Transfektion, Selection, og Colony-picking af menneskeskabte pluripotente stamceller talen målrettet med et GFP-genet i AAVS1 Safe Harbor
Summary
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.
Abstract
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.
Introduction
Evnen til at omprogrammere humane somatiske celler i embryonale stamceller-lignende inducerede pluripotente stamceller (iPSCs) blev først opdaget af Takahashi et al. I 2007 1. Humane dermale fibroblaster transduceret med retrovirus udtrykker fire transkriptionsfaktorer (den såkaldte døbt Yamanaka faktorer Oct3 / 4, Sox2, c-Myc og Klf4) viste sig at være meget lig humane embryonale stamceller (hESCs) baseret på morfologi, spredning, genekspression, og epigenetisk status; afgørende, iPSCs er også i stand til at differentiere til celler af alle tre kimlag 1. Den proliferative potentiale og differentiering kapacitet iPSCs gør dem meget attraktive værktøj; ved at omprogrammere celler fra patienter, der lider af bestemte sygdomme, kan iPSCs anvendes både som in vitro sygdom modelsystemer og som potentielle terapeutiske midler.
Til det sidstnævnte formål, skal en række spørgsmål, der skal løses, før det fulde potentiale i iPSCsi et klinisk miljø kan realiseres; den tumorigent potentiale in vitro dyrkede hESCs og iPSCs, brug af xenogene derivater under omprogrammering og celle vedligeholdelse, og behovet for at spore transplanterede celler in vivo, er alle vigtige hindringer for den kliniske anvendelse af pluripotente stamceller (Anmeldt af Hentze et al. 2). En ideel løsning på behovet for sporing differentierede celler efter transplantation ville indebære en visuelt påviselig markør, der modstår lyddæmpning og variegation uanset ansøgningen. Robust og vedholdende udtryk for integrerede transgener er lettest opnåelige når exogent DNA indføres i safe harbour loci; dvs. genomiske sites, der muliggør tilstrækkelig transskription af en integreret vektor, mens på samme tid at mindske forstyrrelser af ekspression i tilstødende gener 3. Et sådant websted, der har været meget godt karakteriseret siden dens opdagelse er den adenoassocierede virus integration site 1 (AAVS1), i den første intron af proteinphosphatase 1 regulatoriske underenhed 12C (PPP1R12C) genet. Har vist dette locus ikke blot at tillade vedvarende og robust ekspression af integrerede transgener gennem forlænget tid i kultur og in vitro differentiering 3, men også for at beskytte omgivende gener fra transkriptionel perturbation 4; begge funktioner menes at være på grund af tilstedeværelsen af endogene kromatin isolerende elementer flankerer AAVS1 site 5.
Fremskridt i genomet engineering værktøjer over blot det sidste årti har lettet den lethed og effektivitet, hvormed der kan opnås genetiske manipulationer i enhver celletype. Mens tidlige vellykkede eksperimenter påberåbte overordentlig lave niveauer af endogen homolog rekombination (HR) med et indført donor til at opnå genmålretning i økonomiske og sociale råd 6,7, brugen af stedspecifikke nukleaser, såsom zink finger nukleaser (ZFNs), at signiligt fremkalde homolog rekombination ved produktion af en dobbeltstrenget DNA pause i høj grad har øget effektiviteten med sådanne forsøg 8,9. Den nyorientering af både transskription aktivator-lignende effektorer (Tales) vegetabilske patogene xanthomonas slægter og de prokaryote grupperet regelmæssigt spatierede korte palindromiske gentagelser (CRISPR) / Cas9 systemet til effektive stedspecifikke designer nukleaser har gjort genmålretning i pluripotente stamceller en tilgængelig og praktisk metode 10-13.
