Name: transfection, selection, and colony-picking of human induced pluripotent stem cells talen-targeted with a gfp gene into the aavs1 safe harbor
Uploaded: 2015-02-01T14:00:00
Description: Watch this Scientific Journal Video about Transfection, Selection, and Colony-picking of Human Induced Pluripotent Stem Cells TALEN-targeted with a GFP Gene into the AAVS1 Safe Harbor at JoVE.com

This research was supported by the NIH Common Fund and Intramural Research Program of the National Institute of Arthritis, Musculoskeletal, and Skin Diseases.

1. Utarbeidelse av Basement Membran Matrix og Coating av Plast <ol><li> Plasser den frosne basalmembran matriselager fra -20 ° C på is og tine over natten ved 4 ° C. </li><li> Etter tining pipette 2 mg alikvoter av basalmembranmatrise inn i pre-avkjølte Eppendorf rør. Lagre disse ved -20 ° C inntil nødvendig. </li><li> Å forberede basalmembran matrix-belagte plater, tine en delmengde på is inntil den siste biten av is i eppendorf tube forsvinner (vanligvis innen ~ 2 timer). </li><li> Etter tining legge basalm...

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De mest kritiske trinn for vellykket generasjon AAVS1 trygg-havn målrettet menneskelige iPSCs er: (1) effektivt levere Talen og giver plasmider inn iPSCs etter transfeksjon; (2) å optimalisere dissosiasjon av iPSCs i enkeltceller før transfeksjon og plating tettheten etter transfeksjon; (3) å optimalisere dosen og tidspunktet for medikament-seleksjon basert på veksten av IPSC linje; (4) nøye dissekere og plukke målrettede kolonier og overføring til ny plate / brønn. Sammenlignet med lignende metoder som brukes ...

