Name: TALEN-Mediated Targeted Gene Integration: Using Engineered Sequence-Specific Nucleases for the Precise Insertion of Fluorescent Protein Gene into hiPSCs
Uploaded: 2023-04-30T14:56:15
Description: Source: Cerbini, T. et al. Transfection, Selection, and Colony-picking of Human Induced Pluripotent Stem Cells TALEN-targeted with a GFP Gene into the ...

eoe sub articles

EoE Sub Articles

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 thawing, add basement membrane matrix to 12 ml of cold (4 &#176;C) DMEM/F12 to make basement membrane matrix coating solution.</li>
	<li>Add basement membrane matrix solution to the appropriate culture vessel. For a 6-well plate, dispense 1 ml per well. Swirl the plate to make sure the basement membrane matrix solution completely covers each well.</li>
	<li>Parafilm seal the basement membrane matrix-coated plate/dish and incubate at room temperature for 1 hour before use. Alternatively, store basement membrane matrix-coated plates/dishes at 4 &#176;C and use within 2 weeks of coating. 
	NOTE: Add extra DMEM/F12 to the basement membrane matrix-coated plate/dish to prevent drying. Before using 4 &#176;C stored basement membrane matrix-coated plate/dish, place it in a biological safety cabinet and allow it to come to room temperature for at least 30 min.</li>
	<li>Aspirate basement membrane matrix completely before the addition of medium and cells.</li>
</ol>
2. Preparation of E8 medium
<ol>
	<li>Prepare E8 culture medium by thawing E8 supplement overnight at 4 &#176;C.</li>
	<li>Remove 10 ml of E8 basal medium from the 500 ml stock and discard.</li>
	<li>Pipette the entire 10 ml vial of E8 supplement directly into 490 ml of E8 basal medium. Do not warm complete E8 medium in a 37 &#176;C water bath, as repeated temperature fluctuations can degrade the bFGF in the complete E8 medium.</li>
	<li>Use complete E8 medium within 2 weeks of addition of the supplement.</li>
</ol>
3. Thawing of iPSCs
<ol>
	<li>Remove a vial of frozen iPSCs from liquid nitrogen and place on dry ice.</li>
	<li>Rapidly thaw the vial in a 37 &#176;C water bath; swirl the vial in the water bath until only a tiny fragment of ice remains.</li>
	<li>Spray the vial with 70% ethanol and transfer to a biological safety cabinet.</li>
	<li>Add 1 ml of room temperature E8 medium dropwise directly to the vial.</li>
	<li>Using a 2 ml pipette, transfer the cell suspension dropwise into 9 ml of E8 medium in a 15 ml conical tube. Swirl the tube frequently to ensure that the cells and medium mix well quickly.</li>
	<li>Centrifuge the cells at 200 x g for 5 min.</li>
	<li>Aspirate the supernatant and resuspend the cell pellet in an appropriate volume of E8 supplemented with 10 &#956;M Y-27632.</li>
	<li>Add the cells to an appropriate number of basement membrane matrix-coated wells, and place in a 37 &#176;C, 5% CO&#8322; incubator overnight. It is recommended to plate at least 0.2 x 10&#8310; iPSC per well of a 6-well plate to enable quick recovery after thawing.</li>
</ol>
4. Maintenance and Routine Passaging of iPSCs
<ol>
	<li>Refresh E8 medium daily.</li>
	<li>Monitor the morphology and confluency of cells with an inverted microscope. iPSCs of high-quality grow in flat colonies with distinct borders; individual colonies possess a &#8220;cobblestone-like&#8221; appearance.</li>
	<li>Passage the iPSCs cells when they reach ~70% confluency.</li>
	<li>Prepare an EDTA passaging solution by adding 0.9 g NaCl and 500 &#956;l 0.5 M EDTA to 500 ml DPBS. Mix well to dissolve NaCl, and vacuum filter to sterilize. Warm an aliquot of the passaging solution in a 37 &#176;C water bath prior to passaging.</li>
