Name: xenopus laevis as a model to identify translation impairment
Uploaded: 2015-09-27T14:00:00
Description: Watch this Scientific Journal Video about Xenopus laevis as a Model to Identify Translation Impairment at JoVE.com

This research was supported by grant from INSERM, University of Lille 1, University of Lille 2, Regional Hospital Center of Lille (CHR de Lille). MCCH acknowledges supports from the Fondation de France and wishes to thank IRCL, Pr. Sonenberg for the gift of the V5-plasmids, Dr. Dissous (Pasteur Institute, Lille) for the gift of anti-GFP antibodies, UMS 3387 (University of Rennes) where Xenopus oocytes are purchased and Dr Taymans (JPArc, Lille) for critical reading of the manuscript text.

All Xenopus experiments were performed at the animal facility of the Lille 1 University according to the rules of the European Community Council guidelines (86/609/EEC) for laboratory animal experimentation. The animal protocol was approved by the local institutional review board (Comit&#233; d&#8217;Ethique en Experimentation Animale Nord-Pas-De-Calais, CEEA 07/2010).
1. Oocyte Handling
<ol>
	<li>Prepare the anesthetic solution: dissolve 1 g of tricaine methane sulphonate powder in...

<ol>
	<li>Jackson, R. J., Hellen, C. U., Pestova, T. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mechanism+of+eukaryotic+translation+initiation+and+principles+of+its+regulation.">The mechanism of eukaryotic translation initiation and principles of its regulation.</a> Nat Rev Mol Cell Biol. 11 (2), 113-127 (2010).</li><li>Holcik, M., Sonenberg, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translational+control+in+stress+and+apoptosis.">Translational control in stress and apoptosis.</a> Nat Rev Mol Cell Bio. 6 (4), 318-327 (2005).</li><li>Lopez-Lastra, M., Rivas, A., Barria, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+synthesis+in+eukaryotes:+the+growing+biological+relevance+of+cap-independent+translation+initiation.">Protein synthesis in eukaryotes: the growing biological relevance of cap-independent translation initiation.</a> Biol Res. 38 (2-3), 121-146 (2005).</li><li>Terenin, I. M., Andreev, D. E., Dmitriev, S. E., Shatsky, I. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+mechanism+of+eukaryotic+translation+initiation+that+is+neither+m7G-cap-,+nor+IRES-dependent.">A novel mechanism of eukaryotic translation initiation that is neither m7G-cap-, nor IRES-dependent.</a> Nucleic Acids Res. 41 (3), 1807-1816 (2013).</li><li>Sonenberg, N., Hinnebusch, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+translation+initiation+in+eukaryotes:+mechanisms+and+biological+targets.">Regulation of translation initiation in eukaryotes: mechanisms and biological targets.</a> Cell. 136 (4), 731-745 (2009).</li><li>Van der Knaap, M. S., Leegwater, P. A., K&#246;nst, A. A., Visser, A., Naidu, S., Oudejans, C. B., Schutgens, R. B., Pronk, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+in+each+of+the+five+subunits+of+translation+initiation+factor+eIF2B+can+cause+leukoencephalopathy+with+vanishing+white+matter.">Mutations in each of the five subunits of translation initiation factor eIF2B can cause leukoencephalopathy with vanishing white matter.</a> Ann Neurol. 51 (2), 264-270 (2002).