This work is supported in part by funds provided by The University of North Carolina at Charlotte, Wachovia foundation fund for faculty excellence, and a grant from NIGMS (R15GM101571).

1. LSE Preparation
<ol>
 <li>Female frogs (Xenopus laevis) are injected twice for egg collection. The first injection (priming) is 100 U PMSG (Pregnant Mare Serum Gonadotropin) per frog. Frogs must be primed at least two days before inducing egg laying and primed frogs are usable for up to two weeks. To prime frogs, inject PMSG subcutaneously in the dorsal lymph sacs using a 3 ml syringe and 27 G needle.</li>
 <li>To induce egg laying, inject 500 U hCG (human Chorionic Gonadotrophin) per primed frog subcutaneously in the dorsal lymph sacs using a 27 G needle. Incubate injected frogs in separate buckets containing 2 liters of 1x Marc's modified Ringer's solution (MMR). Allow frogs 14-20 hr to lay eggs before collection.</li>
 <li>Remove the frogs from the buckets and pour off the MMR solution until around 100 ml is left. Obtain eggs from the buckets by transferring eggs to a 250 ml beaker.</li>
 <li>Dejelly eggs by adding 100 ml of 2% cysteine (adjust pH to 7.8 with 10 M KOH). Gently swirl the eggs with an inverted glass Pasteur pipet (0.7 cm in diameter) approximately every 30 sec. Decant and replace with fresh cysteine twice during incubation. The dejellying process is complete in about 5-15 min when the eggs form a more condensed layer at the bottom of the beaker.</li>
 <li>Discard the cysteine solution and wash eggs three times with 0.25x MMR. Swirl the eggs in the solution by a glass pipet. "Bad" eggs are determined by simple visual inspection and are "puffy" white in appearance. The "bad" eggs will accumulate at the center of the beaker after swirling. Remove "bad" eggs by a Pasteur pipet.</li>
 <li>Wash eggs with Egg Lysis Buffer (ELB) three times. Remove any additional "bad" eggs by a Pasteur pipet. Pour eggs into a 14 ml Falcon tube.</li>
 <li>Spin the Falcon tube for 55 sec at 188 x g (1,100 rpm) using the CL2 IEC clinical table-top centrifuge with a swinging bucket rotor to compact the eggs. Remove excess buffer from the top of the egg layer. Add 0.5 &mu;l of Aprotinin/Leupeptin stock and 0.5 &mu;l of Cytochalasin B stock per ml of compacted eggs.</li>
 <li>Centrifuge eggs at 16,500 x g (10,000 rpm) for 15 min at 4 &deg;C using the Sorvall RC6 plus superspeed centrifuge with HB6 swinging bucket rotor. After centrifugation, eggs are fractionated into three layers in the tube: lipid, extract, and yolk/pigment from top to bottom, respectively. Puncture the side of the Falcon tube in the lower portion of the middle extract layer with a 21 G needle. Carefully remove the needle as the puncturing needle may be obstructed by plastic. Insert a fresh 21 G needle attached to a 1 ml syringe into the puncture site to collect the extract. Slowly aspirate the extract into syringe to avoid air bubbles and contamination with the lipid and yolk/pigment layers.</li>
 <li>Place the extracts into a chilled 1.5 ml microcentrifuge tube. For each milliliter of egg extract, add the following chemical stock solutions to the indicated final concentration (shown in parentheses): (1) 10 &mu;l Cycloheximide (100 &mu;g/ml); (2) 1 &mu;l Aprotinin/Leupeptin (10 &mu;g/ml each); (3) 1 &mu;l Cytochalasin B (5 &mu;g/ml); (4) 1 &mu;l Dithiothreitol (1 mM); and (5) 0.33 &mu;l Nocodazole (3 &mu;g/ml). Invert the egg extract at least 10 times gently. This is the LSE, which must be made fresh and used within 4 hr. The quality of LSE is compromised after 4 hr or freeze-thaw.</li>
</ol>
2. Treatment of Sperm Chromatin with DNA Damaging Approaches
<ol>
 <li>Prepare normal sperm chromatin according to the method described previously 13.</li>
 <li>Prepare MMS-treated sperm chromatin
 <ol>
 <li>Resuspend normal sperm chromatin in 0.5 ml Buffer X.</li>
 <li>Add ~ 5.5 &mu;l of MMS (9.1 M in stock) to 500 &mu;l resuspended chromatin to a final concentration of 100 mM.</li>
 <li>Incubate sperm chromatin in microcentrifuge tube at room temperature for 30 min with rotation.</li>
 <li>Spin the microcentrifuge tube at 686 x g (2,100 rpm) for 10 min at room temperature using the IEC CL2 clinical centrifuge with swinging bucket rotor and tube adaptors.</li>
