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

1. Preparation of Basement Membrane Matrix and Coating of Plasticware
<ol>
	<li>Place the frozen basement membrane matrix stock from -20 &#176;C onto ice and thaw overnight at 4 &#176;C.</li>
	<li>After thawing, pipette 2 mg aliquots of basement membrane matrix into pre-chilled eppendorf tubes. Store these at -20 &#176;C until needed.</li>
	<li>To prepare basement membrane matrix-coated plates, thaw one aliquot on ice until the last piece of ice in the eppendorf tube disappears (usually within ~2 hr).</li>
	<li>After t...

<ol>
	<li>Takahashi, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+pluripotent+stem+cells+from+adult+human+fibroblasts+by+defined+factors.">Induction of pluripotent stem cells from adult human fibroblasts by defined factors.</a> Cell. 131 (5), 861-872 (2007).</li><li>Hentze, H., Graichen, R., Colman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+therapy+and+the+safety+of+embryonic+stem+cell-derived+grafts.">Cell therapy and the safety of embryonic stem cell-derived grafts.</a> Trends Biotechnol. 25 (1), 24-32 (2007).</li><li>Smith, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Robust,+persistent+transgene+expression+in+human+embryonic+stem+cells+is+achieved+with+AAVS1-targeted+integration.">Robust, persistent transgene expression in human embryonic stem cells is achieved with AAVS1-targeted integration.</a> Stem Cells. 26 (2), 496-504 (2008).</li><li>Lombardo, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Site-specific+integration+and+tailoring+of+cassette+design+for+sustainable+gene+transfer.">Site-specific integration and tailoring of cassette design for sustainable gene transfer.</a> Nat Methods. 8 (10), 861-869 (2011).</li><li>Ogata, T., Kozuka, T., Kanda, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+an+insulator+in+AAVS1,+a+preferred+region+for+integration+of+adeno-associated+virus+DNA.">Identification of an insulator in AAVS1, a preferred region for integration of adeno-associated virus DNA.</a> J Virol. 77 (16), 9000-9007 (2003).</li><li>Zwaka, T. P., Thomson, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homologous+recombination+in+human+embryonic+stem+cells.">Homologous recombination in human embryonic stem cells.</a> Nat Biotechnol. 21 (3), 319-321 (2003).</li><li>Urbach, A., Schuldiner, M., Benvenisty, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+for+Lesch-Nyhan+disease+by+gene+targeting+in+human+embryonic+stem+cells.">Modeling for Lesch-Nyhan disease by gene targeting in human embryonic stem cells.</a> Stem Cells. 22 (4), 635-641 (2004).</li><li>Hockemeyer, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+targeting+of+expressed+and+silent+genes+in+human+ESCs+and+iPSCs+using+zinc-finger+nucleases.">Efficient targeting of expressed and silent genes in human ESCs and iPSCs using zinc-finger nucleases.</a> Nature biotechnology. 27 (9), 851-857 (2009).</li><li>Zou, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+targeting+of+a+disease-related+gene+in+human+induced+pluripotent+stem+and+embryonic+stem+cells.">Gene targeting of a disease-related gene in human induced pluripotent stem and embryonic stem cells.</a> Cell stem cell. 5 (1), 97-110 (2009).</li><li>Hockemeyer, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+engineering+of+human+pluripotent+cells+using+TALE+nucleases.">Genetic engineering of human pluripotent cells using TALE nucleases.</a> Nature. 29 (8), 731-734 (2011).</li><li>Sanjana, N. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+transcription+activator-like+effector+toolbox+for+genome+engineering.">A transcription activator-like effector toolbox for genome engineering.</a> Nature protocols. 7 (1), 171-192 (2012).</li><li>Mali, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-guided+human+genome+engineering+via+Cas9.">RNA-guided human genome engineering via Cas9.</a> Science. 339 (6121), 823-826 (2013).</li><li>Ding, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+efficiency+of+human+pluripotent+stem+cell+genome+editing+through+replacing+TALENs+with+CRISPRs.">Enhanced efficiency of human pluripotent stem cell genome editing through replacing TALENs with CRISPRs.</a> Cell stem cell. 12 (4), 393-394 (2013).</li><li>Luo, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+Enhanced+Green+Fluorescent+Protein+Expression+After+Differentiation+and+Transplantation+of+Reporter+Human.">Stable Enhanced Green Fluorescent Protein Expression After Differentiation and Transplantation of Reporter Human.</a> Induced Pluripotent Stem Cells Generated by AAVS1 Transcription Activator-Like Effector Nucleases. Stem Cells Transl Med. 3 (7), 821-835 (2014).</li><li>Zou, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidase-deficient+neutrophils+from+X-linked+chronic+granulomatous+disease+iPS+cells:+functional+correction+by+zinc+finger+nuclease-mediated+safe+harbor+targeting.">Oxidase-deficient neutrophils from X-linked chronic granulomatous disease iPS cells: functional correction by zinc finger nuclease-mediated safe harbor targeting.</a> Blood. 117 (21), 5561-5572 (2011).</li><li>Luo, Y., Rao, M., Zou, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+GFP+Reporter+Human+Induced+Pluripotent+Stem+Cells+Using+AAVS1+Safe+Harbor+Transcription+Activator-Like+Effector+Nuclease.">Generation of GFP Reporter Human Induced Pluripotent Stem Cells Using AAVS1 Safe Harbor Transcription Activator-Like Effector Nuclease.</a> Curr Protoc Stem Cell Biol. 29, 5A.7.1-5A.7.18 (2014).</li><li>Ran, F. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+engineering+using+the+CRISPR-Cas9+system.">Genome engineering using the CRISPR-Cas9 system.</a> Nat Protoc. 8 (11), 2281-2308 (2013).</li><li>Beers, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Passaging+and+colony+expansion+of+human+pluripotent+stem+cells+by+enzyme-free+dissociation+in+chemically+defined+culture+conditions.">Passaging and colony expansion of human pluripotent stem cells by enzyme-free dissociation in chemically defined culture conditions.</a> Nat Protoc. 7, 2029-2040 (2012).</li></ol>