Muligheten til å omprogrammere menneskelige somatiske celler til embryonale stamcelleliknende induserte pluripotente stamceller (iPSCs) ble først oppdaget av Takahashi et al. I 2007 1. Menneske dermale fibroblaster transdusert med retrovirus uttrykker fire transkripsjonsfaktorer (Den såkalte kalt Yamanaka faktorer Oct3 / 4, Sox2, c-Myc, og Klf4) ble vist å være meget lik den humane embryonale stamceller (hESCs) basert på morfologi, proliferasjon, genekspresjon, og epigenetisk status; avgjørende, iPSCs er også i stand til å differensiere til cellene i alle tre bakterie lag 1. Den proliferativ potensial og differensiering kapasitet på iPSCs gjør dem svært attraktive verktøy; ved å omprogrammere celler fra pasienter som lider av bestemte sykdommer, kan iPSCs brukes både som in vitro sykdom modellsystemer, og som potensielle terapeutiske midler. For sistnevnte formål, må flere problemer løses før det fulle potensialet av iPSCsi en klinisk setting kan realiseres; den karsinogent potensial av in vitro dyrkede hESCs og iPSCs, bruk av xenogene derivater under omprogrammering og celle vedlikehold, og behovet for å spore transplanterte celler in vivo er alle viktige hekk til den kliniske anvendelse av pluripotente stamceller (gjennomgått av Hentze et al. 2). En ideell løsning på behovet for sporing differensierte celler etter transplantasjon ville innebære et visuelt påvisbar markør som motstår stanse og vekslande uavhengig av søknaden. Robust og varig uttrykk for integrerte transgener er mest lett oppnåelig når eksogene DNA er innført i safe-havn loci; som er, genomiske områder som muliggjør tilstrekkelig transkripsjon av en integrert vektor, mens på samme tid begrensende perturbasjoner av ekspresjon i nabogener 3. Et slikt område som har blitt svært godt karakterisert siden oppdagelsen er adenoassosiert virus integrasjon for 1. (AAVS1), i det første intron av proteinfosfatase en regulatorisk subenhet 12C (PPP1R12C) genet. Dette locus har vist seg ikke bare å tillate vedvarende og robust ekspresjon av integrerte transgener gjennom lengre tid i kultur og in vitro differensiering 3, men også for å beskytte omgivelsene gener fra transkripsjons perturbasjon 4; begge funksjonene er tenkt å være på grunn av tilstedeværelsen av endogene kromatin isolator elementer flankerer AAVS1 nettstedet fem. Fremskritt innen genom engineering verktøy enn bare det siste tiåret har i stor grad forenklet letthet og effektivitet som genetiske manipulasjoner i alle celletype kan oppnås. Mens tidligere vellykkede forsøk avhengig av overmåte lave nivåer av endogen homolog rekombinasjon (HR) med en innført donor for å oppnå genet styring i ESCs 6,7, ved bruk av stedspesifikke nukleaser, slik som sinkfinger nukleaser (ZFNs), som signifikantficantly indusere homolog rekombinasjon gjennom generering av en dobbel-strandet DNA pause har betydelig økt effektivitet av slike eksperimenter 8,9. Den gjenbruk av både transkripsjons aktivator lignende effektorer (Tales) av plante patogene xanthomonasgummi slekter og de ​​prokaryote gruppert regelmessig avbrutt av korte palindromiske gjentar (CRISPR) / Cas9 systemet til effektive stedsspesifikke designer nukleaser har gjort genet målretting i pluripotente stamceller en tilgjengelig og praktisk metodikk 10-13. En nyere artikkel beskrev en effektiv metode for stabil integrering av et grønt fluorescerende reporter kassetten inn i AAVS1 trygg-havn locus i menneskelige iPSCs bruker Tale nukleaser (Talens) 14. Disse målrettede iPSCs opprettholdt sin fluorescens selv etter rettet differensiering til kardiomyocytter og transplantasjon i en musemodell for hjerteinfarkt (MI), som gir sterke bevis for nytten av slikestabilt fluorescerende pluripotente stamceller 14. For å oppnå målrettet kolonier, ble et gen-felle metode anvendes hvori en spleising-akseptor (SA), plasserer 2A selv spalte peptidsekvens puromycin N-acetyl-transferase (PAC) genet under kontroll av den endogene promoter PPP1R12C; dermed bare iPSCs som har innarbeidet DNA donor ved AAVS1 locus uttrykke PAC, gjør dem valgbar basert på puromycin-motstand; (Figur 1, 15). Denne protokollen detaljer prosedyrene for å generere AAVS1-GFP iPSCs rapportert i den siste papir 14, herunder prosessen med å trans iPSCs med Talens og en 9.8 kb donor for å integrere en 4.2kb DNA fragment inn i AAVS1 trygg-havn locus, velge iPSCs basert på puromycin-motstand, og plukke kolonier for klonal ekspansjon. De teknikker som er beskrevet her, kan anvendes på mange tekniske genom eksperimenter.