 <li>Discard supernatant and resuspend the pellet in 0.5 ml of Buffer X plus BSA (3%), DTT (0.1 mM) and Aprotinin/Leupeptin (10 &mu;g/ml each).</li>
 <li>Repeat steps (4) and (5) twice to wash the sperm chromatin.</li>
 <li>Determine the concentration of sperm chromatin with a hemocytometer and dilute to 100,000 sperm/&mu;l. Save 5 &mu;l aliquots in -80 &deg;C freezer for further use.</li>
 </ol>
 </li>
 <li>Prepare UV-treated sperm chromatin
 <ol>
 <li>Add desired amount (e.g. 10 &mu;l) of normal sperm chromatin on the surface of a piece of Parafilm.</li>
 <li>Place the Parafilm into a UV crosslinker. Set desired energy parameter, depending on your experiment, and start to damage the sperm chromatin via UV light. For example, it takes approximately 21 sec to reach 1,000 J/m2.</li>
 <li>After UV irradiation, UV-treated sperm chromatin should be added to egg extracts immediately.</li>
 </ol>
 </li>
</ol>
3. Preparation of a DNA Damage-mimicking Structure (AT70)
<ol>
 <li>Dissolve two synthetic oligonucleotides poly-(dA)70 (designated as A70) and poly-(dT)70 (designated as T70) in water to a concentration of 2 &mu;g/&mu;l, respectively.</li>
 <li>Add 100 &mu;l of A70 and 100 &mu;l of T70 to one 1.5 ml microcentrifuge tube. Boil mixed synthetic oligo solution for 5 min at 95 &deg;C in a heatblock.</li>
 <li>Take the dry bath block out (with the tube containing AT mixture in it), and let it cool down to room temperature on a lab bench. This temperature adjustment takes around 45-60 min.</li>
 <li>The cooled mixture is AT70 with a final concentration of 2 &mu;g/&mu;l. Store 10 &mu;l of AT70 aliquots in -20 &deg;C freezer for further use.</li>
</ol>
4. Triggering the DNA Damage Checkpoint in LSE with Damaged Sperm Chromatin or a DNA Damage-mimicking Structure
<ol>
 <li>Induce DNA damage checkpoint in LSE with damaged sperm chromatin
 <ol>
 <li>Add 50 &mu;l of LSE to a 1.5 ml microcentrifuge tube and complement it with 1 &mu;l of Energy Mixture and 2 &mu;l of damaged sperm chromatin (final concentration ~4,000 sperm/&mu;l reaction).</li>
 <li>Incubate the reaction tube at room temperature for 90 min and flick the tube every 10 min.</li>
 <li>Dispense 1 &mu;l of reaction mixture onto a microscope slide supplemented with 1 &mu;l of nuclear dye solution after 30-min incubation. Place a cover slip over the reaction mixture and check for nuclei formation via fluorescence microscope. Typically, round nuclei will form after 30 min of incubation, indicating DNA replication has initiated.</li>
 <li>Take 10 &mu;l of the reaction mixture into 90 &mu;l of sample buffer. Samples are analyzed via immunoblotting using anti-Chk1 P-S344 or anti-Chk1 antibodies.</li>
 </ol>
 </li>
 <li>Induce DNA damage checkpoint in LSE with the AT70
 <ol>
 <li>Add 50 &mu;l of LSE to a 1.5 ml microcentrifuge tube supplemented with 1 &mu;l of Energy Mixture and 1.6 &mu;l of Tautomycin stock.</li>
 <li>Add 1.25 &mu;l of pre-made AT70 (2 &mu;g/&mu;l) or water (as negative control) to each reaction. Incubate the reactions at room temperature for 90 min. Flick the reaction tubes every 10 min.</li>
 <li>Add 10 &mu;l of the reaction mixture into 90 &mu;l of sample buffer. Samples are examined via immunoblotting using anti-Chk1 P-S344 or anti-Chk1 antibodies.</li>
 </ol>
 </li>
</ol>
5. Representative Results
The damaged sperm chromatin or DNA damage-mimicking structure can trigger the ATR/Chk1-mediated DNA damage checkpoint in the Xenopus egg extract system. Figure 2A shows that MMS induces Chk1 phosphorylation at Ser344 (Chk1 P-S344), which is an indicator of ATR kinase activation. Figure 2B shows that AT70, as a DNA damage-mimicking structure, also triggers Chk1 phosphorylation. Total Chk1 samples are used as loading controls in both examples.
<img alt="Figure 1" src="/files/ftp_upload/4449/4449fig1.jpg" /> 
Figure 1. A diagram of the DNA damage checkpoint signaling.
<img alt="Figure 2" src="/files/ftp_upload/4449/4449fig2.jpg" /> 
Figure 2. Chk1 phosphorylation is induced by either MMS or AT70 treatments in Xenopus egg extracts. (A) MMS-damaged sperm chromatin (MMS) or normal sperm chromatin (Con) are incubated in egg extracts for 90 min. Chk1 phosphorylation at Ser344 (Chk1 P-S344) and total Chk1 in egg extracts are examined via immunoblotting. (B) AT70 or water (Con) are added into egg extracts, respectively. Samples are also analyzed via immunoblotting as in (A).

