Team:SYSU-China/Notebookt/Methods

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<!--正 文 部 分 开 始-->
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<DIV class="chapter">
<DIV class="chapter">
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<span>UPDATE <INS>09/18/2013</INS></span>
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<span>Notebookt/Methods</span>
-
<h1>iPSCs</h1>
+
 
-
<h2>What is iPSCs?</h2>
+
<h1>  
-
<h3>A one-way journey?</h3>
+
Methods
 +
</h1>
 +
<h2>  
 +
Introduction
 +
</h2>
 +
 
 +
<p> Our experiments can be divided into two parts, one is the design and construction of genes and vectors, the other is test of parts and circuits with cells, including hepatoma cells, liver cells and iPSCs. So our protocol can be clearly be divided into these two parts, one for molecular operation, the other for cellular tests. Besides, quantitive measurement, like qPCR and western-blotting, is added in the molecular operation parts because it does not need cell operation.
 +
</p>
 +
<h2>  
 +
Molecular Operation
 +
</h2>
 +
 
 +
<p>< In the process of constructing circuit on vectors, PCR, PCR clean up, digestion of restriction endonuclease, gel extraction, ligation, transformation, colony PCR and plasmid extraction is implemented. Besides, for quantitive measurement, qPCR and western-blotting is used..</p>
 +
 
 +
<p><strong> PCR
 +
</strong> </p>
 +
 
 +
<p>
 +
1. Prepare the reaction mix:
 +
</p>
 +
<br><table width="400">
 +
<tr class="first-table-line">
 +
<td>5x Fastpfu buffer </td>
 +
<td> 10ul </td>
 +
</tr>
 +
<tr>
 +
<td>2.5mM dNTPs  </td>
 +
<td>5ul </td>
 +
</tr>
 +
<tr>
 +
<td> Forward primer </td>
 +
<td>  0.8ul </td>
 +
</tr>
 +
<tr>
 +
<td> Reverse primer </td>
 +
<td>0.8ul </td>
 +
</tr>
 +
<tr>
 +
<td> Template DNA </td>
 +
<td> 40-50ng</td>
 +
</tr>
 +
<tr>
 +
<td>  FastPfu    </td>
 +
<td> 1ul </td>
 +
</tr>
 +
<tr>
 +
<td> ddH2O </td>
 +
<td> add to 50ul
 +
</td>
 +
</tr>
 +
</table><br />
 +
 
<p>
<p>
-
During the first half of the 20th century, researchers always believed that the life's one-way journey also applied to cells: Once a cell has developed into a specialized cell, it would be locked into that state, and unable to return to immature, pluripotent stem cell state. Conrad Hal Waddington, a famous developmental biologist in last century, once illustrated the cellular differentiation as an epigenetic landscape in which cells are seen as marbles rolling down in valleys to reach their end-point destinations as differentiated cells. They do not normally move back towards the top of the mountain to get back to the undifferentiated state, and they are not normally crossing into other valleys to develop into unrelated cell lineages<a class="quote">[1]</a>.
+
2. Set the program of thermal cycler
</p>
</p>
-
<img src="https://static.igem.org/mediawiki/2013/8/8b/Introduction_01.jpg" width="500" height="197" />
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<br><table width="400">
 +
 
 +
<tr class="first-table-line">
 +
<td>(1×) </td>
 +
<td> 95℃  </td>
 +
<td>3min </td>
 +
</tr>
 +
<tr>
 +
<td>(25×)  </td>
 +
<td>95℃  </td>
 +
<td> 20s </td>
 +
</tr>
 +
<tr>
 +
<td>  </td>
 +
<td>  A-5℃ </td>
 +
<td> 20s (Tm≈A℃) </td>
 +
</tr>
 +
<tr>
 +
<td> </td>
 +
<td>  72℃</td>
 +
<td>30s for each 1kb </td>
 +
</tr>
 +
<tr>
 +
<td> (1×)</td>
 +
<td> 72℃ </td>
 +
<td>  5min</td>
 +
</tr>
 +
<tr>
 +
<td>(1×)  </td>
 +
<td> 4℃ </td>
 +
<td> forever </td>
 +
</tr>
 +
 
 +
 
 +
</table><br />
 +
<p>
 +
3. Run the PCR program.
 +
</p>
 +
<p>
 +
4. After PCR, load ~2ul to a 1% agarose gel to have a quick run checking the production of desired fragment.
 +
</p>
 +
 
 +
<p><strong> PCR clean up
 +
</strong></p>
 +
 
 +
 
<p>
<p>
-
However, this prevalent view was radically overturned in 1962, when John B. Gurdon demonstrated that the nucleus from a differentiated frog intestinal epithelial cell was capable of generating a fully functional tadpole upon transplantation to an enucleated egg<a class="quote">[2]</a>. This discovery shattered the dogma that cellular differentiation could not be a bidirectional process, but the question remained whether an intact differentiated cell could be fully reprogrammed to become pluripotent again.
+
Use Anxygen PCR clean up kit for PCR clean up. In addition, restriction endonuclease digestion mix can be cleaned if the fragment to be deserted is less than 50bp.
-
</p>  
+
</p>
 +
<p>
 +
1. Add 150ul of buffer PCR-A, vortex.  
 +
</p>
 +
<p>
 +
2. Pipette the liquid to a column, wait for 1min, then centrifuge 1000xg for 1min and 12000xg 30s. </p>
 +
<p>
 +
3. Use Buffer W2(700ul for the first step and 400ul again) to wash, 12000xg for 1min.
 +
</p>
 +
<p>
 +
4. Dry the column on 65°C, warm the elutent in the meantime.
 +
</p>
 +
<p>
 +
5. Use 25-30ul preheated elutent to wash DNA, wait for 1min and centrifuge 12000xg for 1min.
 +
</p>
 +
<p>
 +
6. Use Nanodrop to measure the concentration and quality of the DNA product.
 +
</p>
 +
 
-
<img src="https://static.igem.org/mediawiki/2013/f/f9/Introduction_02.jpg" width="500" height="213" />
+
<p><strong> Digestion of restriction endonuclease
 +
</strong></p>
-
<h3>Life there and back again</h3>
 