</li><li>Sen&#233;e, V., Vattem, K. M., Del&#233;pine, M., Rainbow, L. A., Haton, C., Lecoq, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wolcott-Rallison+Syndrome:+clinical,+genetic,+and+functional+study+of+EIF2AK3+mutations+and+suggestion+of+genetic+heterogeneity.">Wolcott-Rallison Syndrome: clinical, genetic, and functional study of EIF2AK3 mutations and suggestion of genetic heterogeneity.</a> Diabetes. 53 (7), 1876-1883 (2004).</li><li>Chartier-Harlin, M. C., Dachsel, J. C., Vilari&#241;o-G&#252;ell, C., Lincoln, S. J., Lepr&#234;tre, F., Hulihan, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translation+initiator+EIF4G1+mutations+in+familial+Parkinson+disease.">Translation initiator EIF4G1 mutations in familial Parkinson disease.</a> Am J Hum Genet. 89 (3), 398-406 (2011).</li><li>Charlesworth, A., Cox, L. L., MacNicol, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytoplasmic+polyadenylation+Element+(CPE)-+and+CPE-binding+Protein+(CPEB)-independent+mechanisms+regulate+early+class+maternal+mRNA+translational+activation+in+Xenopus+oocytes.">Cytoplasmic polyadenylation Element (CPE)- and CPE-binding Protein (CPEB)-independent mechanisms regulate early class maternal mRNA translational activation in Xenopus oocytes.</a> J Biol Chem. 279 (17), 17650-17659 (2004).</li><li>Dumont, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oogenesis+in+Xenopus+laevis.">Oogenesis in Xenopus laevis.</a> J Morphol. 136, 153-180 (1972).</li><li>Rassa, J. C., Wilson, G. M., Brewer, G. A., Parks, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spacing+constraints+on+reinitiation+of+paramyxovirus+transcription:+the+gene+end+U+tract+acts+as+a+spacer+to+separate+gene+end+from+gene+start+sites.">Spacing constraints on reinitiation of paramyxovirus transcription: the gene end U tract acts as a spacer to separate gene end from gene start sites.</a> Virology. 274, 438-449 (2000).</li><li>Lin, W., Wadlington, N. L., Chen, L., Zhuang, X., Brorson, J. R., Kang, U. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+PINK1+attenuates+HIF-1&#945;+induction+by+preventing+4E-BP1-dependent+switch+in+protein+translation+under+hypoxia.">Loss of PINK1 attenuates HIF-1&#945; induction by preventing 4E-BP1-dependent switch in protein translation under hypoxia.</a> J Neurosci. 34 (8), 3079-3089 (2014).</li><li>Martin, I., Kim, J. W., Lee, B. D., Kang, H. C., Xu, J. C., Jia, H. 2., Stankowski, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ribosomal+protein+s15+phosphorylation+mediates+LRRK2+neurodegeneration+in+Parkinson's+disease.">Ribosomal protein s15 phosphorylation mediates LRRK2 neurodegeneration in Parkinson's disease.</a> Cell. 57 (2), 472-485 (2014).</li><li>Cohen, S., Au, S., Pant&#233;, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+of+Xenopus+laevis+oocytes.">Microinjection of Xenopus laevis oocytes.</a> J Vis Exp. (24), (2009).</li><li>Willis, J., DeStephanis, D., Patel, Y., Gowda, V., Yan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Study+of+the+DNA+damage+checkpoint+using+Xenopus+egg+extracts.">Study of the DNA damage checkpoint using Xenopus egg extracts.</a> J Vis Exp. (69), e4449 (2012).</li><li>Keiper, B. D., Rhoads, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translational+recruitment+of+Xenopus+maternal+mRNAs+in+response+to+poly(A)+elongation+requires+initiation+factor+eIF4G-1.">Translational recruitment of Xenopus maternal mRNAs in response to poly(A) elongation requires initiation factor eIF4G-1.