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analyzing+the+ATR-mediated+checkpoint+using+Xenopus+egg+extracts.">Analyzing the ATR-mediated checkpoint using Xenopus egg extracts.</a> Methods. 41 (2), 222-231 (2007).</li><li>Lupardus, P. J., Byun, T., Yee, M. C., Hekmat-Nejad, M., Cimprich, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+requirement+for+replication+in+activation+of+the+ATR-dependent+DNA+damage+checkpoint.">A requirement for replication in activation of the ATR-dependent DNA damage checkpoint.</a> Genes Dev. 16 (18), 2327-2332 (2002).</li><li>Stokes, M. P., Van Hatten, R., Lindsay, H. D., Michael, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+replication+is+required+for+the+checkpoint+response+to+damaged+DNA+in+Xenopus+egg+extracts.">DNA replication is required for the checkpoint response to damaged DNA in Xenopus egg extracts.</a> J. Cell Biol. 158 (5), 863-872 (2002).</li><li>Kato, K., Strauss, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accumulation+of+an+intermediate+in+DNA+synthesis+by+HEp.2+cells+treated+with+methyl+methanesulfonate.">Accumulation of an intermediate in DNA synthesis by HEp.2 cells treated with methyl methanesulfonate.</a> Proc. Natl. Acad. Sci. U.S.A. 71 (5), 1969-1973 (1974).</li><li>Paulovich, A. G., Hartwell, L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+checkpoint+regulates+the+rate+of+progression+through+S+phase+in+S.+cerevisiae+in+response+to+DNA+damage.">A checkpoint regulates the rate of progression through S phase in S. cerevisiae in response to DNA damage.</a> Cell. 82 (5), 841-847 (1995).</li><li>Guo, Z., Kumagai, A., Wang, S. X., Dunphy, W. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Requirement+for+Atr+in+phosphorylation+of+Chk1+and+cell+cycle+regulation+in+response+to+DNA+replication+blocks+and+UV-damaged+DNA+in+Xenopus+egg+extracts.">Requirement for Atr in phosphorylation of Chk1 and cell cycle regulation in response to DNA replication blocks and UV-damaged DNA in Xenopus egg extracts.</a> Genes Dev. 14 (21), 2745-2756 (2000).</li><li>Jazayeri, A., Balestrini, A., Garner, E., Haber, J. E., Costanzo, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mre11-Rad50-Nbs1-dependent+processing+of+DNA+breaks+generates+oligonucleotides+that+stimulate+ATM+activity.">Mre11-Rad50-Nbs1-dependent processing of DNA breaks generates oligonucleotides that stimulate ATM activity.</a> EMBO. J. 27 (14), 1953-1962 (2008).</li><li>Kumagai, A., Dunphy, W. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Claspin,+a+novel+protein+required+for+the+activation+of+Chk1+during+a+DNA+replication+checkpoint+response+in+Xenopus+egg+extracts.">Claspin, a novel protein required for the activation of Chk1 during a DNA replication checkpoint response in Xenopus egg extracts.</a> Mol. Cell. 6 (4), 839-849 (2000).</li><li>Yan, S., Lindsay, H. D., Michael, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+requirement+for+Xmus101+in+ATR-mediated+phosphorylation+of+Claspin+bound+Chk1+during+checkpoint+signaling.">Direct requirement for Xmus101 in ATR-mediated phosphorylation of Claspin bound Chk1 during checkpoint signaling.</a> J. Cell Biol. 173 (2), 181-186 (2006).</li><li>Shiotani, B., Zou, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-stranded+DNA+orchestrates+an+ATM-to-ATR+switch+at+DNA+breaks.">Single-stranded DNA orchestrates an ATM-to-ATR switch at DNA breaks.</a> Mol. Cell. 33 (5), 547-558 (2009).</li><li>Tutter, A. V., Walter, J. 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No conflicts of interest declared.