<p>
<p>
-
In 2006, Shinya Yamanaka, a Japanese scientist who afterward shared the 2012 Nobel Prize in Physiology or Medicine with John B. Gurdon<a class="quote">[5]</a>, proved that introduction of a small set of transcription factors into a differentiated cell was sufficient to revert the cell into a pluripotent state. In his strikingly bold experiment, what Yamakana did was introducing 24 genes encoding the transcription factors which were considered as candidates to reinstate pluripotency in somatic cells to skin fibroblasts in one step. Surprisingly, he found out that a few of these cells actually generated colonies that showed a remarkable resemblance to ES cells. Then he reduced the number of genes capable of inducing such colonies one by one, and finally, four factors (Myc, Oct3/4, Sox2 and Klf4) were picked out, whose combination were identified to be sufficient to reprogram the skin fibroblasts to pluripotent stem cells<a class="quote">[3]</a>. These synthetical new stem cells were thereafter named by their Japanese father as iPSCs (induced pluripotent stem cells). A year later, using the same four factor combination from the 2006 paper (Myc, Oct4, Sox2 and Klf4), Yamanka's group successfully produced human iPS cells, whereas another laboratory, James Thomson's lab, achieved the same goal with a somewhat different transcription factors combination (Lin28, Nanog, Oct4 and Sox2)<a class="quote">[4]</a>.
+
NEB, Fermentas and Takara restriction enzymes are used. Before use, check the proper buffer for the enzyme, if the activity is low, add twice the amount but prevent the enzyme volume more than 10%, or star activity may rise.
-
</p>
+
-
<img src="https://static.igem.org/mediawiki/2013/2/2a/Introduction_03.jpg" width="500" height="198" />
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-
<h3>A technology with many possibilities
+
-
</h3>
+
-
<p>The groundbreaking work that Yamakana and his group did in 2006 truly opened up a completely new research field to this world. It was a paradigm-shifting fundamental discovery, as it was the first demonstration that an intact differentiated somatic cell could be reprogrammed to become pluripotent. iPSCs technology, a new technology with many possibilities, attracts scientists and researchers all over the world, mainly for its three unique superiorities:
+
</p>
</p>
<p>
<p>
-
<U><EM>- No strong immunological reaction:</EM></U> Since iPS cells can be cultured from the patient's own cells, it is not quite possible to lead to immunological reaction when directionally differentiated iPSCs are transplanted after in vitro culture. While in 2011 several studies argued the weak immune reaction to transgene-free iPSCs, these arguments were subsided later by other scientists and Yamanaka himself, pointing out that "the most prominent study that reported the immunogenicity of the cells examined undifferentiated iPSCs, which will never be used in cell transplantation therapy"<a class="quote">[6]</a>. Recent work by Araki and Guha used syngeneic mouse models to demonstrate that transplanted iPSC-derived embryoid bodies, skin and bone marrow tissues engraft efficiently with almost no signs of rejection<a class="quote">[6]</a>.
+
1. Set up the reaction mix:
</p>
</p>
 +
 +
<br><table width="400">
 +
 +
<tr class="first-table-line">
 +
<td> DNA      </td>
 +
<td>  1ug for insert fragment, 3ug for vector </td>
 +
</tr>
 +
<tr>
 +
<td>10x buffer      </td>
 +
<td>2ul</td>
 +
</tr>
 +
<tr>
 +
<td> Enzymes  </td>
 +
<td> 1ul each </td>
 +
</tr>
 +
<tr>
 +
<td> ddH2O  </td>
 +
<td> add up to 20ul </td>
 +
</tr>
 +
</table><br />
<p>
<p>
-
<U><EM>- "Thank god we don't need embryos anymore!":</EM></U> Simultaneously, the technology of iPSC was also hailed by many commentaries as a milestone advance that solved the ethical and political problems in stem cell research field. In the past, stem cell research relied heavily of embryonic stem cells harvested from embryos. Human embryos are not easy to come by, and many people consider research involving use of human embryonic stem cells to be ethically questionable<a class="quote">[8]</a>. With no further evidence, iPSC technology not only breaks the ethical barrier of relying on using eggs or earlier embryos for deriving stem cells, but also leads to a convenient way of obtaining patient-specific stem cells.
+
2. Carefully mix the reaction mix and put it into 37°C water bath. Control the reaction time 2. Carefully mix the reaction mix and put it into 37°C water bath. Control the reaction time .
</p>
</p>
<p>
<p>
-
<U><EM>- Wide application in different fields:</EM></U> Nowadays, scientists are making progress toward applying iPSCs in many fields. One of the applications - which has already become reality - is to use iPS cells in both analysis of disease mechanisms and investigation of potential new treatments. In the future scientists hope to be able to use iPS cells to culture cells that can be transplanted into the body and replace diseased cells. Two examples are the dopamine-producing cells in the brain that degenerate in patients with Parkinson disease, and the insulin-producing cells that die off in patients with diabetes[13]. So far, more than 100 reports published in the past three years using disease-specific iPSCs<a class="quote">[8]</a>.
+
3. Load 1.5ul mix on 1% agarose gel to have a quick run to check if the reaction is clear.
</p>
</p>
<p>
<p>
-
These advances above together made the iPSC to be the "star cell" which offers immense potential as a source for regenerative therapies. However, the intrinsic qualities of self renewal and pluripotency that make these cells so therapeutically promising are also responsible for an equally fundamental tumorigenic potential, and the the intrinsic shortcomings of the current methods of inducing pluripotency even pose more uncontrollable risks on that. High rates of tumorigenicity has become a most crucial hurdle for large-scale clinical implementation of iPSC technology.
+
4. 75°C water bath for 5min to stop the reaction.
</p>
</p>
-
<h2>Challenges</h2>
+
 
-
<h3>Tumorigenicity as a clinical hurdle </h3>
+
<p><strong> Gel Extraction
 +
</strong></p>
 +
 
