast cells.

Synthetic short nucleic acid polymers, oligonucleotides (oligos), are the most functional and widespread tools of molecular biology. Oligos can be ...

Genomic MRI - a Public Resource for Studying Sequence Patterns within Genomic DNA

Non-coding genomic regions in complex eukaryotes, including intergenic areas, introns, and untranslated segments of exons, are profoundly non-random in their nucleotide composition and consist of a complex mosaic of sequence patterns. These patterns include so-called Mid-Range Inhomogeneity (MRI) regions -- sequences 30-10000 nucleotides in length that are enriched by a particular base or combination of bases (e.g. (G+T)-rich, purine-rich, etc.). MRI regions are associated with unusual (non-B-form) DNA structures that are often involved in regulation of gene expression, recombination, and other genetic processes (Fedorova &amp; Fedorov 2010). The existence of a strong fixation bias within MRI regions against mutations that tend to reduce their sequence inhomogeneity additionally supports the functionality and importance of these genomic sequences (Prakash et al. 2009).
Here we demonstrate a freely available Internet resource -- the Genomic MRI program package -- designed for computational analysis of genomic sequences in order to find and characterize various MRI patterns within them (Bechtel et al. 2008). This package also allows generation of randomized sequences with various properties and level of correspondence to the natural input DNA sequences. The main goal of this resource is to facilitate examination of vast regions of non-coding DNA that are still scarcely investigated and await thorough exploration and recognition.

We present a public computational web site for the analysis of genomic sequences. It detects DNA sequence patterns with various non-random nucleotide compositions. This resource also generates randomized sequences with diverse levels of complexity.

Non-coding genomic regions in complex eukaryotes, including intergenic areas, introns, and untranslated segments of exons, are profoundly non-random in ...

Competitive Genomic Screens of Barcoded Yeast Libraries

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily. The tempo of these advances is accelerating, promising greater depth and breadth. In light of these extraordinary advances, the need for fast, parallel methods to define gene function becomes ever more important. Collections of genome-wide deletion mutants in yeasts and E. coli have served as workhorses for functional characterization of gene function, but this approach is not scalable, current gene-deletion approaches require each of the thousands of genes that comprise a genome to be deleted and verified. Only after this work is complete can we pursue high-throughput phenotyping. Over the past decade, our laboratory has refined a portfolio of competitive, miniaturized, high-throughput genome-wide assays that can be performed in parallel. This parallelization is possible because of the inclusion of DNA 'tags', or 'barcodes,' into each mutant, with the barcode serving as a proxy for the mutation and one can measure the barcode abundance to assess mutant fitness. In this study, we seek to fill the gap between DNA sequence and barcoded mutant collections. To accomplish this we introduce a combined transposon disruption-barcoding approach that opens up parallel barcode assays to newly sequenced, but poorly characterized microbes. To illustrate this approach we present a new Candida albicans barcoded disruption collection and describe how both microarray-based and next generation sequencing-based platforms can be used to collect 10,000 - 1,000,000 gene-gene and drug-gene interactions in a single experiment.

We have developed comprehensive, unbiased genome-wide screens to understand gene-drug and gene-environment interactions. Methods for screening these mutant collections are presented.

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily.  The tempo of these advances is ...