	<li>To passage, aspirate spent culture medium and wash cells once with an equal volume of warm passaging solution. Aspirate, and pipette enough EDTA passaging solution to coat the cells (1 ml per well of a 6-well plate).</li>
	<li>Place the cells under an inverted microscope and observe the iPSC colonies. The appearance of holes within colonies and raised borders should become apparent within 2 to 5 min.</li>
	<li>Carefully aspirate the EDTA passaging solution.</li>
	<li>Using a 10 ml pipette, dispense 4 ml of E8 medium (if using a 6-well plate) under high pressure directly into each well to be passaged.</li>
	<li>Collect the iPSC clumps and split into an appropriate number of wells depending on the split ratio from 1:8 to 1:12. Do not over-pipette, as disaggregation of cell clumps will result in poor viability.</li>
	<li>Place the plate in an incubator, and rock the plate back-and-forth and side-to-side several times to disperse the cells.</li>
</ol>
5. Preparation of MEFs and iPSCs for Transfection
<ol>
	<li>48 hr prior to transfection, passage iPSCs at ~1:6 into four or more wells of a basement membrane matrix-coated 6-well plate, so that they will be 70% confluent two days hence.</li>
	<li>The next day, thaw DR4 MEFs into MEF medium consisting of DMEM (high glucose) supplemented with 10% FBS and 1x MEM-NEAA.</li>
	<li>Plate DR4 MEFs into two 10-cm dishes at ~ 2 x 10&#8308; cells/cm&#178; and incubate overnight at 37 &#176;C.</li>
	<li>On the day of transfection, change MEF medium to E8 supplemented with 10 &#956;M Y-27632 30 min before performing transfection on iPSCs.</li>
	<li>Optional: 4 hr prior to transfection, supplement pre-transfection iPSC culture with Y-27632 at the final concentration of 10 &#956;M.</li>
</ol>
6. Gentle-cell Dissociation Reagent Treatment and Transfection of iPSCs using an Electroporation System
<ol>
	<li>Remove P3 primary cell transfection solution from 4 &#176;C and allow it to come to room temperature for ~30 min. Add the entire 100 &#956;l supplement to the transfection solution prior to use.</li>
	<li>Warm gentle-cell dissociation reagent in a 37 &#176;C water bath.</li>
	<li>Obtain AAVS1 TALENs (pZT-AAVS1-L1 and pZT-AAVS1-R1) and AAVS1-CAG-EGFP donor from -20 &#176;C.</li>
	<li>Remove iPSCs from the incubator and wash once with DPBS.</li>
	<li>Add 1 ml of gentle-cell dissociation reagent per well and incubate iPSCs at 37 &#176;C for 5 min, or until greater than 50% of the cells have dissociated from the culture vessel.</li>
	<li>Pipette the cells up and down a few times using a p1000 pipette to dissociate any remaining cells from the culture vessel, and to break up iPSC clumps.</li>
	<li>Add 2 ml of E8 medium to each well and pipette up and down several times using a 10 ml pipette to further disaggregate cell clumps into single cells. 
	NOTE: Transfection efficiency declines significantly if cell clumps are not sufficiently disaggregated.</li>
	<li>Collect iPSCs in a 15 ml conical tube, and centrifuge at 100 x g for 3 min.</li>
	<li>Aspirate supernatant and resuspend cells in a minimal amount of E8 medium.</li>
	<li>Count the cells using a hemocytometer after application of a vital stain such as 0.4% Trypan blue. Ensure that cells are sufficiently dissociated while counting (1-3 cells per &#8220;clump&#8221;).</li>
	<li>Dispense 3 x 10&#8310; cells into each of two 15 ml conical tubes, and spin down again at 100 x g for 3 min. 
	NOTE: Low speed centrifugation reduces the cell stress and allows easy re-suspension of iPSCs before electroporation.</li>