</a> Dev Biol. 206 (1), 1-14 (1999).</li><li>Wakiyama, M., Imataka, H., Sonenberg, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+of+eIF4G+with+poly(A)-binding+protein+stimulates+translation+and+is+critical+for+Xenopus.oocyte+maturation.">Interaction of eIF4G with poly(A)-binding protein stimulates translation and is critical for Xenopus.oocyte maturation.</a> Curr Biol. 10 (18), 1147-1150 (2000).</li><li>Sheets, M. D., Wu, M., Wickens, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyadenylation+of+c-mos+mRNA+as+a+control+point+in+Xenopus+meiotic+maturation.">Polyadenylation of c-mos mRNA as a control point in Xenopus meiotic maturation.</a> Nature. 374 (6522), 511-516 (1995).</li><li>Gebauer, F., 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=Synthesis+and+function+of+Mos:+the+control+switch+of+vertebrate+oocyte+meiosis.">Synthesis and function of Mos: the control switch of vertebrate oocyte meiosis.</a> Bioessays. 19 (1), 23-28 (1997).</li><li>Le Sommer, C., Lerivray, H., Lesimple, M., Hardy, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenopus+as+a+model+to+study+alternative+splicing+in+vivo.">Xenopus as a model to study alternative splicing in vivo.</a> Biol Cell. 99 (1), 55-65 (2007).</li><li>Au, S., Cohen, S., Pant&#233;, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+of+Xenopus+laevis.oocytes+as+a+system+for+studying+nuclear+transport+of+viruses.">Microinjection of Xenopus laevis.oocytes as a system for studying nuclear transport of viruses.</a> Methods. 51 (1), 114-120 (2010).</li><li>Gotoh, T., Villa, L. M., Capelluto, D. G., Finkielstein, C. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulatory+pathways+coordinating+cell+cycle+progression+in+early+Xenopus+development.">Regulatory pathways coordinating cell cycle progression in early Xenopus development.</a> Results Probl Cell Differ. 53, 171-199 (2011).</li><li>Schmidt, E. K., Clavarino, G., Ceppi, M., Pierre, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SUnSET,+a+nonradioactive+method+to+monitor+protein+synthesis.">SUnSET, a nonradioactive method to monitor protein synthesis.</a> Nat Methods. 6 (4), 275-277 (2009).</li><li>Moreno, J. A., Halliday, M., Molloy, C., Radford, H., Verity, N., Axten, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oral+treatment+targeting+the+unfolded+protein+response+prevents+neurodegeneration+and+clinical+disease+in+prion-infected+mice.">Oral treatment targeting the unfolded protein response prevents neurodegeneration and clinical disease in prion-infected mice.</a> Sci Transl Med. 5 (206), 206ra138 (2013).</li><li>Doyle, K. M., Kennedy, D., Gorman, A. M., Gupta, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unfolded+proteins+and+endoplasmic+reticulum+stress+in+neurodegenerative+disorders.">Unfolded proteins and endoplasmic reticulum stress in neurodegenerative disorders.</a> J Cell Mol Med. 15 (10), 2025-2039 (2011).</li><li>Gandin, V., Sikstr&#246;m, K., Alain, T., Morita, M., McLaughlan, S., Larsson, O., Topisirovic, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polysome+fractionation+and+analysis+of+mammalian+translatomes+on+a+genome-wide+scale.">Polysome fractionation and analysis of mammalian translatomes on a genome-wide scale.</a> J Vis Exp. (87), e51455 (2014).</li></ol>

