המחברים רוצים חברי הודות המעבדה יוסטון לקריאה ביקורתית של כתב היד. המחקר נתמך על ידי המכון הלאומי לבריאות (GM083999) הוענק DWH

<ol>
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C., C, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fertilization+of+cultured+Xenopus+oocytes+and+use+in+studies+of+maternally+inherited+molecules.">Fertilization of cultured Xenopus oocytes and use in studies of maternally inherited molecules.</a> Methods Cell Biol. 36, 213-230 (1991).</li><li>Holwill, S., Heasman, J., Crawley, C., Wylie, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axis+and+germ+line+deficiencies+caused+by+u.v+irradiation+of+Xenopus+oocytes+cultured+in+vitro.">Axis and germ line deficiencies caused by u.v irradiation of Xenopus oocytes cultured in vitro.</a> Development. 100, 735-743 (1987).</li><li>Houston, D. W., King, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+critical+role+for+Xdazl,+a+germ+plasm-localized+RNA,+in+the+differentiation+of+primordial+germ+cells+in+Xenopus.">A critical role for Xdazl, a germ plasm-localized RNA, in the differentiation of primordial germ cells in Xenopus.</a> Development. 127, 447-456 (2000).</li><li>Mir, A., Heasman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+the+mother+can+help:+studying+maternal+Wnt+signaling+by+anti-sense-mediated+depletion+of+maternal+mRNAs+and+the+host+transfer+technique.">How the mother can help: studying maternal Wnt signaling by anti-sense-mediated depletion of maternal mRNAs and the host transfer technique.</a> Methods Mol Biol. 469, 417-429 (2008).</li><li>Rugh, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Experimental Embryology: Techniques and procedures. , (1962).</li><li>Smith, L. D., Ecker, R. E., Subtelny, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+induction+of+physiological+maturation+in+Rana+pipiens+oocytes+removed+from+their+ovarian+follicles.">In vitro induction of physiological maturation in Rana pipiens oocytes removed from their ovarian follicles.</a> Dev Biol. 17, 627-643 (1968).</li><li>Smith, L. D., Xu, W., Varnold, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oogenesis+and+oocyte+isolation.">Oogenesis and oocyte isolation.</a> Methods Cell Biol. 36, 45-60 (1991).</li><li>Woolf, T. M., Jennings, C. G., Rebagliati, M., Melton, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+stability,+toxicity+and+effectiveness+of+unmodified+and+phosphorothioate+antisense+oligodeoxynucleotides+in+Xenopus+oocytes+and+embryos.">The stability, toxicity and effectiveness of unmodified and phosphorothioate antisense oligodeoxynucleotides in Xenopus oocytes and embryos.</a> Nucleic Acids Res. 18, 1763-1769 (1990).</li><li>Wylie, C., Kofron, M., Payne, C., Anderson, R., Hosobuchi, M., Joseph, E., Heasman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maternal+beta-catenin+establishes+a+'dorsal+signal'+in+early+Xenopus+embryos.">Maternal beta-catenin establishes a 'dorsal signal' in early Xenopus embryos.</a> Development. 122, 2987-2996 (1996).</li><li>Zhang, J., Houston, D. W., King, M. L., Payne, C., Wylie, C., Heasman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+maternal+VegT+in+establishing+the+primary+germ+layers+in+Xenopus+embryos.">The role of maternal VegT in establishing the primary germ layers in Xenopus embryos.</a> Cell. 94, 515-524 (1998).</li><li>Zuck, M. V., Wylie, C. C., Heasman, J., Richter, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maternal+mRNAs+in+Xenopus+embryos:+an+antisense+approach..">Maternal mRNAs in Xenopus embryos: an antisense approach..</a> A comparative methods approach to the study of oocytes and embryos. , 341-354 (1998).</li></ol>

ניסויים בבעלי חיים בוצעו בהתאם להנחיות והתקנות שנקבעו על ידי האוניברסיטה של ​​איווה IACUC הוועדה.

העברה מוצלחת תגרום דישון מחשוף נורמלי של 50%&gt; של ביציות (איור 3). בדרך כלל בין 30-60% של ביציות הועבר ישרדו בעבר בשלבים neurula. אנחנו בדרך כלל להעביר 75-150 ביציות לכל קבוצת הניסוי, אשר תניב עוברים מספיק מספר סוגים של ניתוח (באתרו, RT-PCR) וכן לדמיין את הפנוטיפ. השתלת ביציות באופן מש...