<table border="0" cellpadding="0" cellspacing="0" width="1437">
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		<tr height="21">
			<td height="21" style="height:21px;width:588px;">NAME OF MATERIAL/EQUIPMENT</td>
			<td style="width:123px;">COMPANY</td>
			<td style="width:123px;">CATALOG #</td>
			<td style="width:603px;">COMMENTS/DESCRIPTION</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Matrigel Growth Factor Reduced (GFR) Basement Membrane Matrix, *LDEV-Free, 10mL</td>
			<td>Corning</td>
			<td>354230</td>
			<td>Store at -20&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM/F-12</td>
			<td>Life Technologies</td>
			<td>11320-033</td>
			<td>Store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Costar 6 Well Clear TC-Treated Multiple Well Plates</td>
			<td>Corning</td>
			<td>3506</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Essential 8 Medium</td>
			<td>Life Technologies</td>
			<td>A1517001</td>
			<td>Store basal medium at 4&#176;C. Store supplement at -20&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Y-27632 dihydrochloride</td>
			<td>Tocris</td>
			<td>1254</td>
			<td>Store at room temp. Once dissolved in H2O, store at -20&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Chloride</td>
			<td>Sigma</td>
			<td>S5886-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">UltraPure 0.5M EDTA, pH 8.0</td>
			<td>Life Technologies</td>
			<td>15575-020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DPBS, no calcium, no magnesium</td>
			<td>Life Technologies</td>
			<td>14190-250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100mm TC-Treated Culture Dish</td>
			<td>Corning</td>
			<td>430167</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DR4 MEF 2M IRR - Academic</td>
			<td>GlobalStem</td>
			<td>GSC-6204G</td>
			<td>Store in liquid Nitrogen.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM, high glucose, pyruvate</td>
			<td>Life Technologies</td>
			<td>11995-040</td>
			<td>Store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Defined Fetal Bovine Serum, US Origin</td>
			<td>HyClone</td>
			<td>SH30070.03</td>
			<td>Store at -20&#176;C. Thaw at 4&#176;C overnight and aliquot. Store aliquots at -20&#176;C until needed.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MEM Non-Essential Amino Acids Solution (100X)</td>
			<td>Life Technologies</td>
			<td>11140-050</td>
			<td>Store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4D-Nucleofector Core unit&#160;</td>
			<td>Lonza</td>
			<td>AAF-1001B</td>
			<td>part of the electroporation system</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4D-Nucleofector X unit&#160;</td>
			<td>Lonza</td>
			<td>AAF-1001X</td>
			<td>part of the electroporation system</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">P3 Primary Cell 4D-Nucleofector X Kit L (24 RCT)</td>
			<td>Lonza</td>
			<td>V4XP-3024</td>
			<td>Upon arrival, remove Primary Cell Solution and supplement and store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">StemPro Accutase Cell Dissociation Reagent</td>
			<td>Life Technologies</td>
			<td>A1110501</td>
			<td>Store at -20&#176;C. Thaw overnight at 4&#176;C and warm an aliquot in a 37&#176;C water bath before use.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NutriStem XF/FF Culture Medium</td>
			<td>Stemgent</td>
			<td>01-0005</td>
			<td>Store at -20&#176;C. Thaw overnight at 4&#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AAVS1 TALENs (pZT-AAVS1-L1 and pZT-AAVS1-R1)</td>
			<td>Addgene</td>
			<td>52637 and 52638</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AAVS1-CAG-EGFP Homologous Recombination donor</td>
			<td>Addgene</td>
			<td>22212</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Puromycin Dihydrochloride</td>
			<td>Life Technologies</td>
			<td>A11138-03</td>
			<td>Store at -20&#176;C. Prepare working aliquots of 1 mg/mL in ddH2O.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Disposable Borosilicate Glass Pasteur Pipets</td>
			<td>Fisher Scientific</td>
			<td>13-678-20A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sorvall Legend XTR (Refrigerated), 120V 60Hz</td>
			<td>Thermo Scientific</td>
			<td>75-004-521</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TX-750 4 &#215; 750mL Swinging Bucket Rotor</td>
			<td>Thermo Scientific</td>
			<td>75003607</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypan Blue Solution, 0.4%</td>
			<td>Life Technologies</td>
			<td>15250-061</td>
			<td></td>
		</tr>
	</tbody>
</table>

transfection, selection, and colony-picking of human induced pluripotent stem cells talen-targeted with a gfp gene into the aavs1 safe harbor

Targeted transgene addition can provide persistent gene expression while circumventing the gene silencing and insertional mutagenesis caused by viral vector mediated random integration. This protocol describes a universal and efficient transgene targeted addition platform in human iPSCs based on utilization of validated open-source TALENs and a gene-trap-like donor to deliver transgenes into a safe harbor locus. Importantly, effective gene editing is rate-limited by the delivery efficiency of gene editing vectors. Therefore, this protocol first focuses on preparation of iPSCs for transfection to achieve high nuclear delivery efficiency. When iPSCs are dissociated into single cells using a gentle-cell dissociation reagent and transfected using an optimized program, >50% cells can be induced to take up the large gene editing vectors. Because the AAVS1 locus is located in the intron of an active gene (PPP1R12C), a splicing acceptor (SA)-linked puromycin resistant gene (PAC) was used to select targeted iPSCs while excluding random integration-only and untransfected cells. This strategy greatly increases the chance of obtaining targeted clones, and can be used in other active gene targeting experiments as well. Two weeks after puromycin selection at the dose adjusted for the specific iPSC line, clones are ready to be picked by manual dissection of large, isolated colonies into smaller pieces that are transferred to fresh medium in a smaller well for further expansion and genetic and functional screening. One can follow this protocol to readily obtain multiple GFP reporter iPSC lines that are useful for in vivo and in vitro imaging and cell isolation.