There are several advantages in studying the DNA damage checkpoint using Xenopus egg extracts. The use of egg extracts provides a large quantity of cell-free extracts synchronized at interphase of the cell cycle. The egg extracts can be easily and inexpensively made. It is relatively easy to damage DNA or chromatin and to reveal a defect in the DNA damage checkpoint after immunodepleting a target protein from egg extract. Subsequently, a potential function defect can be "rescued" by addback of wild type or muta...

<table border="1">
 <tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Reagents</td>
 </tr>
 <tr>
 <td>Anti-Chk1 P-S344 antibody</td>
 <td>Cell Signaling</td>
 <td>2348L</td>
 <td></td>
 </tr>
 <tr>
 <td>Anti-Chk1 antibody</td>
 <td>Santa Cruz</td>
 <td>SC7898</td>
 <td></td>
 </tr>
 <tr>
 <td>Aprotinin</td>
 <td>MP Biomedicals</td>
 <td>0219115880</td>
 <td></td>
 </tr>
 <tr>
 <td>Cycloheximide</td>
 <td>Sigma</td>
 <td>C7698-5G</td>
 <td></td>
 </tr>
 <tr>
 <td>Cytochalasin B</td>
 <td>EMD</td>
 <td>250233</td>
 <td></td>
 </tr>
 <tr>
 <td>Dithiothreitol (DTT)</td>
 <td>VWR</td>
 <td>JTF780-2</td>
 <td></td>
 </tr>
 <tr>
 <td>hCG</td>
 <td>Sigma</td>
 <td>CG10-10VL</td>
 <td></td>
 </tr>
 <tr>
 <td>L-Cysteine</td>
 <td>Sigma</td>
 <td>C7352-1KG</td>
 <td></td>
 </tr>
 <tr>
 <td>Leupeptin</td>
 <td>VWR</td>
 <td>97063-922</td>
 <td></td>
 </tr>
 <tr>
 <td>Methyl methanesulfonate (MMS)</td>
 <td>Sigma</td>
 <td>129925-5G</td>
 <td></td>
 </tr>
 <tr>
 <td>Nocodazole</td>
 <td>Sigma</td>
 <td>M1404-2MG</td>
 <td></td>
 </tr>
 <tr>
 <td>PMSG</td>
 <td>Calbiochem</td>
 <td>367222</td>
 <td></td>
 </tr>
 <tr>
 <td>Sample buffer</td>
 <td>Sigma </td>
 <td>S3401</td>
 <td></td>
 </tr>
 <tr>
 <td>Tautomycin</td>
 <td>Wako Chemicals USA</td>
 <td>209-12041</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Equipment </td>
 </tr>
 <tr>
 <td>Bucket for egg laying</td>
 <td>Rubbermaid commercial products</td>
 <td>6308</td>
 <td></td>
 </tr>
 <tr>
 <td>CL2 IEC centrifuge with swinging bucket rotor</td>
 <td>Thermo Scientific</td>
 <td>004260F</td>
 <td></td>
 </tr>
 <tr>
 <td>HB6 swinging bucket rotor</td>
 <td>Thermo Scientific</td>
 <td>11860</td>
 <td></td>
 </tr>
 <tr>
 <td>Sorvall RC6 plus superspeed centrifuge </td>
 <td>Thermo Scientific </td>
 <td>46910</td>
 <td></td>
 </tr>
 <tr>
 <td>UV crosslinker</td>
 <td>UVP</td>
 <td>95-0174-01</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Solutions</td>
 </tr>
 <tr>
 <td>1x MMR</td>
 <td></td>
 <td></td>
 <td>100 mM NaCl, 2 mM KCl, 0.5 mM MgSO4, 2.5 mM CaCl2, 5 mM HEPES, adjust pH to 7.8 with 10 M NaOH</td>
 </tr>
 <tr>
 <td>Aprotinin/Leupeptin stock</td>
 <td></td>
 <td></td>
 <td>10 mg/ml each in water. Store 20 &mu;l aliquots at -80 &deg;C. </td>
 </tr>
 <tr>
 <td>Buffer X</td>
 <td></td>
 <td></td>
 <td>0.2 M sucrose, 80 mM KCl, 15 mM NaCl, 5 mM MgCl2, 1 mM EDTA, 10 mM HEPES, adjust pH to 7.5 by HCl</td>
 </tr>
 <tr>
 <td>Cycloheximide stock</td>
 <td></td>
 <td></td>
 <td>10 mg/ml in water. Store 1 ml aliquots at -20 &deg;C. </td>
 </tr>
 <tr>
 <td>Cytochalasin B stock</td>
 <td></td>
 <td></td>
 <td>5 mg/ml in DMSO. Store 20 &mu;l aliquots at -20 &deg;C. </td>
 </tr>
 <tr>
 <td>Dithiothreitol (DTT) stock</td>
 <td></td>
 <td></td>
 <td>1 M in water. Store 1 ml aliquots at -20 &deg;C. </td>
 </tr>
 <tr>
 <td>ELB</td>
 <td></td>
 <td></td>
 <td>0.25 M sucrose, 1 mM DTT, 50 &mu;g/ml cycloheximide, 2.5 mM MgCl2, 50 mM KCl, 10 mM HEPES, pH7.7</td>
 </tr>
 <tr>
 <td>Nocodazole stock</td>
 <td></td>
 <td></td>
 <td>10 mg/ml in DMSO. Store 5 &mu;l aliquots at -80 &deg;C. </td>
 </tr>
 <tr>
 <td>Energy Mixture</td>
 <td></td>
 <td></td>
 <td>375 mM creatine phosphate, 50 mM ATP, and 25 mM MgCl2. Aliquots are saved at -80 &deg;C.</td>
 </tr>
 <tr>
 <td>Nuclear dye solution</td>
 <td></td>
 <td></td>
 <td>0.4 &mu;g/ml Hoechst 33258, 25% glycerol (v/v), in 1x PBS </td>
 </tr>
 <tr>
 <td>Tautomycin stock</td>
 <td></td>
 <td></td>
 <td>100 &mu;M in DMSO. Store 10 &mu;l aliquots at -80 &deg;C. </td>
 </tr>
 </tbody>
 </table>