פתרונות מתכונים: OCM (תרבות בינוני הביצית; שונה מן ויילי et al, 1996). <table border="0" style=";text-align:right;direction:rtl"><tbody><tr><td> 70% L-15 של ליבוביץ בינוני (המכיל L-גלוטמין) </td><td> (Invitrogen 11415-064) </td></tr><tr><td> 0.4 מ&quot;ג / מ&quot;ל ​​אלבומין בסרום שור (BSA, חלק V) </td><td> (Sigma A-9418-50 גרם) </td></tr><tr><td> 1x פניצילין, סטרפטומיצין </td><td> (Sigma P-0781) </td></tr><tr><td colspan="2"> התאם את ה-pH 7.6-7.8 עם 5N NaOH </td></tr><tr><td colspan="2"> בצע טרי בחנות שבועי, בשעה 18 ° C. </td></tr></tbody></table> 10X MMR (פתרון השתנה מארק רינגר;. צוק et al, 1998) <table border="0" style=";text-align:right;direction:rtl"><tbody><tr><td> NaCl 1M </td></tr><tr><td> 20 מ&quot;מ KCl </td></tr><tr><td> 20 מ&quot;מ CaCl 2 </td></tr><tr><td> 10 mM MgCl 2 </td></tr><tr><td> 150 mM HEPES </td></tr><tr><td> להתאים את ה-pH 7.6-7.8 </td></tr><tr><td> חנות ב -4 ° C </td></tr></tbody></table> הרדמה פתרון <table border="0" style=";text-align:right;direction:rtl"><tbody><tr><td> 1 גרם / L 3-Aminobenzoic חומצה קראטין אתיל אסתר (MS222) </td><td> (Sigma A-5040) </td></tr><tr><td colspan="2"> 0.7 g / L נתרן ביקרבונט </td></tr><tr><td colspan="2"> מתמוססים במים Amquel שטופלו </td></tr><tr><td colspan="2"> בצע טרי שבועי </td></tr></tbody></table> הפרוגסטרון מניות <table border="0" style=";text-align:right;direction:rtl"><tbody><tr><td> 10 mM הפרוגסטרון (Sigma P-0130) ב 100% אתנול </td></tr><tr><td> מדולל עד 1 מ&quot;מ ב EtOH 100% במניה 500x / עובדים פתרון </td></tr><tr><td> חנות ב -20 ° C </td></tr></tbody></table> צבעים ויטל (שונה מן Heasman et al., 1991) <table border="0" style=";text-align:right;direction:rtl"><tbody><tr><td> כחול: 0.1% הנילוס הכחול </td><td> (אולדריץ 121479-5G), </td></tr><tr><td> אדום: אדום ניטרלי 0.25% </td><td> (סיגמה N-6634) </td></tr><tr><td> בראון: 1% ביסמרק בראון </td><td> (Sigma B-2759) </td></tr><tr><td colspan="2"> גרין (80 כחול + 80 μl חום μl), </td></tr><tr><td colspan="2"> במוב (80 μl כחול אדום + 80 μl). </td></tr><tr><td colspan="2"> צבע השישי, אורנג&#39; (80 μl אדום + חום μl 80), יכול לשמש גם אבל לעתים קרובות קשה להבחין בין חום. </td></tr></tbody></table> מניות של כחול, אדום וחום בנויים במים deionized ב 50 צינורות מ&quot;ל, מודגרות במשך 20 דקות עם נדנדה וסובב בצנטריפוגה קליני. Supernatants הם aliquotted לתוך צינורות microfuge ומאוחסנים ב -20 ° C.

fertilization of xenopus oocytes using the host transfer method

הלומד את תרומתה של מולקולות בירושה אימהי על התפתחות החולייתנים הראשונים נפגעת לעתים קרובות על ידי הזמן וההוצאות הדרושים כדי ליצור חיות השפעה אימהית מוטנטי. בנוסף, רבים של טכניקות כדי overexpress או לעכב תפקוד הגן באורגניזמים כגון Xenopus ו דג הזברה מצליחים מספיק יעד קריטי מסלולי איתות אימהית, כגון איתות Wnt. ב Xenopus, מניפולציה לתפקד גנים ביציות בתרבית ולאחר מכן זבול אותם ניתן לשפר את הבעיות הללו במידה מסוימת. ביציות הן defolliculated ידני מרקמת השחלה התורם, הזריק או מטופל בתרבות כפי הרצוי, מגורה אז עם פרוגסטרון כדי לזרז הבשלה. לאחר מכן, ביציות הן הציג לתוך חלל הגוף של צפרדע מארח הביוץ הנשי, ואז הם יהיו translocated דרך oviduct של המארח ולרכוש שינויים ומעילים ג&#39;לי הכרחי הפריה. עוברים שהתקבל לאחר מכן ניתן להעלות לבמה הרצוי ניתחו את ההשפעות של כל הפרעות ניסיוני. שיטה זו המארח להעביר כבר יעיל מאוד בחשיפת המנגנונים הבסיסיים של ההתפתחות המוקדמים, ומאפשר מגוון רחב של אפשרויות ניסיוני אינו זמין בכל אורגניזם מודל חוליות אחרים.