<p>
<p>
-
The risks of iPSC tumorigenicity have been widely concerned over the past several years. In the study in 2009, Yamakana tested 55 mice transplanted with SNS (secondary neurospheres) from 11 TTP-iPS cell clones, 46 mice among them died or became weak within 9 weeks after transplantation because of tumors. Whereas in contrast group, the number of the tumor-showing mice transplanted with SNS derived from the ES cell clones was only 3 among 34[11]. This result indicated that the mice transplanted with iPSCs have a much higher rate of tumorigenicity than with ESCs.  
+
Use Omega Gel Extraction Kit to do gel extraction.
</p>
</p>
<p>
<p>
-
<U><EM>- The "fantastic four"are double-edged swords:</EM></U> In the initial study in 2006, Yamanaka has pointed out that among the four transcriptional factors he used, two (Oct-4 and Myc) were oncogenes<a class="quote">[3]</a>. This led to the result that the iPSCs with the four exogenetic oncogenes were more prone to tumorigenesis. However, subsequent studies successively substantiated that both the Oct-4 and Myc are not essentially required for cell self-renewal. For example, in making human iPS cells, Yu et al. used a different set of four factors (Oct-4, Sox-2, Nanog, and Lin-28) to induce pluripotent stem cells from human somatic cells. These results began to raise the scientists' hopes to find the better combination of the cocktails to induce the safer iPSC. However, it turned out to be wishful thinking.  
+
1. Cut the gel slice and weigh, 1ug for 1ul equal volume.  
</p>
</p>
<p>
<p>
-
Scientists figure out that whatever the combination is, almost every inducing/reprogramming factors remaining in the cocktail are oncogenes by definition. Their over-expression has be associated with some forms of cancer. Of particular importance is the MYC transcription factor, which has emerged as one of the fundamental genes shared by iPSCs and cancer. Ectopic activation of OCT4 in somatic cells, induces dysplastic development and features of malignancy. NANOG has a role in the self renewal of CD24+ cancer stem cells in hepatocellular carcinoma. SOX2 has been shown to drive cancer-cell survival and oncogenic fate in several cancer types, including squamous cell carcinomas of the lung and esophagus. Klf-4 has been reported to promote DNA repair checkpoint uncoupling and cellular proliferation in breast cancers by p53 suppression<a class="quote">[7]</a>.
+
2. Add 1x sample volume of Binding Buffer(XP2), add 1x sample volume of isopropanol if DNA is less than 500bp. Add 5ul of 5M pH5.2 NaAc when necessary.(When the color of liquid is orange or red, add it until it turns light yellow)
</p>
</p>
<p>
<p>
-
<U><EM>- Risks from delivery methods:</EM></U> Compared to ESCs, iPSCs are exposed to a number of factors that could promote oncogenic transformation, such as genomic insertion of reprogramming vectors, over expression of oncogenic transcription factors and a global hypomethylation resembling that seen in cancers<a class="quote">[7]</a>.  
+
3. Incubate the mixture in 65°C for 7min or until totally dissolved. Vortex every 2-3min helps.
</p>
</p>
<p>
<p>
-
Methods of diminishing the tumorigenic transformation of iPSCs have mainly focused on a variety of gene delivery vectors that minimize genomic disruption. These strategies can be generally divided into two categories: integrating vectors that can be excised from the host genome and non-integrating vectors. However, all methods can not escape their shortcomings of low transduction efficiency, or even bring about new risks. Here is a table which concludes the oncogenic risks associated with methods of inducing pluripotency in somatic cells<a class="quote">[7]</a>.
+
4. Put it in room temperature to let it cool down to some extent(control the temperature before the liquid become solid). Load it into a column(for more than 700ul, load it in several times). Centrifuge 1000xg for 1min then 10000xg for 30s.
 +
</p>
 +
<p>
 +
5. Add 300ul Binding Buffer to wash the column, 10000xg 1min.
 +
</p>
 +
<p>
 +
6. Wash with buffer SPW, add 700ul, wait 2-3min and centrifuge 10000xg. Repeat 1 time.
 +
</p>
 +
<p>
 +
7. Discard the liquid and centrifuge 10000xg 2-3min to dry.
 +
</p>
 +
<p>
 +
8. 65°C dry heat for elutent and column for totally dry. Wash it by 30-50ul elutent, centrifuge for 1min.
 +
</p>
 +
<p>
 +
9. Use Nanodrop to measure the concentration and quality of the DNA product.
</p>
</p>
-
<table width="536">
+
 