study of the dna damage checkpoint using xenopus egg extracts

On a daily basis, cells are subjected to a variety of endogenous and environmental insults. To combat these insults, cells have evolved DNA damage checkpoint signaling as a surveillance mechanism to sense DNA damage and direct cellular responses to DNA damage. There are several groups of proteins called sensors, transducers and effectors involved in DNA damage checkpoint signaling (Figure 1). In this complex signaling pathway, ATR (ATM and Rad3-related) is one of the major kinases that can respond to DNA damage and replication stress. Activated ATR can phosphorylate its downstream substrates such as Chk1 (Checkpoint kinase 1). Consequently, phosphorylated and activated Chk1 leads to many downstream effects in the DNA damage checkpoint including cell cycle arrest, transcription activation, DNA damage repair, and apoptosis or senescence (Figure 1). When DNA is damaged, failing to activate the DNA damage checkpoint results in unrepaired damage and, subsequently, genomic instability. The study of the DNA damage checkpoint will elucidate how cells maintain genomic integrity and provide a better understanding of how human diseases, such as cancer, develop.
Xenopus laevis egg extracts are emerging as a powerful cell-free extract model system in DNA damage checkpoint research. Low-speed extract (LSE) was initially described by the Masui group1. The addition of demembranated sperm chromatin to LSE results in nuclei formation where DNA is replicated in a semiconservative fashion once per cell cycle.
The ATR/Chk1-mediated checkpoint signaling pathway is triggered by DNA damage or replication stress 2. Two methods are currently used to induce the DNA damage checkpoint: DNA damaging approaches and DNA damage-mimicking structures 3. DNA damage can be induced by ultraviolet (UV) irradiation, &gamma;-irradiation, methyl methanesulfonate (MMS), mitomycin C (MMC), 4-nitroquinoline-1-oxide (4-NQO), or aphidicolin3, 4. MMS is an alkylating agent that inhibits DNA replication and activates the ATR/Chk1-mediated DNA damage checkpoint 4-7. UV irradiation also triggers the ATR/Chk1-dependent DNA damage checkpoint 8. The DNA damage-mimicking structure AT70 is an annealed complex of two oligonucleotides poly-(dA)70 and poly-(dT)70. The AT70 system was developed in Bill Dunphy's laboratory and is widely used to induce ATR/Chk1 checkpoint signaling 9-12.
Here, we describe protocols (1) to prepare cell-free egg extracts (LSE), (2) to treat Xenopus sperm chromatin with two different DNA damaging approaches (MMS and UV), (3) to prepare the DNA damage-mimicking structure AT70, and (4) to trigger the ATR/Chk1-mediated DNA damage checkpoint in LSE with damaged sperm chromatin or a DNA damage-mimicking structure.