	<li>Set the electroporation system to the cell-type specific program for the human embryonic stem cell line H9 (Program CB-150).</li>
	<li>After centrifugation, aspirate the supernatant from the cell pellets. To one pellet, add 10 &#956;g of the HR donor as control sample. To the other pellet add 10 &#956;g of the HR donor, along with 5 &#956;g of each TALEN (pZT-AAVS1-L1 and pZT-AAVS1-R1) as experimental sample.</li>
	<li>Resuspend each cell pellet in 100 &#956;l of P3 primary cell transfection solution, and transfer to a cuvette.</li>
	<li>Perform the transfection and immediately add 500 &#956;l of room temperature E8 medium to each cuvette.</li>
	<li>Transfer the transfected iPSCs dropwise to one 10-cm dish containing DR4 MEFs prepared in step 5.4.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Matrigel Growth Factor Reduced (GFR) Basement Membrane Matrix, *LDEV-Free, 10 ml</td>
			<td>Corning</td>
			<td>354230</td>
			<td>Store at -20 &#176;C.</td>
		</tr>
		<tr>
			<td>DMEM/F-12</td>
			<td>Life Technologies</td>
			<td>11320-033</td>
			<td>Store at 4 &#176;C.</td>
		</tr>
		<tr>
			<td>Costar 6 Well Clear TC-Treated Multiple Well Plates</td>
			<td>Corning</td>
			<td>3506</td>
			<td></td>
		</tr>
		<tr>
			<td>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>
			<td>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>
			<td>Sodium Chloride</td>
			<td>Sigma</td>
			<td>S5886-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>UltraPure 0.5 M EDTA, pH 8.0</td>
			<td>Life Technologies</td>
			<td>15575-020</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS, no calcium, no magnesium</td>
			<td>Life Technologies</td>
			<td>14190-250</td>
			<td></td>
		</tr>
		<tr>
			<td>100 mm TC-Treated Culture Dish</td>
			<td>Corning</td>
			<td>430167</td>
			<td></td>
		</tr>
		<tr>
			<td>DR4 MEF 2M IRR - Academic</td>
			<td>GlobalStem</td>
			<td>GSC-6204G</td>
			<td>Store in liquid Nitrogen.</td>
		</tr>
		<tr>
			<td>DMEM, high glucose, pyruvate</td>
			<td>Life Technologies</td>
			<td>11995-040</td>
			<td>Store at 4 &#176;C.</td>
		</tr>
		<tr>
			<td>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>
			<td>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>
			<td>4D-Nucleofector Core unit</td>
			<td>Lonza</td>
			<td>AAF-1001B</td>
			<td>Part of the electroporation system.</td>
		</tr>
		<tr>
			<td>4D-Nucleofector X unit</td>
			<td>Lonza</td>
			<td>AAF-1001X</td>
			<td>Part of the electroporation system.</td>
		</tr>
		<tr>
			<td>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>
			<td>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>
			<td>NutriStem XF/FF Culture Medium</td>
			<td>Stemgent</td>
			<td>01-2005</td>
			<td>Store at -20 &#176;C. Thaw overnight at 4 &#176;C</td>
		</tr>
		<tr>
			<td>AAVS1 TALENs (pZT-AAVS1-L1 and pZT-AAVS1-R1)</td>
			<td>Addgene</td>
			<td>52637 and 52638</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>AAVS1-CAG-EGFP Homologous Recombination donor</td>
			<td>Addgene</td>
			<td>22212</td>
			<td></td>
		</tr>
		<tr>
			<td>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>
			<td>Disposable Borosilicate Glass Pasteur Pipettes</td>
			<td>Fisher Scientific</td>
			<td>13-678-20A</td>
			<td></td>
		</tr>
		<tr>
			<td>Sorvall Legend XTR (Refrigerated), 120 V 60 Hz</td>
			<td>Thermo Scientific</td>
			<td>75-004-521</td>
			<td></td>
		</tr>
		<tr>
			<td>TX-750 4 &#215; 750 ml Swinging Bucket Rotor</td>
			<td>Thermo Scientific</td>
			<td>75003607</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue Solution, 0.4%</td>
			<td>Life Technologies</td>
			<td>15250-061</td>
			<td></td>
		</tr>
	</tbody>
</table>