-
<caption>Table 1 Oncogenic risks associated with methods of inducing pluripotency in somatic cells</caption>
+
-
<tr>
+
-
<th scope="col">Method of induction</th>
+
-
<th scope="col">Strengths</th>
+
-
<th scope="col">Weaknesses</th>
+
-
</tr>
+
-
<tr class="first-table-line">
+
-
<td>Lentiviral vector</td>
+
-
<td>Robust reprogramming efficiency</td>
+
-
<td>Genomic integration, reactivation of integrated transgenes</td>
+
-
</tr>
+
-
<tr>
+
-
<td>Cre recombinase</td>
+
-
<td>Little genomic disruption</td>
+
-
<td>Low transduction efficiency, integration of LoxP sites into host genome</td>
+
-
</tr>
+
-
<tr>
+
-
<td>PiggyBac transposition</td>
+
-
<td>Minimal risk of genomic disruption</td>
+
-
<td>Low transduction efficiency, risk of uncontrolled rounds of excision and integration</td>
+
-
</tr>
+
-
<tr>
+
-
<td>Adenoviral vector </td>
+
-
<td>Low risk of genomic integration</td>
+
-
<td>Low transduction efficiency, limited transgene expression</td>
+
-
</tr>
+
-
<tr>
+
-
<td>Plasmid transfection</td>
+
-
<td>Minimal risk of genomic disruption</td>
+
-
<td>Very low transduction efficiency typically requires use of oncogenes such as the SV40LT antigen for successful induction of pluripotency</td>
+
-
</tr>
+
-
<tr>
+
-
<td>Minicircle</td>
+
-
<td>Minimal risk of genomic disruption</td>
+
-
<td>Low transduction efficiency</td>
+
-
</tr>
+
-
<tr>
+
-
<td>Sendai virus</td>
+
-
<td>Minimal risk of genomic integration ,relatively high transduction efficiency</td>
+
-
<td>Risk of continuous replication of viral vector in cytoplasm, leading to aberrant silencing of pluripotency transgenes</td>
+
-
</tr>
+
-
<tr>
+
-
<td>Synthetic mRNA</td>
+
-
<td>No risk of genomic integration, ability to control transgene expression</td>
+
-
<td>Variable transduction efficiencies, high technical expertise required</td>
+
-
</tr>
+
-
<tr>
+
-
<td>Protein transduction</td>
+
-
<td>No risk of genomic integration, ability to control transgene expression</td>
+
-
<td>Very low transduction efficiency, labor intensive</td>
+
-
</tr>
+
-
<tr>
+
-
<td>microRNA transfection</td>
+
-
<td>No risk of genomic integration</td>
+
-
<td>Low reprogramming efficiency</td>
+
-
</tr>
+
-
<tr>
+
-
<td>Small molecules</td>
+
-
<td>No risk of genomic integration</td>
+
-
<td>Variable off-target effects</td>
+
-
</tr>
+
-
</table>
+
<p>
<p>
-
From the table above, we can generally realize that these promising strategies for teratoma prevention mostly improve the safety of iPSC generation at the expense of efficiency, which is still another remaining challenge in iPSC technology.  
+
Use Fermentas T4 ligase for ligation.
</p>
</p>
 +
<br><table width="400">
 +
 +
<tr class="first-table-line">
 +
<td> Vector(about 5000bp)  </td>
 +
<td> 40ug </td>
 +
</tr>
 +
<tr>
 +
<td> Insert fragment </td>
 +
<td>  5x chemical amount of vector </td>
 +
</tr>
 +
<tr>
 +
<td> T4 ligase buffer </td>
 +
<td>  3ul </td>
 +
</tr>
 +
<tr>
 +
<td> T4 ligase  </td>
 +
<td>0.3-0.8ul </td>
 +
</tr>
 +
<tr>
 +
<td> ddH2O </td>
 +
<td> add up to 30ul
 +
</td>
 +
</tr>
 +
</table><br />
 +
<p>
<p>
-
In summary, it wouldn't be more perfect if scientists found a method which guaranteed both the safety and efficiency in iPSCs technology. Just as Andrew S Lee wrote on Nature Medicine, "Ideally, the most stringent safety regimes would utilize a flexible, combinatorial approach that may require tailoring for specific PSC lines or graft types. Should these techniques fail to adequately remove enough residual PSCs, retrospective tumor treatments may also be used, including oncologic chemotherapy, radiation and surgery or the incorporation of suicide ablation genes<a class="quote">[7]</a>." Admittedly, just as the saying goes, "You can't have your cake and eat it!" With our knowledge of iPSC biology, it is not likable that such a perfect regime would emerge in several coming years. However, as a group of young pre-scientists like us, maybe, it is our time to look into this problem and try to figure it out in another way.
+
For overnight ligation, use 0.3ul ligase, and for ligation that only proceeds in 2 hour, use 0.8ul ligase. The reaction temperature is room temperature, or a little bit lower than room temperature.(16°C)
</p>
</p>
-
<DIV id="references">
+
<p><strong> Transformation</strong></p>
-
<h2>References</h2>
+
 
-
<p><a class="references">[1]</a>The strategy of genes. CH Waddington& H Kacser. -1957</p>
+
<p>
-
<p><a class="references">[2]</a>Gurdon JB (1962). Developmental Capacity of Nuclei Taken From IntestinalEpithelium Cells of Feeding Tadpoles. J Embryol Exp Morph 10: 622‐640.</p>
+
1. Add 200ul TCM to whole reaction mix(for ligation mix or digestion mix), put on ice for 3-5min. This step is optional when plasmid is transformed.
-
<p><a class="references">[3]</a>Takahashi, K. & Yamanaka, Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors. S. Cell 126, 663–676 (2006).</p>
+
</p>
-
<p><a class="references">[4]</a>James A. Thomson et al, Induced Pluripotent Stem Cell Lines Derived from Human Somatic Cells. Science 21 December 2007: Vol. 318 no. 5858 pp. 1917-1920 </p>
+
<p>
-
<p><a class="references">[5]</a>Scientific Background: Mature cells can be reprogrammed to become pluripotent. Nobelprize.org. Nobel Media 2012  </p>
+
2. Add 50ul chemical competent cells(Top10)and mix gently. Incubate on ice for 30 min
-
<p><a class="references">[6]</a>Shinya Yamanaka, Induced Pluripotent Stem Cells: Past, Present, and Future. Cell Stem Cell 10, June 14, 2012</p>
+
</p>
-
<p><a class="references">[7]</a>Andrew S Lee et al. Tumorigenicity as a clinical hurdle for pluripotent stem cell therapies. Nature Medicine, valume 19, august 2013;</p>
+
<p>
-
<p><a class="references">[8]</a>SHI V. LIU, iPS Cells: A More Critical Review. Stem cell development. 17:391–397 (2008)</p>
+
3. Heat-shock in a 42℃ bath for 60 sec. Put it on ice for 3min.
-
<p><a class="references">[9]</a>Tetsuya Ishii,1,* Renee A. Reijo Pera,2 and Henry T. Greely3, Ethical and Legal Issues Arising in Research on Inducing Human Germ Cells from Pluripotent Stem Cells. Cell Stem Cell 13, August 1, 2013</p>
+
</p>
-
<p><a class="references">[10]</a>Martin F Pera & Kouichi Hasegawa, Simpler and safer cell reprogramming, Nature biotechnology, volume 26, january 2008.</p>
+
<p>
-
<p><a class="references">[11]</a>Shinya Yamanaka et al. Variation in the safety of induced pluripotent stem cell lines, Natue biothchnology, volume 27, august 2009.</p>
+
4. Add 0.5 ml LB/SOC(antibiotic free) medium and incubate at 37.0C for 30-60 min
-
<p><a class="references">[12]</a>Shinya Yamanaka et al. Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors, Cell, 30 November 2007, Pages 861–872</p>
+
</p>
-
<p><a class="references">[13]</a>Gunnar Hargusa et al. Differentiated Parkinson patient-derived induced pluripotent stem cells grow in the adult rodent brain and reduce motor asymmetry in Parkinsonian rats, PNAS, September 7, 2010</p>  
+
<p>
 +
5. Spin at 8,000*g for 3min. Decant most of LB medium but leave –100ul behind to resuspend the bug.
 +
</p>
 +
<p>
 +
6. Plate all of the bug suspensions onto one LB(Amp+) plate. Mark the name of the plate and put it into 37°C incubator for 14-18h.
 +
</p>
 +
 
 +
<p><strong>
 +
Colony PCR
 +
</strong></p>
 +
 
 +
 
 +
<p>
 +
Just like PCR protocol, but use takara rTaq enzyme.
 +
</p>
 +
<p><strong>
 +
Plasmid Extraction
 +
</strong></p>
 +
<p>
 +
Use Omega Endo-free kit for the plasmids for cell.
 +
</p>
 +
<p>
 +
Use Anxygen Plasmid miniprep kit for plasmids for molecular cloning.
 +
</p>
 +
<p>
 +
Follow the protocals provided in the kit. But a 65°C elutent can cause higher efficiency.
 +
</p>
 +
 
 +
 
 +
<h2>
 +
Cellular tests
 +
</h2>
 +
 
 +
 
 +
 
 +
 
 +
<p>
 +
In the process of cellular tests, transient transfection, virus package, selecting stable cell line are included.  
 +
</p>
 +
<p>
 +
<strong> Transient transfection
 +
</strong>
 +
</p>
 +
<p>
 +
1. Seed cells a night before.
 +
</p>
 +
<p>
 +
2. Refresh medium right before transduction. Cell density should be around 60% at the time of transduction.
 +
</p>
 +
<p>
 +
3. For a single well in a 12-well plate, dilute 1.5ug plasmid DNA in 100ul opti-MEM and then add 4ul PEI.
 +
</p>
 +
<p>
 +
4. Gently mix and then incubate for 20min.
 +
</p>
 +
<p>
 +
5. Add the mixture to cells and refresh after 6-8h.
 +
</p>
 +
<p>
 +
6. 1:1000 doxycycline is used to induce the Tet system.
 +
</p>
 +
 
 +
<p>
 +
<strong>
 +
Virus packaging
 +
</strong>
 +
</p>
 +
 
 +
 
 +
<p>
 +
1. seed cells a night before.
 +
</p>
 +
<p>
 +
2. refresh medium right before transduction. Cell density should be 40% at the time of transduction.
 +
</p>
 +
<p>
 +
3. For a single well in a 6-well plate, dilute totally 3ug plasmids in 100 ul CaCl2,gently mix.We use PCGP/VSVG to package retrovirus and PSPAX2/MD2G to package lentivirus.  
 +
</p>
 +
 
 +
<p>
 +
4. Add the mixture to 100ul BBS carefully and incubate for 20min.
 +
</p>
 +
<p>
 +
5. Add the mixture to the cells and refresh after 6-8h.
 +
</p>
 +
<p>
 +
6. collect virus after 48h and store in -80°C.
 +
</p>
 +
<p>
 +
7. For condensed virus, use 10cm culture medium and proportionally increase the reagents as described above. Medium containing virus is sealed by mineral oil and ultracentrifugate at 70000g for 1.5h. Collect the last 1ml medium and resuspend for further experiment.
 +
</p>
 +
<p>
 +
<strong> Selecting stable cell line
 +
 
 +
</strong>
 +
</p>
 +
 
 +
<p>
 +
1. Seed cells a night before.
 +
</p>
 +
<p>
 +
2. Refresh medium first.Add polybrene 8ul/ml final concentration. Gently add 100ul virus to every well in a 24-well plate. .
 +
</p>
 +
<p>
 +
3. Add 1:1000 blastisidin or 1:2000 2mg/ml puromycin to culture medium.
 +
</p>
 +
<p>
 +
4. Three later, refresh culture medium and passage cells into new plates.
 +
</p>
 +
<p>
 +
<strong> Basic cell culturing</strong>
 +
</p>
 +
<p>
 +
Medium: 10% FBS+ 88% DMEM (high-glucose)+1% P/S (5000 mg/ml penicillin and 5000 mg/ml streptomycin)+1% L-glutamine (200 mM). Cells were passaged with 0.25%Trypsin.
 +
</p>
 +
<p>
 +
<p>
 +
<p><strong>
 +
hiPSC culturing
 +
</strong></p>
 +
<p>
 +
<strong>
 +
Introduction
 +
</strong>
 +
</p>
 +
<p>
 +
Culturing human and pluripotent stem cells requires the use of complex media and careful handling techniques. Here, we describe feeder-free and serum free way with the use of Gibco Essential 8 medium in combination with BD matrigel low growth factor to maintain and propagate high quality hIPSCs.</P>
 +
</p>
 +
<p>The following protocol are modified from standard protocols from BD and Gibco </p>
 +
<p>
 +
 