Watch this Scientific Journal Video about Fertilization of Xenopus oocytes using the Host Transfer Method at JoVE.com

Fertilization of Xenopus oocytes using the Host Transfer Method

נוהל זבול<em&gt; Xenopus ביציות</em&gt; לפי שיטת העברת המארח.

הפריה של Xenopus ביציות באמצעות שיטת העברה מארח

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

fertilization-of-xenopus-oocytes-using-the-host-transfer-method

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Fertilization of Xenopus oocytes using the Host Transfer Method

14.4K Views.  University of Iowa. נוהל זבול Xenopus ביציות לפי שיטת העברת המארח.

Video: Fertilization of Xenopus oocytes using the Host Transfer Method

Nuclear Transfer into Mouse Oocytes

Nuclear transfer into an unfertilized oocyte can restore developmental potential to a differentiated cell. This demonstrates that the processes underlying development, differentiation and aging are epigenetic rather than genetic processes. The reversibility of these processes opens exciting perspectives in basic research, and in the more distant future, in regenerative medicine. In the mouse, embryonic stem cells can be derived from cloned preimplantation stage embryos. Such embryonic stem cells have the ability to give rise to all cell types of the adult organism. Importantly, these cells are genetically identical to the donor. If applicable to human, this would allow the derivation of stem cells from a patient. These cells could then be differentiated into the affected cell type of the patient and studied in vitro, or used to replace the damaged or missing cells. The study of nuclear transfer in the mouse remains important as it can inform us about the principles of nuclear reprogramming. This movie and the accompanying protocol are intended to help learning nuclear transfer in the mouse, a method initially developed in the group of Prof. Yanagimachi (WAKAYAMA et al. 1998).

This movie and the protocol are intended to help learning nuclear transfer.

העברת גרעין לתוך ביציות עכבר

Nuclear transfer into an unfertilized oocyte can restore developmental potential to a differentiated cell. This demonstrates that the processes underlying ...

Retrieval of Mouse Oocytes

To date, only a few studies have reported successful manipulations of Peromyscus embryogenesis or reproductive biology.  Together with the Peromyscus Genetic Stock Center (http://stkctr.biol.sc.edu), we are characterizing the salient differences needed to develop this system.  A primary goal has been to optimize oocyte/early embryo retrieval.

This protocol illustrates the technique for extracting oocytes or early-stage fertilized embryos from the oviduct of mice. The ability to identify the infindibulum and insert a blunt end needle into it is essential to correctly performing the procedure.

איחזור של ביציות עכבר

To date, only a few studies have reported successful manipulations of Peromyscus embryogenesis or reproductive biology.  Together with the Peromyscus ...

Patch Clamp Recording of Ion Channels Expressed in  Xenopus Oocytes

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological measurement of single or multiple ion channels in cells. This technique can be applied to ion channels in both their native environment and expressed in heterologous cells, such as oocytes harvested from the African clawed frog, Xenopus laevis. Here, we describe the well-established technique of patch clamp recording from Xenopus oocytes. This technique is used to measure the properties of expressed ion channels either in populations (macropatch) or individually (single-channel recording). We focus on techniques to maximize the quality of oocyte preparation and seal generation. With all factors optimized, this technique gives a probability of successful seal generation over 90 percent. The process may be optimized differently by every researcher based on the factors he or she finds most important, and we present the approach that have lead to the greatest success in our hands.

This is intended as an introduction to patch clamp recording from Xenopus laevis oocytes. It covers vitelline membrane removal, formation of a gigaohm seal (gigaseal), and the optional conversion of the patch to the outside-out topology.

הצמד תיקון הקלטה של ​​תעלות יונים מתבטא ביציות Xenopus

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological ...