talen-mediated targeted gene integration: using engineered sequence-specific nucleases for the precise insertion of fluorescent protein gene into hipscs

Source: Cerbini, T. et al. <a href="https://www.jove.com/v/52504">Transfection, Selection, and Colony-picking of Human Induced Pluripotent Stem Cells TALEN-targeted with a GFP Gene into the AAVS1 Safe Harbor.</a> J. Vis. Exp. (2015)
This video describes a precise genome editing technique in human induced pluripotent stem cells, or hiPSCs, using TALENs to create double-stranded breaks at a targeted locus, inducing homology-directed repair for fluorescence protein gene integration.

TALEN-Mediated Targeted Gene Integration: Using Engineered Sequence-Specific Nucleases for the Precise Insertion of Fluorescent Protein Gene into hiPSCs

Source: Cerbini, T. et al. Transfection, Selection, and Colony-picking of Human Induced Pluripotent Stem Cells TALEN-targeted with a GFP Gene into the ...

Begin with human induced pluripotent stem cell or hiPSC suspension in media. Add a pair of transcription activator-like effector nuclease or TALEN-encoding plasmids. Supplement with a donor plasmid bearing a fluorescent protein gene, coupled to an antibiotic resistance gene, with adjacent homology arms for specific alignment to target loci.
Electroporate the mixture to generate transient pores in the cell membranes, enabling plasmid internalization. Once inside the cells, the TALEN-encoding plasmids get processed, producing TALENs - chimeric proteins composed of a site-specific TALE DNA-binding domain fused to a non-specific FokI restriction endonuclease cleavage domain.
Each TALEN monomer via its DNA-binding domain - comprising tandem amino acid repeat modules - recognizes and binds to target loci, one module per nucleotide, on each DNA strand in opposing orientations, separated by a spacer sequence. FokI domains then dimerize and cleave DNA within the spacer sequence, creating double-strand breaks with overhangs.
In the presence of donor plasmid containing homology arms complementary to regions flanking the cleaved site, homology-directed repair occurs utilizing the homologous sequence as template for DNA repair synthesis to bridge the double-strand breaks. Following ligation of the nicks, the intervening fluorescent protein and antibiotic-resistance genes get integrated into the host genome.
Treat the hiPSCs, seeded on a feeder cell layer to support growth, with antibiotic-containing media. The promoter-less antibiotic resistance gene - driven by an upstream endogenous promoter - facilitates selection of TALEN-edited cells.

talen-mediated-targeted-gene-integration-using-engineered-sequence

Research

JoVE Encyclopedia of Experiments

Biological Techniques

Genome Editing Techniques

Gene knock-in

1.1K Views. Source: Cerbini, T. et al. Transfection, Selection, and Colony-picking of Human Induced Pluripotent Stem Cells TALEN-targeted with a GFP Gene into the AAVS1 Safe Harbor. J. Vis. Exp. (2015) This video describes a precise genome editing technique in human induced pluripotent stem cells, or hiPSCs, using TALENs to create double-stranded breaks at a targeted locus, inducing homology-directed repair for fluorescence protein gene integration. 

Video: TALEN-Mediated Targeted Gene Integration: Using Engineered Sequence-Specific Nucleases for the Precise Insertion of Fluorescent Protein Gene into hiPSCs - Concept

Efficient Generation of hiPSC Neural Lineage Specific Knockin Reporters Using the CRISPR/Cas9 and Cas9 Double Nickase System

Gene targeting is a critical approach for characterizing gene functions in modern biomedical research. However, the efficiency of gene targeting in human cells has been low, which prevents the generation of human cell lines at a desired rate. The past two years have witnessed a rapid progression on improving efficiency of genetic manipulation by genome editing tools such as the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated) system. This manuscript describes a protocol for generating lineage specific human induced pluripotent stem cell (hiPSC) reporters using CRISPR/Cas system assisted homologous recombination. Procedures for obtaining necessary components for making neural lineage reporter lines using the CRISPR/Cas system, focusing on construction of targeting vectors and single guide RNAs, are described. This protocol can be extended to platform establishment and mutation correction in hiPSCs.