Watch this Scientific Journal Video about Study of the DNA Damage Checkpoint using Xenopus Egg Extracts at JoVE.com

Study of the DNA Damage Checkpoint using Xenopus Egg Extracts

Xenopus egg extract is a useful model system to investigate the DNA damage checkpoint. This protocol is for the preparation of Xenopus egg extracts and DNA damage checkpoint inducing reagents. These techniques are adaptable to a variety of DNA damaging approaches in the study of the DNA damage checkpoint signaling.

Study of the DNA Damage Checkpoint using Xenopus Egg Extracts

On a daily basis, cells are subjected to a variety of endogenous and environmental insults. To combat these insults, cells have evolved DNA damage ...

study-of-the-dna-damage-checkpoint-using-xenopus-egg-extracts

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16.6K 조회수.  University of North Carolina at Charlotte. Xenopus 계란 추출물은 DNA 손상 검사 점을 조사 할 수있는 유용한 모델 시스템입니다. 이 프로토콜은 Xenopus의 알 추출물과 DNA 손상 검사 점 유도 시약의 준비입니다. 이 기술은 DNA 손상 검사 점 신호의 연구에서 DNA 손상 방식의 다양한에 적용 할 수 있습니다.

비디오: Study of the DNA Damage Checkpoint using Xenopus Egg Extracts

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

Computer-Generated Animal Model Stimuli

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In particular, understanding the constraints on visual signal design for communication has been of great interest. Traditional methods for investigating animal interactions have used basic observational techniques, staged encounters, or physical manipulation of morphology. Less intrusive methods have tried to simulate conspecifics using crude playback tools, such as mirrors, still images, or models. As technology has become more advanced, video playback has emerged as another tool in which to examine visual communication (Rosenthal, 2000). However, to move one step further, the application of computer-animation now allows researchers to specifically isolate critical components necessary to elicit social responses from conspecifics, and manipulate these features to control interactions. Here, I provide detail on how to create an animation using the Jacky dragon as a model, but this process may be adaptable for other species. In building the animation, I elected to use Lightwave  3D to alter object morphology, add texture, install bones, and provide comparable weight shading that prevents exaggerated movement. The animation is then matched to select motor patterns to replicate critical movement features. Finally, the sequence must rendered into an individual clip for presentation. Although there are other adaptable techniques, this particular method had been demonstrated to be effective in eliciting both conspicuous and social responses in staged interactions.

Computer-generated stimuli using the Jacky dragon as a model.

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In ...

Preparation and Fractionation of Xenopus laevis Egg Extracts

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical analysis. Egg lysis and differential centrifugation are used to prepare the crude extract which in turn in used to prepare fractionated extracts and light membrane preparations.

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical analysis. Egg lysis and differential centrifugation are used to prepare the crude extract which in turn in used to prepare fractionated extracts and light membrane preparations.

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical ...

In Vitro Nuclear Assembly Using Fractionated Xenopus Egg Extracts

Nuclear membrane assembly is an essential step in the cell division cycle; this process can be replicated in the test tube by combining Xenopus sperm chromatin, cytosol, and light membrane fractions. Complete nuclei are formed, including nuclear membranes with pore complexes, and these reconstituted nuclei are capable of normal nuclear processes.