Selective Capture of 5-hydroxymethylcytosine from Genomic DNA

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene expression, maintenance of genome integrity, parental imprinting, X-chromosome inactivation, regulation of development, aging, and cancer1. Recently, the presence of an oxidized 5-mC, 5-hydroxymethylcytosine (5-hmC), was discovered in mammalian cells, in particular in embryonic stem (ES) cells and neuronal cells2-4. 5-hmC is generated by oxidation of 5-mC catalyzed by TET family iron (II)/&alpha;-ketoglutarate-dependent dioxygenases2, 3. 5-hmC is proposed to be involved in the maintenance of embryonic stem (mES) cell, normal hematopoiesis and malignancies, and zygote development2, 5-10. To better understand the function of 5-hmC, a reliable and straightforward sequencing system is essential. Traditional bisulfite sequencing cannot distinguish 5-hmC from 5-mC11. To unravel the biology of 5-hmC, we have developed a highly efficient and selective chemical approach to label and capture 5-hmC, taking advantage of a bacteriophage enzyme that adds a glucose moiety to 5-hmC specifically12.
Here we describe a straightforward two-step procedure for selective chemical labeling of 5-hmC. In the first labeling step, 5-hmC in genomic DNA is labeled with a 6-azide-glucose catalyzed by &beta;-GT, a glucosyltransferase from T4 bacteriophage, in a way that transfers the 6-azide-glucose to 5-hmC from the modified cofactor, UDP-6-N3-Glc (6-N3UDPG). In the second step, biotinylation, a disulfide biotin linker is attached to the azide group by click chemistry. Both steps are highly specific and efficient, leading to complete labeling regardless of the abundance of 5-hmC in genomic regions and giving extremely low background. Following biotinylation of 5-hmC, the 5-hmC-containing DNA fragments are then selectively captured using streptavidin beads in a density-independent manner. The resulting 5-hmC-enriched DNA fragments could be used for downstream analyses, including next-generation sequencing.
Our selective labeling and capture protocol confers high sensitivity, applicable to any source of genomic DNA with variable/diverse 5-hmC abundances. Although the main purpose of this protocol is its downstream application (i.e., next-generation sequencing to map out the 5-hmC distribution in genome), it is compatible with single-molecule, real-time SMRT (DNA) sequencing, which is capable of delivering single-base resolution sequencing of 5-hmC.

Described is a two-step labeling process using &beta;-glucosyltransferase (&beta;-GT) to transfer an azide-glucose to 5-hmC, followed by click chemistry to transfer a biotin linker for easy and density-independent enrichment. This efficient and specific labeling method enables enrichment of 5-hmC with extremely low background and high-throughput epigenomic mapping via next-generation sequencing.

Selektiv opsamling af 5-hydroxymethylcytosine fra genomisk DNA

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene ...

Array Comparative Genomic Hybridization (Array CGH) for Detection of Genomic Copy Number Variants

Array CGH for the detection of genomic copy number variants has replaced G-banded karyotype analysis. This paper describes the technology and its application in a clinical diagnostic service laboratory. DNA extracted from a patient&#8217;s sample (blood, saliva or other tissue types) is labeled with a fluorochrome (either cyanine 5 or cyanine 3). A reference DNA sample is labeled with the opposite fluorochrome. There follows a cleanup step to remove unincorporated nucleotides before the labeled DNAs are mixed and resuspended in a hybridization buffer and applied to an array comprising ~60,000 oligonucleotide probes from loci across the genome, with high probe density in clinically important areas. Following hybridization, the arrays are washed, then scanned and the resulting images are analyzed to measure the red and green fluorescence for each probe. Software is used to assess the quality of each probe measurement, calculate the ratio of red to green fluorescence and detect potential copy number variants.

Array Comparative Genomic Hybridization Array CGH for Detection of Genomic Copy Number Variants

Array CGH for the detection of genomic copy number variants has replaced G-banded karyotype analysis. This paper describes the technology and its application in a diagnostic service laboratory.

Array Comparative Genomisk hybridisering (Array CGH) til påvisning af genomiske Kopiér nummer Varianter

Array CGH for the detection of genomic copy number variants has replaced G-banded karyotype analysis. This paper describes the technology and its ...

Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of diverse libraries of retroviral integration sites using ligation-mediated PCR (LM-PCR) amplification and next-generation sequencing (NGS), (2) mapping the genomic location of each virus-host junction using BEDTools, and (3) analyzing the data for statistical relevance. Genomic DNA extracted from infected cells is fragmented by digestion with restriction enzymes or by sonication. After suitable DNA end-repair, double-stranded linkers are ligated onto the DNA ends, and semi-nested PCR is conducted using primers complementary to both the long terminal repeat (LTR) end of the virus and the ligated linker DNA. The PCR primers carry sequences required for DNA clustering during NGS, negating the requirement for separate adapter ligation. Quality control (QC) is conducted to assess DNA fragment size distribution and adapter DNA incorporation prior to NGS. Sequence output files are filtered for LTR-containing reads, and the sequences defining the LTR and the linker are cropped away. Trimmed host cell sequences are mapped to a reference genome using BLAT and are filtered for minimally 97% identity to a unique point in the reference genome. Unique integration sites are scrutinized for adjacent nucleotide (nt) sequence and distribution relative to various genomic features. Using this protocol, integration site libraries of high complexity can be constructed from genomic DNA in three days. The entire protocol that encompasses exogenous viral infection of susceptible tissue culture cells to integration site analysis can therefore be conducted in approximately one to two weeks. Recent applications of this technology pertain to longitudinal analysis of integration sites from HIV-infected patients.