A visualisering av protokollen er gitt i figur 2, med perioder der iPSCs dyrkes i forskjellige medium markert ved enten grønt eller blått for E8 for NutriStem. Det er viktig å transfektere bare kvalitets iPSCs; undersøke kultur retter gjennom rutinemessig vedlikehold og verifisere at IPSC kulturer inneholder hovedsakelig distinkte kolonier bærer en brostein lignende morfologi (figur 3A); differensierte cellene bør ikke oppta mer enn 10% av kulturen. Transfectability av iPSCs vurde...

Watch this Scientific Journal Video about Transfection, Selection, and Colony-picking of Human Induced Pluripotent Stem Cells TALEN-targeted with a GFP Gene into the AAVS1 Safe Harbor at JoVE.com

Transfection, Selection, and Colony-picking of Human Induced Pluripotent Stem Cells TALEN-targeted with a GFP Gene into the AAVS1 Safe Harbor

TALEN-mediated gene editing at the safe harbor AAVS1 locus enables high-efficiency transgene addition in human iPSCs. This protocol describes the procedures for preparing iPSCs for TALEN and donor vector delivery, transfecting iPSCs, and selecting and isolating iPSC clones to achieve targeted integration of a GFP gene to generate reporter lines.

Transfeksjon, merking og koloni-plukking av menneskeskapte Pluripotent stamceller TALEN målrettet med en GFP Gene inn i AAVS1 Safe Harbor

Targeted transgene addition can provide persistent gene expression while circumventing the gene silencing and insertional mutagenesis caused by viral ...

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Differentiating Chondrocytes from Peripheral Blood-derived Human Induced Pluripotent Stem Cells

Differensiering av kondrocytter fra perifert blod-avledede humane inducerte pluripotente stamceller

In this study, we used peripheral blood cells (PBCs) as seed cells to produce chondrocytes via induced pluripotent stem cells (iPSCs) in an ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

Rask, rettet differensiering av netthinnens Pigment epitelceller fra menneskelige embryonale eller indusert Pluripotent stamceller

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

Using Human Induced Pluripotent Stem Cell-derived Hepatocyte-like Cells for Drug Discovery

Bruker Human indusert Pluripotent Stamcelle-avledet Hepatocyte-lignende celler for medisiner

The ability to differentiate human induced pluripotent stem cells (iPSCs) into hepatocyte-like cells (HLCs) provides new opportunities to study inborn ...

A Familial Hypercholesterolemia Human Liver Chimeric Mouse Model Using Induced Pluripotent Stem Cell-derived Hepatocytes

En familiær hyperkolesterolemi menneskelige leveren Chimeric musemodell med indusert Pluripotent Stamcelle-avledet hepatocytter

Familial hypercholesterolemia (FH) is mostly caused by low-density lipoprotein receptor (LDLR) mutations and results in an increased risk of early-onset ...

RNA-based Reprogramming of Human Primary Fibroblasts into Induced Pluripotent Stem Cells

RNA-baserte omprogrammering av menneskelig primære fibroblaster til indusert Pluripotent stamceller

Induced pluripotent stem cells (iPSCs) have proven to be a valuable tool to study human development and disease. Further advancing iPSCs as a regenerative ...

Efficient Differentiation of Human Pluripotent Stem Cells into Liver Cells

Effektiv differensiering av Human Pluripotent stamceller i leverceller

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver ...

Generation of 3D Skin Organoid from Cord Blood-derived Induced Pluripotent Stem Cells

Generasjon 3D huden Organoid fra ledningen blod-avledet indusert Pluripotent stamceller

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily ...