This work was supported by the Department of Neurosurgery, Memorial Hermann Foundation Staman Ogilvie Fund, the Bentsen Stroke Center at the University of Texas Health Science Center at Houston, and Mission Connect TIRR Foundation.

1. Design and Vector Construction for Targeting Vectors
<ol>
	<li>Once the lineage specific marker is determined, locate the genomic sequence of the gene and design the homology arms accordingly. The length of 5&#8217; homology arm is ~1 kb and of 3&#8217; homology arm is ~1.5 kb (Figure 1).</li>
	<li>Tag the reporter cassette sequentially downstream of the genomic sequence right before the stop codon, by subcloning, so that the endogenous expression is not altered or disrupted. Link the reporter, or dual reporter cassette(s), with self-cleaving 2A peptide sequences (F2A, E2A or T2A) 20,21, or internal ribosome entry site (IRES). 
	Note: Reporters may include fluorescent proteins such as GFP, tdTomato, tagRFP, YFP, luciferase, or antibiotic resistance cassette, a combination of fluorescent protein and antibiotic resistance cassette. An example is shown in Figure 1 for constructing targeting vector for human ALDH1L1 gene, tagged by EGFP and neomycin resistance cassette.</li>
	<li>Add a positive selection cassette downstream of the reporter. The positive selection is a floxed antibiotic resistant cassette which will be excised from the targeted iPSCs after the positive clones are selected and isolated.</li>
	<li>Include a negative selection cassette outside of the 3&#8217; homology arm. Common negative selection cassettes are thymidine kinase, TK, and diphtheria toxin A (DTA) 22.</li>
	<li>Obtain a human BAC clone containing the sequence of the lineage marker and verify the clone by PCR amplification. Construct the targeting vector in pKD46 containing-DH5&#945; using recombineering to pull down the targeting site 23. Apply multisite gateway, Golden gate strategies23,24 when necessary.</li>
</ol>
2. Design and Vector Construction of sgRNAs for CRISPR/Cas9 System.
<ol>
	<li>Determine the lineage specific marker that will be targeted to make the reporter. Copy the cDNA sequence from NCBI database. Go to the human BLAT search to obtain genomic DNA alignment.</li>
	<li>Click the tab &#8220;browser&#8221; to view the assembly of the gene. Under &#8220;View&#8221; tab, click &#8220;DNA&#8221; to obtain the full genomic DNA sequence in FASTA format.</li>
	<li>Locate the stop codon within the DNA sequence, where the 2A sequence and the reporter cassette will be tagged.</li>
	<li>Search for the protospacer adjacent motif (PAM, i.e., NGG) within the vinicity (~100 bp flanking area) of the stop codon. Scan both strands to locate the appropriate NGG sequence. Consider the specificity of the upstream sequences to avoid potential off-target events. Use open source tools such as BLAST and CasOT (see Step 5 for details).</li>
	<li>Copy the sequence of 20 bases immediately upstream of NGG and paste it into the web designing tool ZiFit to design oligos with overhangs compatible with human-gRNA-expression vector (with U6 promoter) MLM3636.</li>
	<li>Synthesize both forward and reverse oligos 7.</li>
	<li>To make double-stranded sgRNA, denature both oligos at 70 &#176;C for 15 min, and gradually cool down to RT. For a 50 &#181;l reaction, add 22.5 &#181;l of forward and reverse oligos (stock concentration is 0.1 mM) respectively, and 5 &#181;l of annealing buffer (100 mM Tris-HCl, 10 mM EDTA, 1.5 M NaCl, pH 8.0).</li>
	<li>Mix and ligate the oligos obtained from step 2.7 with BsmBI-digested human-gRNA-expression vector (with U6 promoter) MLM3636. For a 20 &#181;l reaction, add 1 &#181;l of oligos from Step 2.7, 100 ng of MLM3636, and 1 &#181;l (400 U) of T4 ligase. Incubate at RT O/N.</li>
	<li>Transform DH5&#945; competent cells with the ligation product by heat shock at 42&#176;C for 30 sec. Grow the bacteria in LB agar Ampicillin (100 &#956;g/ml) plate at 37&#176;C O/N. Pick 3~5 colonies for plasmid miniprep according to manufacturer&#8217;s protocol. Sequence the plasmid DNA to confirm using commercial vendors. Maxiprep the correct plasmid according to manufacturer&#8217;s protocol.</li>
	<li>Evaluate sgRNA by T7 endonuclease 1 (T7E1) assay (see Step 4 for details). Transfect hiPSC with the plasmid and sgRNA will be expressed under the built-in U6 promoter in the MLM3636 backbone (See step 6 for details).</li>
</ol>
3. Design and Construction of Cas9n Double Nickase sgRNA
<ol>
	<li>Obtain double nickase Cas9n D10A vector pX335-U6-Chimeric_BB-CBh-hSpCas9n (D10A).</li>
	<li>Design a pair of forward and reverse oligos for each targeting locus. Copy the sequence of 20 bases immediately upstream of NGG as described in Step 2 and paste it into web designing tool ZiFit</li>
	<li>To obtain oligos with overhangs compatible with pX335-U6-Chimeric_BB-CBh-hSpCas9n (D10A), remove the first and last base of both forward and reverse oligos that are shown by the ZiFit designing tool.</li>
	<li>Synthesize forward and reverse oligos7. Name these oligos as f(n)-F, f(n)-R for PAMs on one side of the target site, r(n)-F, r(n)-R for PAMs on the other side of the target site. Of these, n is the number to designate different PAMs, lowercase f and r represent the location of the PAMs either at sense (f) or antisense (r) strand,, F and R represent forward and reverse oligos.
	<ol>
		<li>Test multiple pairs (e.g., r5 and f2) using the T7E1 assay (detailed in Step 4) to identify the optimized pair of sgRNAs to be used for Cas9n.</li>
	</ol>
	</li>
	<li>To make double-stranded sgRNA, denature both oligos at 70 &#176;C for 15 min, and gradually cool down to RT. For a 50 &#181;l reaction, add 22.5 &#181;l of forward and of reverse oligos (stock concentration is 0.1 mM) respectively, and 5 &#181;l of annealing buffer (100 mM Tris-HCl, 10 mM EDTA, 1.5 M NaCl, pH 8.0).</li>
	<li>Mix and ligate the oligos obtained from step 3.5 with BbsI-digested pX335-U6-Chimeric_BB-CBh-hSpCas9n (D10A). For a 20 &#181;l reaction, add 1 &#181;l of sgRNA from Step 3.5, 100 ng of Cas9n (D10A), 1 &#181;l (400 U) of T4 ligase. Incubate at RT O/N.</li>
	<li>Transform DH5&#945; competent cells with the ligation product by heat shock at 42 &#176;C for 30 sec. Grow the bacteria in LB agar Ampicillin (100 &#956;g/ml) plate at 37 &#176;C O/N. Pick 3~5 colonies for plasmid miniprep according to manufacturer&#8217;s protocol. Sequence the plasmid DNA to confirm using commercial vendors. Maxiprep the correct plasmid according to manufacturer&#8217;s protocol.</li>
	<li>Evaluate sgRNA by T7E1 assay (see Step 4 for details). Transfect hiPSC with the plasmid and sgRNA will be expressed under the built-in U6 promoter in the Cas9n (D10A) backbone (See step 6 for details).</li>
</ol>
4. Evaluation of sgRNAs by T7 Endonuclease1 (T7E1) Assay
<ol>
	<li>For one 24-well plate, plate 2 x 105 293FT cells per well one day prior (day -1).</li>
	<li>On the day of transfection (day 0), co-transfect 293FT cells with sgRNA plasmid and Cas9 expression vector. Add 0.5 &#181;g of sgRNA vector, 0.5 &#181;g of Cas9 expression vector JDS246 (Cas9-003), and 1.5 &#181;l of Lipofectamine 2000.
	<ol>
		<li>For negative control, add 0.5 &#181;g of GFP expression vector (e.g., CMV promoter driven GFP in a pcDNA3 backbone) instead of sgRNA vectors.</li>
	</ol>
	</li>
	<li>After 72 hr (day 3), harvest cells by adding 100 &#181;l of DNA quick extraction buffer and triturating vigorously.</li>
	<li>Incubate the mixture at 68 &#176;C for 15 min, then 95 &#176;C for 8 min. This is the template for the subsequent PCR experiments.</li>
	<li>Design both forward and reverse PCR primers to amplify the sequence flanking PAM. Usually the targeting PCR product is around 400-500 bp in length. Synthesize the oligos7.</li>
	<li>For a 50 &#181;l PCR reaction, add 1 &#181;l of template from Step 4.5, 0.5 &#181;l of Herculase II, 0.5 &#181;l of DMSO, 10 &#181;l of 5x buffer, 0.5 &#181;l of dNTP mix, and 1 &#181;l of forward and reverse primer mix.</li>
	<li>Use the following PCR parameters: 98 &#176;C for 2 min, 35 cycles of 98 &#176;C for 20 sec, 59 &#176;C for 20 sec, 72 &#176;C for 1 min, with a final extension at 72 &#176;C for 5 min. 
	Note: The PCR parameters described here yield specific products for several genes tested. However, these conditions are dependent upon user-chosen genes of interest. PCRs using genomic DNA as template often require optimization to ensure that no extra bands appear. These extra bands can be confused with cleavage products after the T7E1 assay.</li>
	<li>Measure DNA concentration of the PCR products using a spectrometer at A260.</li>
	<li>Add 400 ng of PCR products to 2 &#181;l of Digestion buffer 2. Add ddH2O to a final reaction volume of 19 &#181;l. Incubate in boiling water for 5 min. Let the reaction sit on the bench and naturally cool down to RT (45-60 min).</li>
	<li>Add 1 &#181;l (10 units/&#181;l) of T7E1, incubate at 37 &#176;C for 15 min. Stop the reaction by adding 2 &#181;l of 0.25 M EDTA mixed with 6x DNA Loading Dye.</li>
	<li>Dissolve 2.5 g of agarose powder to 100 ml of 1xTAE. Microwave the mixture and pour gel onto a gel box. When gel is formed, load the whole 20 &#181;l reaction to the 2.5% agarose gel. Run the gel at 100-150 V for 30 min.</li>
	<li>Inspect the gel under UV light (Figure 3). T7E1 cuts at the mismatched sites. For sgRNAs that have caused non-homologous end joining, the PCR products will show additional fast migrating bands (smaller in size) compared to negative control (described in step 4.3).</li>
	<li>Estimate and rank sgRNA cutting efficiency and insertion and deletion percentage (indel%) by comparing the band density using Image J software.
	<ol>
		<li>For each sample that run on the gel, designate the gel bands as uncut band, cut band 1 and cut band 2.</li>
		<li>Measure the density of each band using ImageJ according to the software handbook.</li>
		<li>Calculate fcut using the following formula: fcut = (cut band 1 + cut band 2) / (uncut band + cut band 1 + cut band 2)</li>
		<li>Calculate Indel% using the following formula: Indel=1- &#8730;(1-fcut).</li>
	</ol>
	</li>
</ol>
5. In Silico Prediction of Potential Off-target Sites
Note: Potential off target sites can be predicted using an online open source tool called CasOT17, which is a Perl based program.
<ol>
	<li>For a Windows system, download the CasOT program and the human genome database files to local hard drive.</li>
	<li>Prepare the target file of the designed sgRNA in the FASTA format.</li>
	<li>Open the file named &#8220;casot&#8221;. A command window appears. Leave it as is for now.</li>
	<li>Open the file named &#8220;opt-generator&#8221; to generate option string. Paste the path and name of the target file into the dialog of &#8220;-t&#8221; or &#8220;-target&#8221; option.</li>
	<li>Paste the path of the genome sequence file into the dialog of &#8220;-g&#8221; or &#8220;-genome&#8221; option.</li>
	<li>Input values of &#8211;s or &#8211;seed (default value is 2) and &#8211;n or -nonseed (default value is 255). The option string is generated and will appear in the box at the top of the page.</li>
	<li>Copy the option string and paste it into the CasOT command window described in Step 5.3. 
	Note: It might take 30-90 min to run the CasOT program depending on the options and the computer used. Once completed, a folder named &#8216;&#60;input_target_file&#62;-&#60;genome_file&#62;-&#60;off-target_related_options&#62;&#8217; will be generated. Two files will be included in the folder, a &#8220;.stat&#8221; file describing the statistics and a .csv (comma-separated values) file containing the detailed search results.&#60;/off-target_related_options&#62;&#60;/genome_file&#62;&#60;/input_target_file&#62;</li>
	<li>Open the .csv file with a spreadsheet program. The following information is provided in the file for every off target site: chromosomal and genomic location, sequence of the site, mismatch (Mm) type, total number of mismatched bases, and exon information relating to the off target site.</li>
	<li>Design PCR primers for the sites that have &#60;2 Mm in either the seed or the non-seed sequence.</li>
	<li>Perform PCR for selected positive hiPSC clone using the following conditions: 98 &#176;C for 2 min, 35 cycles of 98 &#176;C for 20 sec, 59 &#176;C for 20 sec, 72 &#176;C for 1 min, with a final extension at 72 &#176;C for 5 min. 
	Note: The PCR parameters described here yield specific products for several genes tested in our lab. However, these conditions are dependent upon user-chosen genes of interest. PCRs using genomic DNA as template often require optimization to ensure that no extra bands appear.</li>
	<li>Sequence the PCR products, align sequencing results with predicted sequences and search for mutations. If mutations exist, then off-target events are identified.</li>
</ol>
6. Electroporation of hiPSCs to Make Neural Lineage Reporters
<ol>
	<li>Culture hiPSCs in TeSR-E8 medium on Geltrex coated dishes. Change medium to MEF-CM (Mouse embryonic fibroblasts conditioned medium) medium two days before electroporation. Incubate in 2 ml of Accutase for 5 min and collect cells.</li>
	<li>Electroporate hiPSCs with linearized targeting vector, Cas9-003 expression vector, and sgRNA vector using the following conditions: 1 x 107 hiPSCs, co-transfect 2.5 &#181;g of sgRNA, 2.5 &#181;g of Cas9 and 50 &#181;g of targeting vector. For double nickase strategy, add 50 &#181;g of targeting vector, 2.5 &#181;g of each sgRNA (with Cas9n co-expressed by the vector).</li>
	<li>Select clones by positive and negative selections as described24,25.</li>
	<li>Identify positive clones by Southern blot analysis or long range genomic PCR24,25.</li>
</ol>

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The authors have nothing to disclose.

Gene targeting is an essential tool in characterizing gene functions. However, the relatively low efficiency demands labor-intensive and time-consuming work. Recently development on genome editing tools such as the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated) system has greatly improved the targeting efficiency. This manuscript describes a protocol for generating lineage specific human induced pluripotent stem cell (hiPSC) reporter using CRISPR/Cas system assisted homologous recombination. Steps ...

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated) system, together with other genome editing tools, has revolutionized the human genome manipulation in recent years1-7. Major components in the CRISPR/Cas9 system are single guide RNA (sgRNA) and Cas9. Cas9 is an RNA-guided type II DNA endonuclease. It has two nuclease domains, HNH and RuvC, which are responsible for making DNA double-stranded breaks (DSBs) at a specific genomic region next to a protospacer adjacent motif (PAM)7-10.An sgRNA has a combined function of that of crRNA and tracrRNA, two adaptive immunity RNA molecules identified in bacteria (such as Streptococcus pyogenes) and archaea to fight against DNA invasion of exogenous sources9-11. When appropriately designed and co-introduced to mammalian cells, the sgRNA recognizes the PAM sequence, base-pairs with the complementary strand of target DNA, and guides and transactivates Cas9 to cleave at both DNA strands immediately upstream of PAM7. Subsequently, homologous directed recombination (HDR, in the presence of a targeting vector) or non-homologous end joining (NHEJ) will occur to repair the DSBs. Transgenes such as cDNAs, reporter cassettes, or antibiotic resistant fragments can be integrated, and transgenic or knockin lines made.
Although the CRISPR/Cas9 system is highly efficient and relatively specific, undesired off-target activities have been reported12-15. To minimize off-target events, Cas9 nickases (Cas9n) have also been engineered9,10,14,16,17. Cas9n (Cas9 D10A or Cas9 H840A) is Cas9 enzyme with mutations at one of the two nuclease domains, which only elicits a nick at one strand of DNA. To achieve cleavage at both strands, two sgRNAs are designed to guide a pair of nickases, which cuts separately at nearby loci of the two different DNA strands. The &#8220;double nicking&#8221; strategy requires a more stringent design than the conventional Cas9 platform, and offers both high efficiency and high specificity for gene editing experiments.
This manuscript describes a protocol for generating lineage specific human induced pluripotent stem cell (hiPSC) reporter using CRISPR/Cas system assisted homologous recombination. Steps for obtaining the necessary components for making neural lineage reporter lines using the CRISPR/Cas system mediated genome editing are described, with a focus on construction of targeting vectors and sgRNAs. This protocol has been successfully repeated in multiple hiPSC lines including those derived from healthy individuals and from patients with CNS diseases. Genes targeted in our laboratory include OLIG2, HB9 (MNX1), NEUROG2, SOX1, ALDH1L1 and SOD1. Targeting efficiency from CRISPR/Cas system mediated homologous recombination in hiPSCs ranges from 20-40%, which is consistent with previous reports1,18,19. Compared to a typical efficiency of 1-2% in conventional gene targeting experiments, the targeting efficiency is significantly improved.

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			<td height="20" style="height:20px;width:259px;">Name of Material/Equipment</td>
			<td style="width:265px;">Vendor</td>
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			<td height="20" style="height:20px;width:259px;">ROCK inhibitor Y-27632</td>
			<td style="width:265px;">Millipore</td>
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			<td height="20" style="height:20px;width:259px;">SMC4 reagent&#160;</td>
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			<td style="width:141px;">PMEF-NL</td>
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</table>

efficient generation of hipsc neural lineage specific knockin reporters using the crispr/cas9 and cas9 double nickase system

基因靶向是现代生物医学研究表征基因功能的一个重要方法。然而，基因在人类细胞靶向效率一直较低，从而防止人细胞系的产生在所需的速率。近两年来，在提高遗传操纵基因组编辑工具效率的快速进展，如CRISPR（聚集定期短相互间隔重复回文）/ CAS（CRISPR相关）系统。这份手稿描述了一个协议，用于使用辅助同源重组的CRISPR / CAS系统沿袭特定的人类诱导多能干细胞（hiPSC）记者。为获得必要的组件制造神经系记者行使用CRISPR / CAS系统，重点建设目标向量和引导单RNA的过程，进行了描述。该协议可以扩展到平台建立和突变校正在人iPS细胞。

Targeting vectors with all necessary components including homology arms, reporter cassette, positive and negative selection cassettes are constructed (Figure 1). Based on the endogenous genomic locus, multiple (2 to 3) sgRNAs (if using the Cas9 system) or sgRNA pairs (if using the Cas9n double nickase strategy) are designed and constructed into human-gRNA-expression vector MLM3636 or pX335-U6-Chimeric_BB-CBh-hSpCas9n (D10A) backbones (Figure 2). Before transfecting hiPSCs, the sgRNAs are...

Watch this Scientific Journal Video about Efficient Generation of hiPSC Neural Lineage Specific Knockin Reporters Using the CRISPR/Cas9 and Cas9 Double Nickase System at JoVE.com

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

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.

的hiPSC神经细胞系特异性敲开记者代高效使用CRISPR / Cas9和Cas9双切口酶系统

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

efficient-generation-hipsc-neural-lineage-specific-knockin-reporters

Research

JoVE Journal

Developmental Biology

11.0K 次观看.  The University of Texas Health Science Center at Houston. 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.

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

Alginate Encapsulation of Pluripotent Stem Cells Using a Co-axial Nozzle

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening. However, the development of a mass culture system would be required for using PS cells in these applications. Suspension culture is one promising culture method for the mass production of PS cells, although some issues such as controlling aggregation and limiting shear stress from the culture medium are still unsolved. In order to solve these problems, we developed a method of calcium alginate (Alg-Ca) encapsulation using a co-axial nozzle. This method can control the size of the capsules easily by co-flowing N2 gas. The controllable capsule diameter must be larger than 500 &#181;m because too high a flow rate of N2 gas causes the breakdown of droplets and thus heterogeneous-sized capsules. Moreover, a low concentration of Alg-Na and CaCl2 causes non-spherical capsules. Although an Alg-Ca capsule without a coating of Alg-PLL easily dissolves enabling the collection of cells, they can also potentially leak out from capsules lacking an Alg-PLL coating. Indeed, an alginate-PLL coating can prevent cellular leakage but is also hard to break. This technology can be used to research the stem cell niche as well as the mass production of PS cells because encapsulation can modify the micro-environment surrounding cells including the extracellular matrix and the concentration of secreted factors.

We established a method of encapsulating pluripotent stem cells (PS cells) into alginate hydrogel capsules using a co-axial nozzle. This prevents cells from aggregating excessively and limits the shear stress experienced by cells in suspension culture. The technique is applicable to the mass production of PS cells as well as research on stem cell niche.

多能干细胞的海藻酸钠封装使用同轴喷嘴

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening.  However, the ...

科研

发育生物学

Stable and Efficient Genetic Modification of Cells in the Adult Mouse V-SVZ for the Analysis of Neural Stem Cell Autonomous and Non-autonomous Effects

Relatively quiescent somatic stem cells support life-long cell renewal in most adult tissues. Neural stem cells in the adult mammalian brain are restricted to two specific neurogenic niches: the subgranular zone of the dentate gyrus in the hippocampus and the ventricular-subventricular zone (V-SVZ; also called subependymal zone or SEZ) in the walls of the lateral ventricles. The development of in vivo gene transfer strategies for adult stem cell populations (i.e. those of the mammalian brain) resulting in long-term expression of desired transgenes in the stem cells and their derived progeny is a crucial tool in current biomedical and biotechnological research. Here, a direct in vivo method is presented for the stable genetic modification of adult mouse V-SVZ cells that takes advantage of the cell cycle-independent infection by LVs and the highly specialized cytoarchitecture of the V-SVZ niche. Specifically, the current protocol involves the injection of empty LVs (control) or LVs encoding specific transgene expression cassettes into either the V-SVZ itself, for the in vivo targeting of all types of cells in the niche, or into the lateral ventricle lumen, for the targeting of ependymal cells only. Expression cassettes are then integrated into the genome of the transduced cells and fluorescent proteins, also encoded by the LVs, allow the detection of the transduced cells for the analysis of cell autonomous and non-autonomous, niche-dependent effects in the labeled cells and their progeny.

Here we describe a procedure based on the use of lentiviral particles for the long-term genetic modification of neural stem cells and/or their adjacent ependymal cells in the adult ventricular-subventricular neurogenic niche which allows the separate analysis of cell autonomous and non-autonomous, niche-dependent effects on neural stem cells.

在成年小鼠V-SVZ细胞的神经分析的稳定，高效的基因修饰干细胞自主和非自主的影响

Relatively quiescent somatic stem cells support life-long cell renewal in most adult tissues. Neural stem cells in the adult mammalian brain are ...

A Method for Lineage Tracing of Corneal Cells Using Multi-color Fluorescent Reporter Mice

Lineage tracing experiments define the origin, fate and behavior of cells in a specific tissue or organism. This technique has been successfully applied for many decades, revealing seminal findings in developmental biology. More recently, it was adopted by stem cell biologists to identify and track different stem cell populations with minimal experimental intervention. The recent developments in mouse genetics, the availability of a large number of mouse strains, and the advancements in fluorescent microscopy allow the straightforward design of powerful lineage tracing systems for various tissues with basic expertise, using commercially available tools. We have recently taken advantage of this powerful methodology to explore the origin and fate of stem cells at the ocular surface using R26R-Confetti mouse. This model offers a multi-color genetic system, for the expression of 4 fluorescent genes in a random manner. Here we describe the principles of this methodology and provide an adaptable protocol for designing lineage tracing experiments; specifically for the corneal epithelium as well as for other tissues.

In this article we describe the principles for designing and performing a multi-color lineage tracing experiment using R26R-Confetti mice. We provide a specific protocol for tracking corneal epithelial cells which can be modified for other tissues of interest.

方法对角膜细胞的谱系追踪使用多色荧光记者小鼠

Lineage tracing experiments define the origin, fate and behavior of cells in a specific tissue or organism. This technique has been successfully applied ...

Generating CRISPR/Cas9 Mediated Monoallelic Deletions to Study Enhancer Function in Mouse Embryonic Stem Cells

Enhancers control cell identity by regulating tissue-specific gene expression in a position and orientation independent manner. These enhancers are often located distally from the regulated gene in intergenic regions or even within the body of another gene. The position independent nature of enhancer activity makes it difficult to match enhancers with the genes they regulate. Deletion of an enhancer region provides direct evidence for enhancer activity and is the gold standard to reveal an enhancer's role in endogenous gene transcription. Conventional homologous recombination based deletion methods have been surpassed by recent advances in genome editing technology which enable rapid and precisely located changes to the genomes of numerous model organisms. CRISPR/Cas9 mediated genome editing can be used to manipulate the genome in many cell types and organisms rapidly and cost effectively, due to the ease with which Cas9 can be targeted to the genome by a guide RNA from a bespoke expression plasmid. Homozygous deletion of essential gene regulatory elements might lead to lethality or alter cellular phenotype whereas monoallelic deletion of transcriptional enhancers allows for the study of cis-regulation of gene expression without this confounding issue. Presented here is a protocol for CRISPR/Cas9 mediated deletion in F1 mouse embryonic stem (ES) cells (Mus musculus129 x Mus castaneus). Monoallelic deletion, screening and expression analysis is facilitated by single nucleotide polymorphisms (SNP) between the two alleles which occur on average every 125 bp in these cells.

Experimental validation of enhancer activity is best approached by loss-of-function analysis. Presented here is an efficient protocol that uses CRISPR/Cas9 mediated deletion to study allele-specific regulation of gene transcription in F1 ES cells which contain a hybrid genome (Mus musculus129 x Mus castaneus).

生成CRISPR / Cas9介导的单等位基因缺失来研究小鼠胚胎干细胞功能增强

Enhancers control cell identity by regulating tissue-specific gene expression in a position and orientation independent manner. These enhancers are often ...

Co-localization of Cell Lineage Markers and the Tomato Signal

The cell lineage tracing system has been used predominantly in developmental biology studies. The use of Cre recombinase allows for the activation of the reporter in a specific cell line and all progeny. Here, we used the cell lineage tracing technique to demonstrate that chondrocytes directly transform into osteoblasts and osteocytes during long bone and mandibular condyle development using two kinds of Cre, Col10a1-Cre and Aggrecan-CreERT2 (Agg-CreERT2), crossed with Rosa26tdTomato. Both Col10 and aggrecan are well-recognized markers for chondrocytes.
On this basis, we developed a new method-cell lineage tracing in conjunction with fluorescent immunohistochemistry-to define cell fate by analyzing the expression of specific cell markers. Runx2 (a marker for early-stage osteogenic cells) and Dentin matrix protein1 (DMP1; a marker for late-stage osteogenic cells) were used to identify chondrocyte-derived bone cells and their differentiation status. This combination not only broadens the application of cell lineage tracing, but also simplifies the generation of compound mice. More importantly, the number, location, and differentiation statuses of parent cell progeny are displayed simultaneously, providing more information than cell lineage tracing alone. In conclusion, the co-application of cell lineage tracing techniques and immunofluorescence is a powerful tool for investigating cell biology in vivo.

We developed two sets of tracing combinations in Rosa26tdtomato (ubiquitously expressed in all cells)/Cre (specifically expressed in chondrocytes) mice: one with 2.3Col1a1-GFP (specific to osteoblasts) and one with immunofluorescence (specific to bone cells). The data demonstrate the direct transformation of chondrocytes into bone cells.

细胞系标记的共定位和番茄信号

The cell lineage tracing system has been used predominantly in developmental biology studies. The use of Cre recombinase allows for the activation of the ...

Cell Lineage Analyses and Gene Function Studies Using Twin-spot MARCM

Mosaic analysis with a repressible cell marker (MARCM) is a positive mosaic labeling system that has been widely applied in Drosophila neurobiological studies to depict intricate morphologies and to manipulate the function of genes in subsets of neurons within otherwise unmarked and unperturbed organisms. Genetic mosaics generated in the MARCM system are mediated through site-specific recombination between homologous chromosomes within dividing precursor cells to produce both marked (MARCM clones) and unmarked daughter cells during mitosis. An extension of the MARCM method, called twin-spot MARCM (tsMARCM), labels both of the twin cells derived from a common progenitor with two distinct colors. This technique was developed to enable the retrieval of useful information from both hemi-lineages. By comprehensively analyzing different pairs of tsMARCM clones, the tsMARCM system permits high-resolution neural lineage mapping to reveal the exact birth-order of the labeled neurons produced from common progenitor cells. Furthermore, the tsMARCM system also extends gene function studies by permitting the phenotypic analysis of identical neurons of different animals. Here, we describe how to apply the tsMARCM system to facilitate studies of neural development in Drosophila.

Here, we present a protocol for a mosaic labeling technique that permits the visualization of neurons derived from a common progenitor cell in two distinct colors. This facilitates neural lineage analysis with the capability of birth-dating individual neurons and studying gene function in the same neurons of different individuals.

细胞谱系分析和基因功能研究使用双点MARCM

Mosaic analysis with a repressible cell marker (MARCM) is a positive mosaic labeling system that has been widely applied in Drosophila neurobiological ...

CRISPR/Cas9 Technology in Restoring Dystrophin Expression in iPSC-Derived Muscle Progenitors

Duchenne muscular dystrophy (DMD) is a severe progressive muscle disease caused by mutations in the dystrophin gene, which ultimately leads to the exhaustion of muscle progenitor cells. Clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9) gene editing has the potential to restore the expression of the dystrophin gene. Autologous induced pluripotent stem cells (iPSCs)-derived muscle progenitor cells (MPC) can replenish the stem/progenitor cell pool, repair damage, and prevent further complications in DMD without causing an immune response. In this study, we introduce a combination of CRISPR/Cas9 and non-integrated iPSC technologies to obtain muscle progenitors with recovered dystrophin protein expression. Briefly, we use a non-integrating Sendai vector to establish an iPSC line from dermal fibroblasts of Dmdmdx mice. We then use the CRISPR/Cas9 deletion strategy to restore dystrophin expression through a non-homologous end joining of the reframed dystrophin gene. After PCR validation of exon23 depletion in three colonies from 94 picked iPSC colonies, we differentiate iPSC into MPC by doxycycline (Dox)-induced expression of MyoD, a key transcription factor playing a significant role in regulating muscle differentiation. Our results show the feasibility of using CRISPR/Cas9 deletion strategy to restore dystrophin expression in iPSC-derived MPC, which has significant potential for developing future therapies for the treatment of DMD.

Here, we present a Cas9-based exon23 deletion protocol to restore dystrophin expression in iPSC from Dmdmdx mouse-derived skin fibroblasts and directly differentiate iPSCs into myogenic progenitor cells (MPC) using the Tet-on MyoD activation system.

CRISPR/Cas9在iPSC衍生肌肉预祖中恢复肌营养不良素表达的技术

Duchenne muscular dystrophy (DMD) is a severe progressive muscle disease caused by mutations in the dystrophin gene, which ultimately leads to the ...