En nylig papir beskrevet en effektiv metode til stabil integration af et grønt fluorescerende reporter kassette i AAVS1 safe harbour locus i menneskelige iPSCs hjælp af fortælling nukleaser (Talens) 14. Disse målrettede iPSCs opretholdt deres fluorescens selv efter rettet differentiering til cardiomyocytter og transplantation i en musemodel for myokardieinfarkt (MI), som giver stærke beviser for anvendeligheden af sådannestabilt fluorescerende pluripotente stamceller 14. For at opnå målrettede kolonier blev en gen-trap metode hvor en splejsning-acceptor (SA), 2A selvspaltende peptidsekvens placerer puromycin N-acetyl-transferase (PAC) genet under kontrol af den endogene PPP1R12C promotor; Således er det kun iPSCs der har inkorporeret DNA-donoren på AAVS1 locus udtrykke PAC, gør dem valgbare baseret på puromycin-resistens; (Figur 1, 15). Denne protokol beskriver de procedurer generere AAVS1-GFP iPSCs rapporteret i den seneste papir 14, herunder processen med transfektion iPSCs med Talens og en 9,8 kb donor til at integrere et 4,2 kb DNA-fragment i AAVS1 safe harbour locus, vælge iPSCs baseret på puromycin-resistens, og plukke kolonier for klonal ekspansion. De heri beskrevne teknikker kan anvendes til mange genom engineering eksperimenter.
Protocol
Representative Results
Discussion
De mest kritiske trin for en vellykket generation af AAVS1 safe harbour målrettet menneskelige iPSCs er: (1) effektivt at levere talen og donorplasmider i iPSCs ved transfektion; (2) at optimere dissociation af iPSCs i enkeltceller før transfektion og udpladningsdensitet efter transfektion; (3) optimering dosis og tidspunkt for lægemiddel-selektion baseret på væksten af iPSC linje; (4) forsigtigt dissekere og plukke målrettede kolonier og overførsel til ny plade / godt. Sammenlignet med lignende metoder, de…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
This research was supported by the NIH Common Fund and Intramural Research Program of the National Institute of Arthritis, Musculoskeletal, and Skin Diseases.
Materials
| NAME OF MATERIAL/EQUIPMENT | COMPANY | CATALOG # | COMMENTS/DESCRIPTION |
| Matrigel Growth Factor Reduced (GFR) Basement Membrane Matrix, *LDEV-Free, 10mL | Corning | 354230 | Store at -20°C. |
| DMEM/F-12 | Life Technologies | 11320-033 | Store at 4°C. |
| Costar 6 Well Clear TC-Treated Multiple Well Plates | Corning | 3506 | |
| Essential 8 Medium | Life Technologies | A1517001 | Store basal medium at 4°C. Store supplement at -20°C. |
| Y-27632 dihydrochloride | Tocris | 1254 | Store at room temp. Once dissolved in H2O, store at -20°C. |
| Sodium Chloride | Sigma | S5886-500G | |
| UltraPure 0.5M EDTA, pH 8.0 | Life Technologies | 15575-020 | |
| DPBS, no calcium, no magnesium | Life Technologies | 14190-250 | |
| 100mm TC-Treated Culture Dish | Corning | 430167 | |
| DR4 MEF 2M IRR – Academic | GlobalStem | GSC-6204G | Store in liquid Nitrogen. |
| DMEM, high glucose, pyruvate | Life Technologies | 11995-040 | Store at 4°C. |
| Defined Fetal Bovine Serum, US Origin | HyClone | SH30070.03 | Store at -20°C. Thaw at 4°C overnight and aliquot. Store aliquots at -20°C until needed. |
| MEM Non-Essential Amino Acids Solution (100X) | Life Technologies | 11140-050 | Store at 4°C. |
| 4D-Nucleofector Core unit | Lonza | AAF-1001B | part of the electroporation system |
| 4D-Nucleofector X unit | Lonza | AAF-1001X | part of the electroporation system |
| P3 Primary Cell 4D-Nucleofector X Kit L (24 RCT) | Lonza | V4XP-3024 | Upon arrival, remove Primary Cell Solution and supplement and store at 4°C. |
| StemPro Accutase Cell Dissociation Reagent | Life Technologies | A1110501 | Store at -20°C. Thaw overnight at 4°C and warm an aliquot in a 37°C water bath before use. |
| NutriStem XF/FF Culture Medium | Stemgent | 01-0005 | Store at -20°C. Thaw overnight at 4°C |
| AAVS1 TALENs (pZT-AAVS1-L1 and pZT-AAVS1-R1) | Addgene | 52637 and 52638 | |
| AAVS1-CAG-EGFP Homologous Recombination donor | Addgene | 22212 | |
| Puromycin Dihydrochloride | Life Technologies | A11138-03 | Store at -20°C. Prepare working aliquots of 1 mg/mL in ddH2O. |
| Disposable Borosilicate Glass Pasteur Pipets | Fisher Scientific | 13-678-20A | |
| Sorvall Legend XTR (Refrigerated), 120V 60Hz | Thermo Scientific | 75-004-521 | |
| TX-750 4 × 750mL Swinging Bucket Rotor | Thermo Scientific | 75003607 | |
| Trypan Blue Solution, 0.4% | Life Technologies | 15250-061 |
Referencias
- Takahashi, K., et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. 131 (5), 861-872 (2007).