Nuclear membrane assembly is an essential step in the cell division cycle; this process can be replicated in the test tube by combining Xenopus sperm chromatin, cytosol, and light membrane fractions. Complete nuclei are formed, including nuclear membranes with pore complexes, and these reconstituted nuclei are capable of normal nuclear processes.

Nuclear membrane assembly is an essential step in the cell division cycle; this process can be replicated in the test tube by combining Xenopus sperm ...

Fertilization of Xenopus oocytes using the Host Transfer Method

Studying the contribution of maternally inherited molecules to vertebrate early development is often hampered by the time and expense necessary to generate maternal-effect mutant animals. Additionally, many of the techniques to overexpress or inhibit gene function in organisms such as Xenopus and zebrafish fail to sufficiently target critical maternal signaling pathways, such as Wnt signaling. In Xenopus, manipulating gene function in cultured oocytes and subsequently fertilizing them can ameliorate these problems to some extent. Oocytes are manually defolliculated from donor ovary tissue, injected or treated in culture as desired, and then stimulated with progesterone to induce maturation. Next, the oocytes are introduced into the body cavity of an ovulating host female frog, whereupon they will be translocated through the host's oviduct and acquire modifications and jelly coats necessary for fertilization. The resulting embryos can then be raised to the desired stage and analyzed for the effects of any experimental perturbations. This host-transfer method has been highly effective in uncovering basic mechanisms of early development and allows a wide range of experimental possibilities not available in any other vertebrate model organism.

Fertilization of Xenopus oocytes using the Host Transfer Method

Procedure for fertilizing Xenopus oocytes by the host transfer method.

Studying the contribution of maternally inherited molecules to vertebrate early development is often hampered by the time and expense necessary to ...

Two- and Three-Dimensional Live Cell Imaging of DNA Damage Response Proteins

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate mutations and chromosomal aberrations1. To prevent this trauma from catalyzing genomic instability, it is crucial for cells to detect DSBs, activate the DNA damage response (DDR), and repair the DNA. When stimulated, the DDR works to preserve genomic integrity by triggering cell cycle arrest to allow for repair to take place or force the cell to undergo apoptosis. The predominant mechanisms of DSB repair occur through nonhomologous end-joining (NHEJ) and homologous recombination repair (HRR) (reviewed in2). There are many proteins whose activities must be precisely orchestrated for the DDR to function properly. Herein, we describe a method for 2- and 3-dimensional (D) visualization of one of these proteins, 53BP1.
The p53-binding protein 1 (53BP1) localizes to areas of DSBs by binding to modified histones3,4, forming foci within 5-15 minutes5. The histone modifications and recruitment of 53BP1 and other DDR proteins to DSB sites are believed to facilitate the structural rearrangement of chromatin around areas of damage and contribute to DNA repair6. Beyond direct participation in repair, additional roles have been described for 53BP1 in the DDR, such as regulating an intra-S checkpoint, a G2/M checkpoint, and activating downstream DDR proteins7-9. Recently, it was discovered that 53BP1 does not form foci in response to DNA damage induced during mitosis, instead waiting for cells to enter G1 before localizing to the vicinity of DSBs6. DDR proteins such as 53BP1 have been found to associate with mitotic structures (such as kinetochores) during the progression through mitosis10.
In this protocol we describe the use of 2- and 3-D live cell imaging to visualize the formation of 53BP1 foci in response to the DNA damaging agent camptothecin (CPT), as well as 53BP1's behavior during mitosis. Camptothecin is a topoisomerase I inhibitor that primarily causes DSBs during DNA replication. To accomplish this, we used a previously described 53BP1-mCherry fluorescent fusion protein construct consisting of a 53BP1 protein domain able to bind DSBs11. In addition, we used a histone H2B-GFP fluorescent fusion protein construct able to monitor chromatin dynamics throughout the cell cycle but in particular during mitosis12. Live cell imaging in multiple dimensions is an excellent tool to deepen our understanding of the function of DDR proteins in eukaryotic cells.

This protocol describes a method for visualizing a DNA double-strand break signaling protein activated in response to DNA damage as well as its localization during mitosis.

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate ...