 +
<strong>
 +
Materials required
 +
</strong>
 +
</p>
 +
<p>
 +
Essential 8 medium, consisting of DMEM/F12(AM)1:1 and Essential 8 Supplement(50*)</p>
 +
<p>
 +
0.5M EDTA, pH8.0</p><p>
 +
37°C water bath</p><p>
 +
BD matrigel low growh factor and Gibco knockout MEM</p><p>
 +
DPBS without Calcium and Magnesium</p><p>
 +
Approprate tissue culture plates and supplies</p><p>
 +
<strong> Preparing Media and Materials
 +
</strong>
 +
</p>
 +
<p>
 +
<strong> Essential 8 Medium (500 mL of complete medium)
 +
</strong>
 +
</p>
 +
<p>
 +
1. Thaw Essential 8 Supplement (50X) at 2–8°C overnight instead of at 37°C. </p><p>
 +
2.  Mix the following component into 500 mL of complete Essential 8 Medium s: </p><p>
 +
DMEM/F-12 (HAM) 1:1            490 mL </p><p>
 +
Essential 8  Supplement (50X)      10 mL </p><p>
 +
Note: Before use, warm complete medium at room temperature until it is no longer cool to the touch. Do not warm the medium at 37°C.  </p><p>
 +
<strong>0.5 mM EDTA in DPBS (50 mL)
 +
</strong>
 +
</p><p>
 +
1. To prepare 50 mL of 0.5 mM EDTA in DPBS Mix the following components in a 50-mL conical tube in a biological safety cabinet to prepare 50 mL of 0.5 mM EDTA in DPBS: </p><p>
 +
DPBS without Calcium and Magnesium  50 mL </p><p>
 +
0.5 M EDTA                        50 µL </p><p>
 +
2.  Filter sterilize the solution. The solution can be stored at room temperature for up to six months. </p><p>
 +
<strong> Coating Culture Vessels with BD matrigel with low growth factor
 +
</strong>
 +
</p><p>
 +
BD Matrigel low growth factor Matrix should be aliquoted and frozen. Consult the Certificate of Analysis supplied with the BD Matrigel™ for the recommended aliquot size (“Dilution Factor”) to make up 25 mL of diluted matrix. Make sure to always keep BD Matrigel on ice when thawing and handling to prevent it from gelling. </p><p>
 +
Note: Use tissue culture-treated cultureware (e.g. 6-well plates, BD Catalog #353046). </p><p>
 +
1.  Thaw one aliquot of BD Matrigel on ice. </p><p>
 +
2.  Dispense 25 mL of cold dilution medium (DMEM/F-12; Catalog #36254) into a 50 mL conical tube and keep on ice. </p><p>
 +
3.  Add thawed BD Matrigel to the cold dilution medium (in the 50 mL tube) and mix well. The vial may be washed with cold medium if desired. </p><p>
 +
4.  Immediately use the diluted BD Matrigel solution to coat tissue culture-treated cultureware. for recommended coating volumes.  </p><p>
 +
<strong> Passaging iPSCs
 +
</strong>
 +
</p><p>
 +
In general, split cells when one of the following occurs: </p><p>
 +
•  PSC colonies are becoming too dense or too large.  </p><p>
 +
•  PSC colonies are showing increased differentiation. </p><p>
 +
•  The colonies cover approximately 85% of the surface area of the culture vessel, usually every 4 days. Even if the colonies are sparse and small, it is important to split the culture every 4 to 5 days. </p><p>
 +
The split ratio can vary, though it is generally between 1:2 and 1:4 for early passages and between 1:3 and </p><p>
 +
1:12 for established cultures. Occasionally, cells will grow at a different rate and the split ratio will need to be adjusted. </p><p>
 +
•  A general rule is to observe the last split ratio and adjust the ratio according to the appearance of the PSC colonies. If the cells look healthy and colonies have enough space, split using the same ratio. If they are overly dense and crowding, increase the ratio. If the cells are sparse, decrease the ratio.  </p><p>
 +
<strong> Passaging PSC Colonies using EDTA
 +
</strong>
 +
</p><p>
 +
Note: Newly derived PSC lines may contain a fair amount of differentiation through passage 4. It is not necessary to remove differentiated material prior to passaging. By propagating/splitting the cells the overall culture health should improve throughout the early passages. </p><p>
 +
1.  Prior to starting, equilibrate your matrigel coated dishes to room temperature in the hood (this takes about one hour). Pre-warm the required volume of Essential 8 Medium at room temperature until it is no longer cool to the touch. </p><p>
 +
Note: Do not warm medium in a 37°C water bath. </p><p>
 +
2. Aspirate the spent medium from the vessel containing PSCs with a Pasteur pipette, and rinse the vessel twice with Dulbecco’s PBS (DPBS) without Calcium and Magnesium. Refer to Table 2 for the recommended volumes. </p><p>
 +
3.  Add 0.5 mM EDTA in DPBS to the vessel containing PSCs. Adjust the volume of EDTA for various dish sizes (refer to Table 2). Swirl the dish to coat the entire cell surface. </p><p>
 +
4.  Incubate the vessel at room temperature for 5–8 minutes or 37°C for 4–5 minutes. When the cells start to separate and round up, and the colonies will appear to have holes in them when viewed under a microscope, they are ready to be removed from the vessel. </p><p>
 +
Note: In larger vessels or with certain cell lines, this may take longer than 5 minutes. </p><p>
 +
5.  Aspirate the EDTA solution with a Pasteur pipette.  </p><p>
 +
6.  Add pre-warmed complete Essential 8 Medium to the dish. </p><p>
 +
7. Remove the cells from the well(s) by gently squirting medium and pipetting the colonies up using a 5-mL glass pipette. Avoid creating bubbles. Collect cells in a 15-mL conical tube. </p><p>
 +
For 6-well plate, 1ml/well 0.5nM EDTA in DPBS is used to digest cells and 2ml/well complete Essential 8 Medium is used for culturing. </p><p>
 +
Note: Do not scrape the cells from the dish. There may be obvious patches of cells that were not dislodged and left behind. Do not attempt to recover them through scraping. </p><p>
 +
Note: Little or no extra pipetting is required to break up cell clumps after EDTA treatment. </p><p>
 +
Note: Depending upon the cell line, work with no more than one to three wells at a time, and work quickly to remove cells after adding Essential 8 Medium to the well(s). The initial effect of the EDTA will be neutralized quickly by the medium. Some lines re-adhere very rapidly after medium addition, and must be removed 1 well at a time. Others are slower to re-attach, and may be removed 3 wells at a time. </p><p>
 +
8.  Aspirate residual matrigel solution from the pre-coated dish. </p><p>
 +
9.  Add an appropriate volume of pre-warmed Essential 8 Medium to each well of a coated 6-well plate so that each well contains 2 mL medium after the cell suspension has been added. Refer to Table 2 for volumes for other culture vessels. </p><p>
 +
10.  Move the vessel in several quick figure eight motions to disperse cells across the surface of the vessels. </p><p>
 +
11. Place dish gently into the 37°C, 5% CO 2 incubator and incubate the cells overnight. </p><p>
 +
12.  Feed PSC cells beginning the second day after splitting. Replace spent medium daily. </p><p>
 +
Note: It is normal to see cell debris and small colonies after passage.  </p><p>
 +
 
 +
<p>
</DIV>
</DIV>

Latest revision as of 01:29, 29 October 2013

ipsc

Notebookt/Methods

Methods

Introduction

Our experiments can be divided into two parts, one is the design and construction of genes and vectors, the other is test of parts and circuits with cells, including hepatoma cells, liver cells and iPSCs. So our protocol can be clearly be divided into these two parts, one for molecular operation, the other for cellular tests. Besides, quantitive measurement, like qPCR and western-blotting, is added in the molecular operation parts because it does not need cell operation.

Molecular Operation

< In the process of constructing circuit on vectors, PCR, PCR clean up, digestion of restriction endonuclease, gel extraction, ligation, transformation, colony PCR and plasmid extraction is implemented. Besides, for quantitive measurement, qPCR and western-blotting is used..