Translation is a mechanism involved in the physiopathology of numerous human disorders including several neurodegenerative diseases. For instance in Parkinson&#8217;s disease several reports suggested the impairment in translation associated with hereditary mutations8,12,13.
Several cellular models are available to study translation. Here, in order to study the translational consequences of a mutation in eIF4G1 that acts as dominant negative mutation reducing the interaction between...

Proteins are essential elements of cellular life and thus at larger-scale of the organism. They ensure the majority of cellular functions including structure, transport, reaction catalysis, regulation, gene expression etc.&#160;Their expression is the result of a complex mechanism of translation allowing the conversion of an mRNA into protein. The translation is subjected to various controls to adapt and to regulate gene expression according to the cell needs, during development and differentiation, aging, physiological stresses or pathological manifestations.
Translation is divided into 3 phases (initiation, elongation and termination) and presents 3 initiation translation systems in order to respond to these needs: cap-dependent, cap-independent via Internal Ribosome Entry Segment (IRES) structures and cap-Independent Translation Enhancers (CITE).
Most eukaryotic mRNA are translated in a cap-dependent manner via the 7-methylguanosine 5&#39;-triphosphate cap that serves as a recognition feature during protein synthesis. This cap binds to eIF4E, a component of eIF4F complex with eIF4G1 and eIF4A. Associated with other partners like poly(A) Binding Protein (PABP), eIF2-GTP-Met-tRNAMet, these translation initiation factors allow to circularize mRNA and improve its accessibility to forms 43S complex until the AUG initiation codon recognition1. This event corresponds to the end of the translation initiation i.e., the first step of translation.
Cap-independent translation is used by mRNA encoding for essential proteins under stressed conditions that induce for instance cell proliferation and apoptosis. This mechanism involves secondary structures in mRNA 5&#39;- untranslated region (UTR) called IRES, the carboxy-terminal end of eIF4G1 associated with eIF4A and the 43S complex. The binding of this 43S pre-initiation complex to IRES initiates the cap independent translation without the need for eIF4E factor2,3.
Finally, another translation mechanism still not well understood supports this cap-independent translation activity under stressed conditions via CITE structures located within mRNA UTR 4.
Through these various modes of translation differing by their initiation steps, translation plays a critical role in cellular homeostasis and any change in one of these processes would thus impact the organism with small to large scale effects. Indeed, the initiation is a rate limiting step governing the correct translation processes of mRNA into proteins and is thus the target of numerous controls and regulation points5. Whether it is for the latter or for components of these processes, if one turns out to be defective, it will perturb the established balance in the cell and thus could lead to pathologic conditions. In this context, mutations in translation factors have been involved in several disorders including neurodegenerative disorders such as `leukoencephalopathy with vanishing white matter&#180; (eIF2B1-5 subunit)6, in Walcott-Rallison syndrome (EIF2AK3 gene encoding for PERK)7, potentially in Parkinson&#39;s disease (eIF4G1 p.R1205H)8. It is therefore important to conduct cellular and molecular studies of these mutant proteins to increase our knowledge on disease development and on the general process of translation initiation.
To carry out these studies, it is essential to choose the most adequate models to observe the consequences of these mutations. Xenopus laevis oocytes are particularly well adapted due to their physiological and biochemical properties: physiological synchronicity (blocked in phase G2 of the cell cycle), high capacity of protein synthesis (200-400 ng/day/oocyte), high number of extracted oocytes from a same animal (800-1,000 oocytes/female) and cell size (1.2-1.4 mm in diameter) which facilitates their manipulation. Microinjection of Xenopus oocytes with synthesized mRNA can easily be performed to dissect translation steps. In this view it presents other advantages. Given the speed of meiosis progression and of translation after mRNA microinjection (~24 hr), Xenopus oocyte represents a fast system compared to reconstituted cellular systems (extracted from E.coli, wheat germs or rabbit reticulocyte...) in which an mRNA is translated with a reduced translation rate and at a lower speed. So, the effects of a mutation introduced in an mRNA will be quickly observable and easily studied in several oocytes. Another advantage of Xenopus oocytes is that maternal mRNAs are latent and protein translation is blocked before progesterone stimulation. Addition of progesterone is thus a good means of controlling the translation induction. Cytoplasmic polyadenylation does not occur during oogenesis. It begins during oocyte maturation in progesterone-stimulated oocytes in a temporal order and continues throughout early development and could be used to study the different steps of translation.