Production of Xenopus tropicalis Egg Extracts to Identify Microtubule-associated RNAs

Many organisms localize mRNAs to specific subcellular destinations to spatially and temporally control gene expression. Recent studies have demonstrated that the majority of the transcriptome is localized to a nonrandom position in cells and embryos. One approach to identify localized mRNAs is to biochemically purify a cellular structure of interest and to identify all associated transcripts. Using recently developed high-throughput sequencing technologies it is now straightforward to identify all RNAs associated with a subcellular structure. To facilitate transcript identification it is necessary to work with an organism with a fully sequenced genome. One attractive system for the biochemical purification of subcellular structures are egg extracts produced from the frog Xenopus laevis. However, X. laevis currently does not have a fully sequenced genome, which hampers transcript identification. In this article we describe a method to produce egg extracts from a related frog, X. tropicalis, that has a fully sequenced genome. We provide details for microtubule polymerization, purification and transcript isolation. While this article describes a specific method for identification of microtubule-associated transcripts, we believe that it will be easily applied to other subcellular structures and will provide a powerful method for identification of localized RNAs.

Production of Xenopus tropicalis Egg Extracts to Identify Microtubule-associated RNAs

We describe the collection of unfertilized Xenopus tropicalis eggs and production of a meiosis II-arrested egg extract. This egg extract can be used to purify microtubules and microtubule-associated RNAs.

Many  organisms localize mRNAs to specific subcellular destinations to spatially and  temporally control gene expression. Recent studies have demonstrated ...

Reconstitution Of &#946;-catenin Degradation In Xenopus Egg Extract

Xenopus laevis egg extract is a well-characterized, robust system for studying the biochemistry of diverse cellular processes. Xenopus egg extract has been used to study protein turnover in many cellular contexts, including the cell cycle and signal transduction pathways1-3. Herein, a method is described for isolating Xenopus egg extract that has been optimized to promote the degradation of the critical Wnt pathway component, &beta;-catenin. Two different methods are described to assess &beta;-catenin protein degradation in Xenopus egg extract. One method is visually informative ([35S]-radiolabeled proteins), while the other is more readily scaled for high-throughput assays (firefly luciferase-tagged fusion proteins). The techniques described can be used to, but are not limited to, assess &beta;-catenin protein turnover and identify molecular components contributing to its turnover. Additionally, the ability to purify large volumes of homogenous Xenopus egg extract combined with the quantitative and facile readout of luciferase-tagged proteins allows this system to be easily adapted for high-throughput screening for modulators of &beta;-catenin degradation.

Reconstitution Of &amp;#946;-catenin Degradation In Xenopus Egg Extract

A method is described for analyzing protein degradation using radiolabeled and luciferase-fusion proteins in Xenopus egg extract and its adaptation for high-throughput screening for small molecule modulators of protein degradation.

Xenopus laevis egg extract is a well-characterized, robust system for studying the biochemistry of diverse cellular processes. Xenopus egg extract has ...

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3&#8217;-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.

Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

Detection and Analysis of DNA Damage in Mouse Skeletal Muscle In Situ Using the TUNEL Method

Terminal deoxynucleotidyl transferase (TdT) deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) is the method of using the TdT enzyme to covalently attach a tagged form of dUTP to 3&#8217; ends of double- and single-stranded DNA breaks in cells. It is a reliable and useful method to detect DNA damage and cell death in situ. This video describes dissection, tissue processing, sectioning, and fluorescence-based TUNEL labeling of mouse skeletal muscle. It also describes a method of semi-automated TUNEL signal quantitation. Inherent normal tissue features and tissue processing conditions affect the ability of the TdT enzyme to efficiently label DNA. Tissue processing may also add undesirable autofluorescence that will interfere with TUNEL signal detection. Therefore, it is important to empirically determine tissue processing and TUNEL labeling methods that will yield the optimal signal-to-noise ratio for subsequent quantitation. The fluorescence-based assay described here provides a way to exclude autofluorescent signal by digital channel subtraction. The TUNEL assay, used with appropriate tissue processing techniques and controls, is a relatively fast, reproducible, quantitative method for detecting apoptosis in tissue. It can be used to confirm DNA damage and apoptosis as pathological mechanisms, to identify affected cell types, and to assess the efficacy of therapeutic treatments in vivo.

Detection and Analysis of DNA Damage in Mouse Skeletal Muscle In Situ Using the TUNEL Method

This video describes dissection, tissue processing, sectioning, and fluorescence-based TUNEL labeling of mouse skeletal muscle. It also describes a method for semi-automated analysis of TUNEL labeling.

Terminal deoxynucleotidyl transferase (TdT) deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) is the method of using the TdT enzyme to covalently ...