We describe a protocol for amplifying retroviral integration sites from the genomic DNA of infected cells, sequencing the amplified virus-host junctions, and then mapping these sequences to a reference genome. We also describe techniques to quantify the distribution of integration sites relative to various genomic annotations using BEDTools.

Forstærkning, Næste generations sekventering, og Genomisk DNA Kortlægning af retroviral integration steder

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of ...

Targeted RNA Sequencing Assay to Characterize Gene Expression and Genomic Alterations

RNA sequencing (RNAseq) is a versatile method that can be utilized to detect and characterize gene expression, mutations, gene fusions, and noncoding RNAs. Standard RNAseq requires 30 - 100 million sequencing reads and can include multiple RNA products such as mRNA and noncoding RNAs. We demonstrate how targeted RNAseq (capture) permits a focused study on selected RNA products using a desktop sequencer. RNAseq capture can characterize unannotated, low, or transiently expressed transcripts that may otherwise be missed using traditional RNAseq methods. Here we describe the extraction of RNA from cell lines, ribosomal RNA depletion, cDNA synthesis, preparation of barcoded libraries, hybridization and capture of targeted transcripts and multiplex sequencing on a desktop sequencer. We also outline the computational analysis pipeline, which includes quality control assessment, alignment, fusion detection, gene expression quantification and identification of single nucleotide variants. This assay allows for targeted transcript sequencing to characterize gene expression, gene fusions, and mutations.

We describe a targeted RNA sequencing-based method that includes preparation of indexed cDNA libraries, hybridization and capture with custom probes and data analysis to interrogate selected transcripts for gene expression, mutations, and gene fusions. Targeted RNAseq permits cost-effective, rapid evaluation of selected transcripts on a desktop sequencer.

Målrettet RNA Sekventering Assay til karakterisere genekspression og Genomisk Alterations

RNA sequencing (RNAseq) is a versatile method that can be utilized to detect and characterize gene expression, mutations, gene fusions, and noncoding ...

Preparing and Injecting Embryos of Culex Mosquitoes to Generate Null Mutations using CRISPR/Cas9

Culex mosquitoes are the major vectors of several diseases that negatively impact human and animal health including West Nile virus and diseases caused by filarial nematodes such as canine heartworm and elephantasis. Recently, CRISPR/Cas9 genome editing has been used to induce site-directed mutations by injecting a Cas9 protein that has been complexed with a guide RNA (gRNA) into freshly laid embryos of several insect species, including mosquitoes that belong to the genera Anopheles and Aedes. Manipulating and injecting Culex mosquitoes is slightly more difficult as these mosquitoes lay their eggs upright in rafts rather than individually like other species of mosquitoes. Here we describe how to design gRNAs, complex them with Cas9 protein, induce female mosquitoes of Culex pipiens to lay eggs, and how to prepare and inject newly laid embryos for microinjection with Cas9/gRNA. We also describe how to rear and screen injected mosquitoes for the desired mutation. The representative results demonstrate that this technique can be used to induce site-directed mutations in the genome of Culex mosquitoes and, with slight modifications, can be used to generate null-mutants in other mosquito species as well.&#160;

Preparing and Injecting Embryos of Culex Mosquitoes to Generate Null Mutations using CRISPR/Cas9

CRISPR/Cas9 is increasingly used to characterize gene function in non-model organisms. This protocol describes how to generate knock-out lines of Culex pipiens, from preparing injection mixes, to obtaining and injecting mosquito embryos, as well as how to rear, cross, and screen injected mosquitoes and their progeny for desired mutations.

Forberedelse og injektion af embryoner af Culex-myg til generering af Null-mutationer ved hjælp af CRISPR/Cas9

Culex mosquitoes are the major vectors of several diseases that negatively impact human and animal health including West Nile virus and diseases caused by ...