The polyadenylation of mos mRNA is among the first to occur and it belongs with Aurora A/Eg2, Histone-Like B4 mRNA to the class of &#8220;early maturation&#8221; genes as defined in Charlesworth et al. (2004)9. The translational induction of &#8220;late&#8221; mRNA such as Cyclin A1 and Cyclin B1 occurs around the time of germinal vesicle breakdown (GVBD). Mos mRNA encodes a serine/threonine-protein kinase. Its translation is crucial since it induces the MAP kinase cascade that indirectly activates the oocyte maturation. Indeed, in response to progesterone, polyadenylation of mos mRNA is enhanced via a process involving Aurora A/Eg2 regulatory proteins and other RNA binding proteins with the 3&#8217;UTR of mos mRNA. This increased polyadenylation of mos mRNA leads to an increase of mos protein level, which in turn activates MEK1. This process mediates the activation of the extracellular signaling-regulated kinase 2 (ERK2) (Figure 1). This signaling cascade can then trigger the maturation M-phase promoting factors, a complex formed by Cyclin B and Cdc2 kinase, and eventually results in meiotic resumption.
Therefore in Xenopus laevis oocytes, the study of maternal mRNA such as mos could easily be used to test their translatability with several endpoints from their efficient polyadenylation to translation of several mos signaling components, including also the determination of the GVBD rate. This system is therefore interesting to evaluate the first consequences of mutations in translation initiation factors without interference of newly transcribed mRNA or of transfection efficiency, problems often occurring with eukaryotic cell studies.
Here, a protocol is established where mutant eIF4G1 mRNAs are microinjected in Xenopus laevis oocytes and the translation of maternal mRNA is tested. In the presence of a defect in GVBD progression, mos mRNA polyadenylation which is essential for progression through the oocyte meiotic cell cycle and for the subsequent translation of early and late class mRNAs is ascertained. The phosphorylation Aurora A/Eg2 and ERK is also studied to confirm the consequence of mos deregulation.Thus, Xenopus oocytes represent a simple way to analyse different steps of mRNA translation.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Tricaine methane sulphonate</td>
			<td>Sandoz</td>
			<td>MS-222</td>
			<td rowspan="8">oocytes handling</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Moria</td>
			<td>Dumont MC40</td>
		</tr>
		<tr>
			<td>Streptomycin/penicillin</td>
			<td>Sigma</td>
			<td>781</td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Sigma</td>
			<td>P2256</td>
		</tr>
		<tr>
			<td>Soybean trypsin inhibitor</td>
			<td>Sigma</td>
			<td>T9128</td>
		</tr>
		<tr>
			<td>Tetracyclin</td>
			<td>Sigma</td>
			<td>T7660</td>
		</tr>
		<tr>
			<td>Veterinarian absorbable Vicryl thread</td>
			<td>Johnson&#38;Johson Intl</td>
			<td>JV1205</td>
		</tr>
		<tr>
			<td>Collagenase A</td>
			<td>Roche diagnostics</td>
			<td>10103586001</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PmeI</td>
			<td>New England Biolabs</td>
			<td>R0560S</td>
			<td rowspan="14">Preparation of synthetic mRNA</td>
		</tr>
		<tr>
			<td>DNAse/RNAse free H2O</td>
			<td>Life Technologies</td>
			<td>10977</td>
		</tr>
		<tr>
			<td>Sodium Acetate</td>
			<td>Merck</td>
			<td>6268</td>
		</tr>
		<tr>
			<td>Absolute ethanol</td>
			<td>Sigma</td>
			<td>02854</td>
		</tr>
		<tr>
			<td>Nano Drop</td>
			<td>Thermo Scientific</td>
			<td></td>
		</tr>
		<tr>
			<td>TBE 10X</td>
			<td>Eppendorf</td>
			<td>0032006.507</td>
		</tr>
		<tr>
			<td>Ethidium bromide 10 mg/mL</td>
			<td>Life Technologies</td>
			<td>15585-011</td>
		</tr>
		<tr>
			<td>mMESSAGE mMACHINE&#174; Kit</td>
			<td>Ambion by Life Technologies</td>
			<td>AM1344</td>
		</tr>
		<tr>
			<td>MOPS</td>
			<td>Sigma</td>
			<td>M1254</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Fluka</td>
			<td>03609</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Life Technologies</td>
			<td>16500-500</td>
		</tr>
		<tr>
			<td>Formaldehyde 37%</td>
			<td>Merck</td>
			<td>1.04003.1000</td>