PCR

1. Prepare the reaction mix:


5x Fastpfu buffer 10ul
2.5mM dNTPs 5ul
Forward primer 0.8ul
Reverse primer 0.8ul
Template DNA 40-50ng
FastPfu 1ul
ddH2O add to 50ul

2. Set the program of thermal cycler


(1×) 95℃ 3min
(25×) 95℃ 20s
A-5℃ 20s (Tm≈A℃)
72℃ 30s for each 1kb
(1×) 72℃ 5min
(1×) 4℃ forever

3. Run the PCR program.

4. After PCR, load ~2ul to a 1% agarose gel to have a quick run checking the production of desired fragment.

PCR clean up

Use Anxygen PCR clean up kit for PCR clean up. In addition, restriction endonuclease digestion mix can be cleaned if the fragment to be deserted is less than 50bp.

1. Add 150ul of buffer PCR-A, vortex.

2. Pipette the liquid to a column, wait for 1min, then centrifuge 1000xg for 1min and 12000xg 30s.

3. Use Buffer W2(700ul for the first step and 400ul again) to wash, 12000xg for 1min.

4. Dry the column on 65°C, warm the elutent in the meantime.

5. Use 25-30ul preheated elutent to wash DNA, wait for 1min and centrifuge 12000xg for 1min.

6. Use Nanodrop to measure the concentration and quality of the DNA product.

Digestion of restriction endonuclease

NEB, Fermentas and Takara restriction enzymes are used. Before use, check the proper buffer for the enzyme, if the activity is low, add twice the amount but prevent the enzyme volume more than 10%, or star activity may rise.

1. Set up the reaction mix:


DNA 1ug for insert fragment, 3ug for vector
10x buffer 2ul
Enzymes 1ul each
ddH2O add up to 20ul

2. Carefully mix the reaction mix and put it into 37°C water bath. Control the reaction time 2. Carefully mix the reaction mix and put it into 37°C water bath. Control the reaction time .

3. Load 1.5ul mix on 1% agarose gel to have a quick run to check if the reaction is clear.

4. 75°C water bath for 5min to stop the reaction.

Gel Extraction

Use Omega Gel Extraction Kit to do gel extraction.

1. Cut the gel slice and weigh, 1ug for 1ul equal volume.

2. Add 1x sample volume of Binding Buffer(XP2), add 1x sample volume of isopropanol if DNA is less than 500bp. Add 5ul of 5M pH5.2 NaAc when necessary.(When the color of liquid is orange or red, add it until it turns light yellow)

3. Incubate the mixture in 65°C for 7min or until totally dissolved. Vortex every 2-3min helps.

4. Put it in room temperature to let it cool down to some extent(control the temperature before the liquid become solid). Load it into a column(for more than 700ul, load it in several times). Centrifuge 1000xg for 1min then 10000xg for 30s.

5. Add 300ul Binding Buffer to wash the column, 10000xg 1min.

6. Wash with buffer SPW, add 700ul, wait 2-3min and centrifuge 10000xg. Repeat 1 time.

7. Discard the liquid and centrifuge 10000xg 2-3min to dry.

8. 65°C dry heat for elutent and column for totally dry. Wash it by 30-50ul elutent, centrifuge for 1min.

9. Use Nanodrop to measure the concentration and quality of the DNA product.

Use Fermentas T4 ligase for ligation.


Vector(about 5000bp) 40ug
Insert fragment 5x chemical amount of vector
T4 ligase buffer 3ul
T4 ligase 0.3-0.8ul
ddH2O add up to 30ul

For overnight ligation, use 0.3ul ligase, and for ligation that only proceeds in 2 hour, use 0.8ul ligase. The reaction temperature is room temperature, or a little bit lower than room temperature.(16°C)

Transformation

1. Add 200ul TCM to whole reaction mix(for ligation mix or digestion mix), put on ice for 3-5min. This step is optional when plasmid is transformed.

2. Add 50ul chemical competent cells(Top10)and mix gently. Incubate on ice for 30 min

3. Heat-shock in a 42℃ bath for 60 sec. Put it on ice for 3min.

4. Add 0.5 ml LB/SOC(antibiotic free) medium and incubate at 37.0C for 30-60 min

5. Spin at 8,000*g for 3min. Decant most of LB medium but leave –100ul behind to resuspend the bug.

6. Plate all of the bug suspensions onto one LB(Amp+) plate. Mark the name of the plate and put it into 37°C incubator for 14-18h.

Colony PCR

Just like PCR protocol, but use takara rTaq enzyme.

Plasmid Extraction

Use Omega Endo-free kit for the plasmids for cell.

Use Anxygen Plasmid miniprep kit for plasmids for molecular cloning.

Follow the protocals provided in the kit. But a 65°C elutent can cause higher efficiency.

Cellular tests

In the process of cellular tests, transient transfection, virus package, selecting stable cell line are included.

Transient transfection

1. Seed cells a night before.

2. Refresh medium right before transduction. Cell density should be around 60% at the time of transduction.

3. For a single well in a 12-well plate, dilute 1.5ug plasmid DNA in 100ul opti-MEM and then add 4ul PEI.

4. Gently mix and then incubate for 20min.

5. Add the mixture to cells and refresh after 6-8h.

6. 1:1000 doxycycline is used to induce the Tet system.

Virus packaging

1. seed cells a night before.

2. refresh medium right before transduction. Cell density should be 40% at the time of transduction.

3. For a single well in a 6-well plate, dilute totally 3ug plasmids in 100 ul CaCl2,gently mix.We use PCGP/VSVG to package retrovirus and PSPAX2/MD2G to package lentivirus.

4. Add the mixture to 100ul BBS carefully and incubate for 20min.

5. Add the mixture to the cells and refresh after 6-8h.

6. collect virus after 48h and store in -80°C.

7. For condensed virus, use 10cm culture medium and proportionally increase the reagents as described above. Medium containing virus is sealed by mineral oil and ultracentrifugate at 70000g for 1.5h. Collect the last 1ml medium and resuspend for further experiment.

Selecting stable cell line

1. Seed cells a night before.

2. Refresh medium first.Add polybrene 8ul/ml final concentration. Gently add 100ul virus to every well in a 24-well plate. .