Genome editing tools such as the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated) system have greatly improved gene targeting efficiency in human induced pluripotent stem cells (hiPSCs). This manuscript describes a protocol for generating lineage specific hiPSC reporter using CRISPR/Cas system assisted homologous recombination.

Gene targeting is a critical approach for characterizing gene functions in modern biomedical research. However, the efficiency of gene targeting in human ...

JoVE Journal

Developmental Biology

Establishment of Genome-edited Human Pluripotent Stem Cell Lines: From Targeting to Isolation

Genome-editing of human pluripotent stem cells (hPSCs) provides a genetically controlled and clinically relevant platform from which to understand human development and investigate the pathophysiology of disease. By employing site-specific nucleases (SSNs) for genome editing, the rapid derivation of new hPSC lines harboring specific genetic alterations in an otherwise isogenic setting becomes possible. Zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 are the most commonly used SSNs. All of these nucleases function by introducing a double stranded DNA break at a specified site, thereby promoting precise gene editing at a genomic locus. SSN-meditated genome editing exploits two of the cell&#39;s endogenous DNA repair mechanisms, non-homologous end joining (NHEJ) and homology directed repair (HDR), to either introduce insertion/deletion mutations or alter the genome using a homologous repair template at the site of the double stranded break. Electroporation of hPSCs is an efficient means of transfecting SSNs and repair templates that incorporate transgenes such as fluorescent reporters and antibiotic resistance cassettes. After electroporation, it is possible to isolate only those hPSCs that incorporated the repair construct by selecting for antibiotic resistance. Mechanically separating hPSC colonies and confirming proper integration at the target site through genotyping allows for the isolation of correctly targeted and genetically homogeneous cell lines. The validity of this protocol is demonstrated here by using all three SSN platforms to incorporate EGFP and a puromycin resistance construct into the AAVS1 safe harbor locus in human pluripotent&#160;stem cells.

Genome editing of human pluripotent stem cells (hPSCs) can be done quickly and efficiently. Presented here is a robust experimental procedure to genetically engineer hPSCs as exemplified by editing the AAVS1 safe harbor locus to express EGFP and introduce antibiotic resistance.

Genome-editing of human pluripotent stem cells (hPSCs) provides a genetically controlled and clinically relevant platform from which to understand human ...

Genome Editing in Astyanax mexicanus Using Transcription Activator-like Effector Nucleases (TALENs)

Identifying alleles of genes underlying evolutionary change is essential to understanding how and why evolution occurs. Towards this end, much recent work has focused on identifying candidate genes for the evolution of traits in a variety of species. However, until recently it has been challenging to functionally validate interesting candidate genes. Recently developed tools for genetic engineering make it possible to manipulate specific genes in a wide range of organisms. Application of this technology in evolutionarily relevant organisms will allow for unprecedented insight into the role of candidate genes in evolution. Astyanax mexicanus (A. mexicanus) is a species of fish with both surface-dwelling and cave-dwelling forms. Multiple independent lines of cave-dwelling forms have evolved from ancestral surface fish, which are interfertile with one another and with surface fish, allowing elucidation of the genetic basis of cave traits. A. mexicanus has been used for a number of evolutionary studies, including linkage analysis to identify candidate genes responsible for a number of traits. Thus, A. mexicanus is an ideal system for the application of genome editing to test the role of candidate genes. Here we report a method for using transcription activator-like effector nucleases (TALENs) to mutate genes in surface A. mexicanus. Genome editing using TALENs in A. mexicanus has been utilized to generate mutations in pigmentation genes. This technique can also be utilized to evaluate the role of candidate genes for a number of other traits that have evolved in cave forms of A. mexicanus.

Genome Editing in Astyanax mexicanus Using Transcription Activator-like Effector Nucleases TALENs

Gene-targeting mutagenesis is now possible in a wide range of organisms using genome editing techniques. Here, we demonstrate a protocol for targeted gene mutagenesis using transcription activator like effector nucleases (TALENs) in Astyanax mexicanus, a species of fish that includes surface fish and cavefish.

Identifying alleles of genes underlying evolutionary change is essential to understanding how and why evolution occurs.  Towards this end, much recent ...