		</tr>
		<tr>
			<td>Formamide</td>
			<td>Fluka</td>
			<td>47671</td>
		</tr>
		<tr>
			<td>Gel Doc Imager</td>
			<td>Biorad</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Oocyte Pipet with 100 8&#34; Glass Capillaries</td>
			<td>Drummond Scientific Company</td>
			<td>3-000-510-X</td>
			<td rowspan="4">microinjection</td>
		</tr>
		<tr>
			<td>Replacement Glass 8&#34;</td>
			<td>Drummond Scientific Company</td>
			<td>3-000-210-G8</td>
		</tr>
		<tr>
			<td>0,45 &#181;m filter</td>
			<td>Millipore</td>
			<td>SLHA025NB</td>
		</tr>
		<tr>
			<td>Progesterone</td>
			<td>Sigma</td>
			<td>P0130</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hepes</td>
			<td>Sigma</td>
			<td>H3375</td>
			<td rowspan="32">Western Blot</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S5886</td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>Biorad</td>
			<td>161-0301</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma</td>
			<td>A3294</td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma</td>
			<td>A4612</td>
		</tr>
		<tr>
			<td>leupeptin</td>
			<td>Sigma</td>
			<td>L8511</td>
		</tr>
		<tr>
			<td>aprotinin</td>
			<td>Sigma</td>
			<td>A1153</td>
		</tr>
		<tr>
			<td>benzamidine</td>
			<td>Sigma</td>
			<td>B6506</td>
		</tr>
		<tr>
			<td>PMSF</td>
			<td>Sigma</td>
			<td>P7626</td>
		</tr>
		<tr>
			<td>sodium vanadate</td>
			<td>Sigma</td>
			<td>S6508</td>
		</tr>
		<tr>
			<td>Laemmli buffer</td>
			<td>Biorad</td>
			<td>161-0737</td>
		</tr>
		<tr>
			<td>NuPAGE Novex 4-12% Bis-Tris Protein Gels, 1.0 mm, 15 well</td>
			<td>Life Technologies</td>
			<td>NP0323BOX</td>
		</tr>
		<tr>
			<td>SDS-PAGE electrophoresis, mini-Protean TGX</td>
			<td>Biorad</td>
			<td>456-1036 and -1096</td>
		</tr>
		<tr>
			<td>horizontal semi-dry blotting system</td>
			<td>W.E.P. Compagny</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Biorad</td>
			<td>161-0718</td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>Biorad</td>
			<td>161-0719</td>
		</tr>
		<tr>
			<td>Hybond ECL Membrane</td>
			<td>Amersham Life Science</td>
			<td>10401180</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>VWR</td>
			<td>20846-292</td>
		</tr>
		<tr>
			<td>Ponceau Red (0,5%)</td>
			<td>Sigma</td>
			<td>P3504</td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma</td>
			<td>P2287</td>
		</tr>
		<tr>
			<td>anti-GFP</td>
			<td>Life Technologies</td>
			<td>A11122</td>
		</tr>
		<tr>
			<td>anti-V5</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-58052</td>
		</tr>
		<tr>
			<td>anti-Eg2</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-27884</td>
		</tr>
		<tr>
			<td>anti-Eg2-P</td>
			<td>Cell Signaling</td>
			<td>C39D8</td>
		</tr>
		<tr>
			<td>anti-ERK2</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-1647</td>
		</tr>
		<tr>
			<td>anti-ERK2-P (Tyr204)</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-7976</td>
		</tr>
		<tr>
			<td>anti-Rsk</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-231</td>
		</tr>
		<tr>
			<td>anti-mos</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-86</td>
		</tr>
		<tr>
			<td>anti-mouse horseradish peroxidase labeled secondary antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-2005</td>
		</tr>
		<tr>
			<td>anti-rabbit horseradish peroxidase labeled secondary antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-2812</td>
		</tr>
		<tr>
			<td>anti-goat horseradish peroxidase labeled secondary antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-2378</td>
		</tr>
		<tr>
			<td>Advanced ECL Detection System</td>
			<td>Amershan Life Science</td>
			<td>RPN2135</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PBS 1x</td>
			<td>Sigma</td>
			<td>P4417</td>
			<td rowspan="11">polyadenylation assay</td>
		</tr>
		<tr>
			<td>TRIZOL (Qiazol Lysis Reagent)</td>
			<td>Qiagen</td>
			<td>79306</td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Sigma</td>
			<td>31998-8</td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>Sigma</td>
			<td>278475-1L</td>
		</tr>
		<tr>
			<td>RNeasy mini kit</td>
			<td>Qiagen</td>
			<td>74106</td>
		</tr>
		<tr>
			<td>RTL buffer&#160;</td>
			<td>Qiagen</td>
			<td>79216</td>
		</tr>
		<tr>
			<td>RNAse free DNAse set</td>
			<td>Qiagen</td>
			<td>79254</td>
		</tr>
		<tr>
			<td>Primers</td>
			<td>Eurogentec</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 RNA ligase 1&#160;</td>
			<td>New England Biolabs</td>
			<td>M0204S</td>
		</tr>
		<tr>
			<td>High capacity c-DNA Reverse Transcription kit</td>
			<td>Applied Biosystems, Life Technologies</td>
			<td>4368813</td>
		</tr>
		<tr>
			<td>Taq polymerase</td>
			<td>Life Technologies</td>
			<td>10342020</td>
		</tr>
	</tbody>
</table>