3. Add 1:1000 blastisidin or 1:2000 2mg/ml puromycin to culture medium.

4. Three later, refresh culture medium and passage cells into new plates.

Basic cell culturing

Medium: 10% FBS+ 88% DMEM (high-glucose)+1% P/S (5000 mg/ml penicillin and 5000 mg/ml streptomycin)+1% L-glutamine (200 mM). Cells were passaged with 0.25%Trypsin.

hiPSC culturing

Introduction

Culturing human and pluripotent stem cells requires the use of complex media and careful handling techniques. Here, we describe feeder-free and serum free way with the use of Gibco Essential 8 medium in combination with BD matrigel low growth factor to maintain and propagate high quality hIPSCs.

The following protocol are modified from standard protocols from BD and Gibco

Materials required

Essential 8 medium, consisting of DMEM/F12(AM)1:1 and Essential 8 Supplement(50*)

0.5M EDTA, pH8.0

37°C water bath

BD matrigel low growh factor and Gibco knockout MEM

DPBS without Calcium and Magnesium

Approprate tissue culture plates and supplies

Preparing Media and Materials

Essential 8 Medium (500 mL of complete medium)

1. Thaw Essential 8 Supplement (50X) at 2–8°C overnight instead of at 37°C.

2. Mix the following component into 500 mL of complete Essential 8 Medium s:

DMEM/F-12 (HAM) 1:1 490 mL

Essential 8 Supplement (50X) 10 mL

Note: Before use, warm complete medium at room temperature until it is no longer cool to the touch. Do not warm the medium at 37°C.

0.5 mM EDTA in DPBS (50 mL)

1. To prepare 50 mL of 0.5 mM EDTA in DPBS Mix the following components in a 50-mL conical tube in a biological safety cabinet to prepare 50 mL of 0.5 mM EDTA in DPBS:

DPBS without Calcium and Magnesium 50 mL

0.5 M EDTA 50 µL

2. Filter sterilize the solution. The solution can be stored at room temperature for up to six months.

Coating Culture Vessels with BD matrigel with low growth factor

BD Matrigel low growth factor Matrix should be aliquoted and frozen. Consult the Certificate of Analysis supplied with the BD Matrigel™ for the recommended aliquot size (“Dilution Factor”) to make up 25 mL of diluted matrix. Make sure to always keep BD Matrigel on ice when thawing and handling to prevent it from gelling.

Note: Use tissue culture-treated cultureware (e.g. 6-well plates, BD Catalog #353046).

1. Thaw one aliquot of BD Matrigel on ice.

2. Dispense 25 mL of cold dilution medium (DMEM/F-12; Catalog #36254) into a 50 mL conical tube and keep on ice.

3. Add thawed BD Matrigel to the cold dilution medium (in the 50 mL tube) and mix well. The vial may be washed with cold medium if desired.

4. Immediately use the diluted BD Matrigel solution to coat tissue culture-treated cultureware. for recommended coating volumes.

Passaging iPSCs

In general, split cells when one of the following occurs:

• PSC colonies are becoming too dense or too large.

• PSC colonies are showing increased differentiation.

• The colonies cover approximately 85% of the surface area of the culture vessel, usually every 4 days. Even if the colonies are sparse and small, it is important to split the culture every 4 to 5 days.

The split ratio can vary, though it is generally between 1:2 and 1:4 for early passages and between 1:3 and

1:12 for established cultures. Occasionally, cells will grow at a different rate and the split ratio will need to be adjusted.

• A general rule is to observe the last split ratio and adjust the ratio according to the appearance of the PSC colonies. If the cells look healthy and colonies have enough space, split using the same ratio. If they are overly dense and crowding, increase the ratio. If the cells are sparse, decrease the ratio.

Passaging PSC Colonies using EDTA

Note: Newly derived PSC lines may contain a fair amount of differentiation through passage 4. It is not necessary to remove differentiated material prior to passaging. By propagating/splitting the cells the overall culture health should improve throughout the early passages.

1. Prior to starting, equilibrate your matrigel coated dishes to room temperature in the hood (this takes about one hour). Pre-warm the required volume of Essential 8 Medium at room temperature until it is no longer cool to the touch.

Note: Do not warm medium in a 37°C water bath.

2. Aspirate the spent medium from the vessel containing PSCs with a Pasteur pipette, and rinse the vessel twice with Dulbecco’s PBS (DPBS) without Calcium and Magnesium. Refer to Table 2 for the recommended volumes.

3. Add 0.5 mM EDTA in DPBS to the vessel containing PSCs. Adjust the volume of EDTA for various dish sizes (refer to Table 2). Swirl the dish to coat the entire cell surface.

4. Incubate the vessel at room temperature for 5–8 minutes or 37°C for 4–5 minutes. When the cells start to separate and round up, and the colonies will appear to have holes in them when viewed under a microscope, they are ready to be removed from the vessel.

Note: In larger vessels or with certain cell lines, this may take longer than 5 minutes.

5. Aspirate the EDTA solution with a Pasteur pipette.

6. Add pre-warmed complete Essential 8 Medium to the dish.

7. Remove the cells from the well(s) by gently squirting medium and pipetting the colonies up using a 5-mL glass pipette. Avoid creating bubbles. Collect cells in a 15-mL conical tube.

For 6-well plate, 1ml/well 0.5nM EDTA in DPBS is used to digest cells and 2ml/well complete Essential 8 Medium is used for culturing.

Note: Do not scrape the cells from the dish. There may be obvious patches of cells that were not dislodged and left behind. Do not attempt to recover them through scraping.

Note: Little or no extra pipetting is required to break up cell clumps after EDTA treatment.

Note: Depending upon the cell line, work with no more than one to three wells at a time, and work quickly to remove cells after adding Essential 8 Medium to the well(s). The initial effect of the EDTA will be neutralized quickly by the medium. Some lines re-adhere very rapidly after medium addition, and must be removed 1 well at a time. Others are slower to re-attach, and may be removed 3 wells at a time.

8. Aspirate residual matrigel solution from the pre-coated dish.

9. Add an appropriate volume of pre-warmed Essential 8 Medium to each well of a coated 6-well plate so that each well contains 2 mL medium after the cell suspension has been added. Refer to Table 2 for volumes for other culture vessels.

10. Move the vessel in several quick figure eight motions to disperse cells across the surface of the vessels.

11. Place dish gently into the 37°C, 5% CO 2 incubator and incubate the cells overnight.

12. Feed PSC cells beginning the second day after splitting. Replace spent medium daily.

Note: It is normal to see cell debris and small colonies after passage.

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