The most critical steps for the successful generation of AAVS1 safe-harbor targeted human iPSCs are: (1) efficiently delivering TALEN and donor plasmids into iPSCs by transfection; (2) optimizing dissociation of iPSCs into single cells before transfection and plating density after transfection; (3) optimizing dose and time of drug-selection based on the growth of iPSC line; (4) carefully dissecting and picking targeted colonies and transferring to new plate/well. Compared to similar methods used in Hockemeyer&#8217;s pap...

The ability to reprogram human somatic cells into embryonic stem cell-like induced pluripotent stem cells (iPSCs) was first discovered by Takahashi et al. in 20071. Human dermal fibroblasts transduced with retroviruses expressing four transcription factors (The so-dubbed Yamanaka factors Oct3/4, Sox2, c-Myc, and Klf4) were shown to be highly similar to human embryonic stem cells (hESCs) based on morphology, proliferation, gene expression, and epigenetic status; crucially, iPSCs are also capable of differentiating into cells of all three germ layers1. The proliferative potential and differentiation capacity of iPSCs makes them very attractive tools; by reprogramming cells from patients suffering from specific diseases, iPSCs can be used both as in vitro disease model systems and as potential therapeutics.
For the latter purpose, several issues must be addressed before the full potential of iPSCs in a clinical setting can be realized; the tumorigenic potential of in vitro cultured hESCs and iPSCs, the use of xenogenic derivatives during reprogramming and cell maintenance, and the need to track transplanted cells in vivo are all crucial hurdles to the clinical application of pluripotent stem cells (Reviewed by Hentze et at.2). An ideal solution to the need for tracking differentiated cells post-transplantation would involve a visually detectable marker that resists silencing and variegation regardless of the application. Robust and sustained expression of integrated transgenes is most readily achievable when exogenous DNA is introduced into safe-harbor loci; that is, genomic sites that enable sufficient transcription of an integrated vector while at the same time mitigating perturbations of expression in neighboring genes3. One such site that has been very well characterized since its discovery is the adeno-associated virus integration site 1 (AAVS1), in the first intron of the protein phosphatase 1 regulatory subunit 12C (PPP1R12C) gene. This locus has been shown not only to permit sustained and robust expression of integrated transgenes through extended time in culture and in vitro differentiation3, but also to protect surrounding genes from transcriptional perturbation4; both features are thought to be due to the presence of endogenous chromatin insulator elements flanking the AAVS1 site5.
Advances in genome engineering tools over just the past decade have greatly facilitated the ease and efficiency with which genetic manipulations in any cell type can be achieved. While early successful experiments relied on exceedingly low levels of endogenous homologous recombination (HR) with an introduced donor to achieve gene targeting in ESCs6,7, the use of site-specific nucleases, such as zinc finger nucleases (ZFNs), that significantly induce homologous recombination through the generation of a double-stranded DNA break has greatly increased the efficiency of such experiments8,9. The repurposing of both transcription activator-like effectors (TALEs) of plant pathogenic xanthomonas genera and the prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system into efficient site-specific designer nucleases has made gene targeting in pluripotent stem cells an accessible and practicable methodology10-13.
A recent paper described an efficient method for the stable integration of a green fluorescent reporter cassette into the AAVS1 safe-harbor locus in human iPSCs using TALE nucleases (TALENs)14. These targeted iPSCs maintained their fluorescence even after directed differentiation to cardiomyocytes and transplantation into a mouse model of myocardial infarction (MI), providing strong evidence for the utility of such stably fluorescent pluripotent stem cells14. To obtain targeted colonies, a gene-trap method was used wherein a splicing-acceptor (SA), 2A self-cleaving peptide sequence places the puromycin N-acetyl-transferase (PAC) gene under the control of the endogenous PPP1R12C promoter; thus, only iPSCs that have incorporated the DNA donor at the AAVS1 locus express PAC, rendering them selectable based on puromycin-resistance; (Figure 1,15). This protocol details the procedures of generating AAVS1-GFP iPSCs reported in the recent paper14, including the process of transfecting iPSCs with TALENs and a 9.8 kb donor to integrate a 4.2kb DNA fragment into the AAVS1 safe-harbor locus, selecting iPSCs based on puromycin-resistance, and picking colonies for clonal expansion. The techniques described herein can be applied to many genome engineering experiments.

<table border="0" cellpadding="0" cellspacing="0" width="1437">
	<tbody>
		<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 visualization of the protocol is provided in Figure 2, with periods during which iPSCs are cultured in different medium highlighted by either green for E8 or blue for NutriStem. It is important to transfect only high-quality iPSCs; examine culture dishes throughout routine maintenance and verify that iPSC cultures contain mainly distinct colonies bearing a cobblestone-like morphology (Figure 3A); differentiated cells should not occupy more than 10% of the culture. Transfectability of i...

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.

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

transfection-selection-colony-picking-human-induced-pluripotent-stem

Research

JoVE Journal

Developmental Biology

19.2K visualizações.  National Institutes of Health. 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.