xenopus laevis as a model to identify translation impairment

Protein synthesis is a fundamental process to gene expression impacting diverse biological processes notably adaptation to environmental conditions. The initiation step, which involves the assembly of the ribosomal subunits at the mRNA initiation codon, involved initiation factor including eIF4G1. Defects in this rate limiting step of translation are linked to diverse disorders. To study the potential consequences of such deregulations, Xenopus laevis oocytes constitute an attractive model with high degrees of conservation of essential cellular and molecular mechanisms with human. In addition, during meiotic maturation, oocytes are transcriptionally repressed and all necessary proteins are translated from preexisting, maternally derived mRNAs. This inexpensive model enables exogenous mRNA to become perfectly integrated with an effective translation. Here is described a protocol for assessing translation with a factor of interest (here eIF4G1) using stored maternal mRNA that are the first to be polyadenylated and translated during oocyte maturation as a physiological readout. At first, mRNA synthetized by in vitro transcription of plasmids of interest (here eIF4G1) are injected in oocytes and kinetics of oocyte maturation by Germinal Vesicle Breakdown detection is determined. The studied maternal mRNA target is the serine/threonine-protein-kinase mos. Its polyadenylation and its subsequent translation are investigated together with the expression and phosphorylation of proteins of the mos signaling cascade involved in oocyte maturation. Variations of the current protocol to put forward translational defects are also proposed to emphasize its general applicability. In light of emerging evidence that aberrant protein synthesis may be involved in the pathogenesis of neurological disorders, such a model provides the opportunity to easily assess this impairment and identify new targets.

Kinetic maturation of Xenopus oocytes and determination of the percentage oocyte GVBD after 24 hr of PG stimulation (Figures 2B, 2C):
In order to study the translational consequences of the eIF4G1-DN mutation, the response to PG in Xenopus laevis oocytes microinjected with cRNA eIF4G1-DN is compared to eIF4G1-WT and to other control conditions (H2O, GFP). The controls enable to assess the incidence of oocyte microinjection regardless of the nature ...

Watch this Scientific Journal Video about Xenopus laevis as a Model to Identify Translation Impairment at JoVE.com

Xenopus laevis as a Model to Identify Translation Impairment

Protein synthesis control occurs mainly at the translation initiation step, deficiencies in which are linked to diverse disorders. To better understand their etiology, we described here a protocol using Xenopus laevis oocytes assessing the translation of mos transcript in the presence of a mutant of translation initiation factor eIF4G1.

Xenopus laevis as a Model to Identify Translation Impairment

Protein synthesis is a fundamental process to gene expression impacting diverse biological processes notably adaptation to environmental conditions. The ...

xenopus-laevis-as-a-model-to-identify-translation-impairment

Research

JoVE Journal

Biology

Dissection of Organizer and Animal Pole Explants from Xenopus laevis Embryos and Assembly of a Cell Adhesion Assay

This video demonstrates the technique used for preparation of organizer and animal pole explants from Xenopus laevis embryos, including the use of the eyebrow knife - a specialized dissection tool made of one's eyebrow. The protocol for assembling an adhesion assay is also given, which probes for the presence of key adhesion molecules present on the surface organizer or animal pole cells that are critical for proper development.

Plastic Embedding and Sectioning of Xenopus laevis Embryos

Plastic sections maintain true tissue morphology in thin sections of tissue that can be immunostained with fluorescent secondary antibodies, making this method more useful than paraffin-embedded or frozen sections for many types of tissue. The method for staining, plastic embedding, and sectioning is demonstrated in this video.

Obtaining Eggs from Xenopus laevis Females

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, collect and dejelly the eggs, and sort the eggs to remove any damaged eggs.

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, collect and dejelly the eggs, and sort the eggs to remove any damaged eggs.

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, ...

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 ...

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

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.