Three-dimensional Angiogenesis Assay System using Co-culture Spheroids Formed by Endothelial Colony Forming Cells and Mesenchymal Stem Cells

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more than 50 different pathological conditions, such as rheumatoid arthritis, oculopathy, cardiovascular diseases, and tumor metastasis. During angiogenesis drug development, it is crucial to use in vitro assay systems with appropriate cell types and proper conditions to reflect the physiologic angiogenesis process. To overcome limitations of current in vitro angiogenesis assay systems using mainly endothelial cells, we developed a 3-dimensional (3D) co-culture spheroid sprouting assay system. Co-culture spheroids were produced by two human vascular cell precursors, endothelial colony forming cells (ECFCs) and mesenchymal stem cells (MSCs) with a ratio of 5 to 1. ECFCs+MSCs spheroids were embedded into type I collagen matrix to mimic the in vivo extracellular environment. A real-time cell recorder was utilized to continuously observe the progression of angiogenic sprouting from spheroids for 24 h. Live cell fluorescent labeling technique was also applied to tract the localization of each cell type during sprout formation. Angiogenic potential was quantified by counting the number of sprouts and measuring the cumulative length of sprouts generated from the individual spheroids. Five randomly-selected spheroids were analyzed per experimental group. Comparison experiments demonstrated that ECFCs+MSCs spheroids showed greater sprout number and cumulative sprout length compared with ECFCs-only spheroids. Bevacizumab, an FDA-approved angiogenesis inhibitor, was tested with the newly-developed co-culture spheroid assay system to verify its potential to screen anti-angiogenic drugs. The IC50 value for ECFCs+MSCs spheroids compared to the ECFCs-only spheroids was closer to the effective plasma concentration of bevacizumab obtained from the xenograft tumor mouse model. The present study suggests that the 3D ECFCs+MSCs spheroid angiogenesis assay system is relevant to physiologic angiogenesis, and can predict an effective plasma concentration in advance of animal experiments.

Three-dimensional co-culture spheroid angiogenesis assay system is designed to mimic the physiologic angiogenesis. Co-culture spheroids are formed by two human vascular cell precursors, ECFCs and MSCs, and embedded in collagen gel. The new system is effective for evaluating angiogenic modulators, and provides more relevant information to the in vivo study.

使用由内皮结肠形成细胞和间质干细胞形成的共培养球体的三维血管生成测定系统

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more ...

Efficient Neural Differentiation using Single-Cell Culture of Human Embryonic Stem Cells

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular levels and provided cells for use in regenerative applications. Standard approaches for hESC culture using colony type culture to maintain undifferentiated hESCs and embryoid body (EB) and rosette formation for differentiation into different germ layers are inefficient and time-consuming. Presented here is a single-cell culture method using hESCs instead of a colony-type culture. This method allows maintenance of the characteristic features of undifferentiated hESCs, including expression of hESC markers at levels comparable to colony type hESCs. In addition, the protocol presents an efficient method for neural progenitor cell (NPC) generation from single-cell type hESCs that produces NPCs within 1 week. These cells highly express several NPC marker genes and can differentiate into various neural cell types, including dopaminergic neurons and astrocytes. This single-cell culture system for hESCs will be useful in investigating the molecular mechanisms of these processes, studies of certain diseases, and drug discovery screens.

Presented here is a protocol for the generation of a single-cell culture of human embryonic stem cells and their subsequent differentiation into neural progenitor cells. The protocol is simple, robust, scalable, and suitable for drug screening and regenerative medicine applications.

利用人类胚胎干细胞的单细胞培养进行高效神经分化

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular ...

An Optimized O9-1/Hydrogel System for Studying Mechanical Signals in Neural Crest Cells

Neural crest cells (NCCs) are vertebrate embryonic multipotent cells that can migrate and differentiate into a wide array of cell types that give rise to various organs and tissues. Tissue stiffness produces mechanical force, a physical cue that plays a critical role in NCC differentiation; however, the mechanism remains unclear. The method described here provides detailed information for the optimized generation of polyacrylamide hydrogels of varying stiffness, the accurate measurement of such stiffness, and the evaluation of the impact of mechanical signals in O9-1 cells, a NCC line that mimics in vivo NCCs.
Hydrogel stiffness was measured using atomic force microscopy (AFM) and indicated different stiffness levels accordingly. O9-1 NCCs cultured on hydrogels of varying stiffness showed different cell morphology and gene expression of stress fibers, which indicated varying biological effects caused by mechanical signal changes. Moreover, this established that varying the hydrogel stiffness resulted in an efficient in vitro system to manipulate mechanical signaling by altering gel stiffness and analyzing the molecular and genetic regulation in NCCs. O9-1 NCCs can differentiate into a wide range of cell types under the influence of the corresponding differentiation media, and it is convenient to manipulate chemical signals in vitro. Therefore, this in vitro system is a powerful tool to study the role of mechanical signaling in NCCs and its interaction with chemical signals, which will help researchers better understand the molecular and genetic mechanisms of neural crest development and diseases.

Detailed step-by-step protocols are described here for studying mechanical signals in vitro using multipotent O9-1 neural crest cells and polyacrylamide hydrogels of varying stiffness.

一种优化的O9-1/水凝胶系统，用于研究神经嵴细胞中的机械信号

Neural crest cells (NCCs) are vertebrate embryonic multipotent cells that can migrate and differentiate into a wide array of cell types that give rise to ...