- Hentze, H., Graichen, R., Colman, A. Cell therapy and the safety of embryonic stem cell-derived grafts. Trends Biotechnol. 25 (1), 24-32 (2007).
- Smith, J. R., et al. Robust, persistent transgene expression in human embryonic stem cells is achieved with AAVS1-targeted integration. Stem Cells. 26 (2), 496-504 (2008).
- Lombardo, A., et al. Site-specific integration and tailoring of cassette design for sustainable gene transfer. Nat Methods. 8 (10), 861-869 (2011).
- Ogata, T., Kozuka, T., Kanda, T. Identification of an insulator in AAVS1, a preferred region for integration of adeno-associated virus DNA. J Virol. 77 (16), 9000-9007 (2003).
- Zwaka, T. P., Thomson, J. A. Homologous recombination in human embryonic stem cells. Nat Biotechnol. 21 (3), 319-321 (2003).
- Urbach, A., Schuldiner, M., Benvenisty, N. Modeling for Lesch-Nyhan disease by gene targeting in human embryonic stem cells. Stem Cells. 22 (4), 635-641 (2004).
- Hockemeyer, D., et al. Efficient targeting of expressed and silent genes in human ESCs and iPSCs using zinc-finger nucleases. Nature biotechnology. 27 (9), 851-857 (2009).
- Zou, J., et al. Gene targeting of a disease-related gene in human induced pluripotent stem and embryonic stem cells. Cell stem cell. 5 (1), 97-110 (2009).
- Hockemeyer, D., et al. Genetic engineering of human pluripotent cells using TALE nucleases. Nature. 29 (8), 731-734 (2011).
- Sanjana, N. E., et al. A transcription activator-like effector toolbox for genome engineering. Nature protocols. 7 (1), 171-192 (2012).
- Mali, P., et al. RNA-guided human genome engineering via Cas9. Science. 339 (6121), 823-826 (2013).
- Ding, Q., et al. Enhanced efficiency of human pluripotent stem cell genome editing through replacing TALENs with CRISPRs. Cell stem cell. 12 (4), 393-394 (2013).
- Luo, Y., et al. Stable Enhanced Green Fluorescent Protein Expression After Differentiation and Transplantation of Reporter Human. Induced Pluripotent Stem Cells Generated by AAVS1 Transcription Activator-Like Effector Nucleases. Stem Cells Transl Med. 3 (7), 821-835 (2014).
- Zou, J., et al. Oxidase-deficient neutrophils from X-linked chronic granulomatous disease iPS cells: functional correction by zinc finger nuclease-mediated safe harbor targeting. Blood. 117 (21), 5561-5572 (2011).
- Luo, Y., Rao, M., Zou, J. Generation of GFP Reporter Human Induced Pluripotent Stem Cells Using AAVS1 Safe Harbor Transcription Activator-Like Effector Nuclease. Curr Protoc Stem Cell Biol. 29, 5A.7.1-5A.7.18 (2014).
- Ran, F. A., et al. Genome engineering using the CRISPR-Cas9 system. Nat Protoc. 8 (11), 2281-2308 (2013).
- Beers, J., et al. Passaging and colony expansion of human pluripotent stem cells by enzyme-free dissociation in chemically defined culture conditions. Nat Protoc. 7, 2029-2040 (2012).