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

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes in cell surface area and cell height, and where cells undergo mitosis and migrate. Furthermore, epithelial cells can also regulate morphogenetic movements in adjacent tissues1. A traditional method to study epithelial cells and tissues involve chemical fixation and histological methods to determine cell morphology or localization of particular proteins of interest. These approaches continue to be useful and provide "snapshots" of cell shapes and tissue architecture, however, much remains to be understood about how cells acquire specific shapes, how various proteins move or localize to specific positions, and what paths cells follow toward their final differentiated fate. High resolution live imaging complements traditional methods and also allows more direct investigation into the dynamic cellular processes involved in the formation, maintenance, and morphogenesis of multicellular epithelial sheets. Here we demonstrate experimental methods from the isolation of animal cap tissues from Xenopus laevis embryos to confocal imaging of epithelial cells and simple measurement approaches that together can augment molecular and cellular studies of epithelial morphogenesis.

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Xenopus embryonic epithelia are an ideal model system to study cell behaviors such as polarity development and shape change during epithelial morphogenesis. Traditional histology of fixed samples is increasingly being complemented by live-cell confocal imaging. Here we demonstrate methods to isolate frog tissues and visualize live epithelial cells and their cytoskeleton using live-cell confocal microscopy.

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes ...

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 ...

Facial Transplants in Xenopus laevis Embryos

Craniofacial birth defects occur in 1 out of every 700 live births, but etiology is rarely known due to limited understanding of craniofacial development. To identify where signaling pathways and tissues act during patterning of the developing face, a 'face transplant' technique has been developed in embryos of the frog Xenopus laevis. A region of presumptive facial tissue (the "Extreme Anterior Domain" (EAD)) is removed from a donor embryo at tailbud stage, and transplanted to a host embryo of the same stage, from which the equivalent region has been removed. This can be used to generate a chimeric face where the host or donor tissue has a loss or gain of function in a gene, and/or includes a lineage label. After healing, the outcome of development is monitored, and indicates roles of the signaling pathway within the donor or surrounding host tissues. Xenopus is a valuable model for face development, as the facial region is large and readily accessible for micromanipulation. Many embryos can be assayed, over a short time period since development occurs rapidly. Findings in the frog are relevant to human development, since craniofacial processes appear conserved between Xenopus and mammals.

Facial Transplants in Xenopus laevis Embryos

A technique for transplanting "Extreme Anterior Domain" facial tissue between Xenopus laevis embryos has been developed. Tissue can be moved from one gene expression background into another, allowing the study of local requirements for craniofacial development and for signaling interactions between facial regions.

Craniofacial birth defects occur in 1 out of every 700 live births, but etiology is rarely known due to limited understanding of craniofacial development. ...

Using plusTipTracker Software to Measure Microtubule Dynamics in Xenopus laevis Growth Cones

Microtubule (MT) plus-end-tracking proteins (+TIPs) localize to the growing plus-ends of MTs and regulate MT dynamics1,2. One of the most well-known and widely-utilized +TIPs for analyzing MT dynamics is the End-Binding protein, EB1, which binds all growing MT plus-ends, and thus, is a marker for MT polymerization1. Many studies of EB1 behavior within growth cones have used time-consuming and biased computer-assisted, hand-tracking methods to analyze individual MTs1-3. Our approach is to quantify global parameters of MT dynamics using the software package, plusTipTracker4, following the acquisition of high-resolution, live images of tagged EB1 in cultured embryonic growth cones5. This software is a MATLAB-based, open-source, user-friendly package that combines automated detection, tracking, visualization, and analysis for movies of fluorescently-labeled +TIPs. Here, we present the protocol for using plusTipTracker for the analysis of fluorescently-labeled +TIP comets in cultured Xenopus laevis growth cones. However, this software can also be used to characterize MT dynamics in various cell types6-8.

Using plusTipTracker Software to Measure Microtubule Dynamics in Xenopus laevis Growth Cones

The MATLAB-based, open source software package, plusTipTracker, can be used to analyze image series of fluorescently-labeled +TIPs to quantify microtubule dynamics.

Microtubule (MT) plus-end-tracking proteins (+TIPs) localize to the growing plus-ends of MTs and regulate MT dynamics1,2. One of the most well-known and ...