Microinjection of Xenopus Laevis Oocytes

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic transport. Because of its large nucleus and high density of nuclear pore complexes (NPCs), nuclear transport can be easily visualized in the Xenopus oocyte. Much insight into the mechanisms of nuclear import and export has been gained through use of this system (reviewed by Pant&eacute;, 2006). In addition, we have used microinjection of Xenopus oocytes to dissect the nuclear import pathways of several viruses that replicate in the host nucleus. 
Here we demonstrate the cytoplasmic microinjection of Xenopus oocytes with a nuclear import substrate. We also show preparation of the injected oocytes for visualization by thin-sectioning EM, including dissection, dehydration, and embedding of the oocytes into an epoxy embedding resin. Finally, we provide representative results for oocytes that have been microinjected with the capsid of the baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV) or the parvovirus Minute Virus of Mice (MVM), and discuss potential applications of the technique.

Here we demonstrate cytoplasmic microinjection of Xenopus laevis oocytes with a nuclear import substrate, as well as preparation of the injected oocytes for visualization by thin-sectioning electron microscopy.

Microinjection של ביציות Xenopus Laevis

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic ...

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982).  We have introduced two changes to the original protocol that both streamline the procedure and increase sample uniformity.  First, we normalize all sperm volumes using freezing media that does not contain the cryoprotectant. Second, cryopreserved sperm are stored in cryovials instead of capillary tubes.  The rates of sperm freezing and thawing (&delta;&deg;C/time) are probably the two most critical variables to control in this procedure.  For this reason, do not substitute different tubes for those specified.  Working in teams of 2 it is possible to freeze the sperm of 100 males per team in ~2 hrs.  Sperm cryopreserved using this protocol has an average of 25% fertility (measured as the number of viable embryos generated in an in vitro fertilization divided by the total number of eggs fertilized) and this percent fertility is stable over many years.

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

This is a high-throughput sperm cryopreservation protocol for zebrafish. Sperm cryopreserved using this protocol has an average of 25% fertility in subsequent vitro fertilization and is stable over many years.

שיטת תפוקה גבוהה עבור cryopreservation זרע דג הזברה במבחנה הפריה

This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982).  We have introduced two changes to ...

Visualizing RNA Localization in Xenopus Oocytes

RNA localization is a conserved mechanism of establishing cell polarity. Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to spatially restrict gene expression of Vg1 protein. Tight control of Vg1 distribution in this manner is required for proper germ layer specification in the developing embryo. RNA sequence elements in the 3' UTR of the mRNA, the Vg1 localization element (VLE) are required and sufficient to direct transport. To study the recognition and transport of Vg1 mRNA in vivo, we have developed an imaging technique that allows extensive analysis of trans-factor directed transport mechanisms via a simple visual readout. 
To visualize RNA localization, we synthesize fluorescently labeled VLE RNA and microinject this transcript into individual oocytes. After oocyte culture to allow transport of the injected RNA, oocytes are fixed and dehydrated prior to imaging by confocal microscopy. Visualization of mRNA localization patterns provides a readout for monitoring the complete pathway of RNA transport and for identifying roles in directing RNA transport for cis-acting elements within the transcript and trans-acting factors that bind to the VLE (Lewis et al., 2008, Messitt et al., 2008). We have extended this technique through co-localization with additional RNAs and proteins (Gagnon and Mowry, 2009, Messitt et al., 2008), and in combination with disruption of motor proteins and the cytoskeleton (Messitt et al., 2008) to probe mechanisms underlying mRNA localization.

Visualizing RNA Localization in Xenopus Oocytes

Visualization of in vivo RNA transport is accomplished by microinjection of fluorescently labeled RNA transcripts into Xenopus oocytes, followed by confocal microscopy.

הדמיה ב-RNA לוקליזציה Xenopus ביציות

RNA localization is a conserved mechanism of establishing cell polarity.  Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to ...

Tissue Determination Using the Animal Cap Transplant (ACT) Assay in Xenopus laevis

Many proteins play a dual role in embryonic development. Those that regulate cell fate determination in a specific tissue can also affect the development of a larger region of the embryo. This makes defining its role in a particular tissue difficult to analyze. For example, noggin overexpression in Xenopus laevis embryos causes the expansion of the entire anterior region, including the eye1,2. From this result, it is not known if Noggin plays a direct role in eye determination or that by causing an expansion of neural tissue, Noggin indirectly affects eye formation. Having this complex phenotype makes studying its eye-specific role in cell fate determination difficult to analyze. We have developed an assay that overcomes this problem. Taking advantage of the pluripotent nature of the Xenopus laevis animal cap 3, we have developed an assay to test the ability of gene product(s), like noggin or the eye field transcription factors (EFTFs), to transform caps into particular tissue or cell types by transplanting this tissue onto the side of the embryo 4. While we have found either Noggin protein treatment or a collection of transcription factors can determine retinal cell fate in animal caps, this procedure could be used to identify gene product(s) involved in specifying other tissues as well.

Tissue Determination Using the Animal Cap Transplant ACT Assay in Xenopus laevis

Animal caps overexpressing gene product(s) are transplanted to the flank of developing Xenopus laevis embryos in order to establish whether tissue is determined.

רקמות קביעת שווי שימוש בבעלי חיים להשתלות (ACT) Assay ב Xenopus laevis</em

Many proteins play a dual role in embryonic development. Those that regulate cell fate determination in a specific tissue can also affect the development ...

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may also be used to study the structure-function relationship for other ion channels and neurotransmitter receptors1. BK channels are widely expressed in different tissues and have been implicated in many physiological functions, including regulation of smooth muscle contraction, frequency tuning of inner hair cells and regulation of neurotransmitter release2-6. BK channels are activated by membrane depolarization and by intracellular Ca2+ and Mg2+ 6-9. Therefore, the protocol is designed to control both the membrane voltage and the intracellular solution. In this protocol, messenger RNA of BK channels is injected into Xenopus laevis oocytes (stage V-VI) followed by 2-5 days of incubation at 18&deg;C10-13. Membrane patches that contain single or multiple BK channels are excised with the inside-out configuration using patch clamp techniques10-13. The intracellular side of the patch is perfused with desired solutions during recording so that the channel activation under different conditions can be examined. To summarize, the mRNA of BK channels is injected into Xenopus laevis oocytes to express channel proteins on the oocyte membrane; patch clamp techniques are used to record currents flowing through the channels under controlled voltage and intracellular solutions.

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Ionic current of BK channels is recorded using patch clamp techniques. BK channels are expressed in Xenopus oocytes by injecting messenger RNA. The intracellular solution during patch clamp recordings is controlled by a perfusion system.

הצמד תיקון וטכניקות זלוף עבור לימוד תעלות יונים מתבטא Xenopus ביציות

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...

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.

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.

חקר מחסום הניזק לדנ&quot;א באמצעות<em&gt; Xenopus</em&gt; תמציות ביצה

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

The MultiBac Protein Complex Production Platform at the EMBL

Proteomics research revealed the impressive complexity of eukaryotic proteomes in unprecedented detail. It is now a commonly accepted notion that proteins in cells mostly exist not as isolated entities but exert their biological activity in association with many other proteins, in humans ten or more, forming assembly lines in the cell for most if not all vital functions.1,2 Knowledge of the function and architecture of these multiprotein assemblies requires their provision in superior quality and sufficient quantity for detailed analysis. The paucity of many protein complexes in cells, in particular in eukaryotes, prohibits their extraction from native sources, and necessitates recombinant production. The baculovirus expression vector system (BEVS) has proven to be particularly useful for producing eukaryotic proteins, the activity of which often relies on post-translational processing that other commonly used expression systems often cannot support.3 BEVS use a recombinant baculovirus into which the gene of interest was inserted to infect insect cell cultures which in turn produce the protein of choice. MultiBac is a BEVS that has been particularly tailored for the production of eukaryotic protein complexes that contain many subunits.4 A vital prerequisite for efficient production of proteins and their complexes are robust protocols for all steps involved in an expression experiment that ideally can be implemented as standard operating procedures (SOPs) and followed also by non-specialist users with comparative ease. The MultiBac platform at the European Molecular Biology Laboratory (EMBL) uses SOPs for all steps involved in a multiprotein complex expression experiment, starting from insertion of the genes into an engineered baculoviral genome optimized for heterologous protein production properties to small-scale analysis of the protein specimens produced.5-8 The platform is installed in an open-access mode at EMBL Grenoble and has supported many scientists from academia and industry to accelerate protein complex research projects.

Protein complexes catalyze key cellular functions. Detailed functional and structural characterization of many essential complexes requires recombinant production. MultiBac is a baculovirus/insect cell system particularly tailored for expressing eukaryotic proteins and their complexes. MultiBac was implemented as an open-access platform, and standard operating procedures developed to maximize its utility.

פלטפורמת הפקת MultiBac החלבון המורכבת בEMBL

Proteomics  research revealed the impressive complexity of eukaryotic proteomes in  unprecedented detail. It is now a commonly accepted notion that ...