Name: using mouse oocytes to assess human gene function during meiosis i
Uploaded: 2018-04-10T15:00:00
Description: Watch this Scientific Journal Video about Using Mouse Oocytes to Assess Human Gene Function During Meiosis I at JoVE.com

Dit werk werd ondersteund door een onderzoek subsidie van de American Society for Reproductive Medicine en van de Charles en Johanna Busch Memorial Fund bij Rutgers, The State University van NJ aan K.S. A.L.N. werd gesteund door een subsidie van de N.I.H. (F31 HD0989597).

1. moleculaire klonen
<ol><li>Verkrijgen van full-length codering sequentie van het gen van belang en de plasmide in vitro transcriptie (pIVT)11vector. Opmerking: Full-length cDNA klonen zijn commercieel verkrijgbaar bij verschillende leveranciers of via omgekeerde transcriptie polymerasekettingreactie (RT-PCR) kunnen worden gegenereerd. Het nationaal centrum voor biotechnologische informatie (NCBI) onlinebron biedt transcript sequenties van genen van de Nucleotide en Expressed seque...

<ol>
	<li>Thoma, M. E., 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=Prevalence+of+infertility+in+the+United+States+as+estimated+by+the+current+duration+approach+and+a+traditional+constructed+approach.">Prevalence of infertility in the United States as estimated by the current duration approach and a traditional constructed approach.</a> Fertil Steril. 99 (5), 1324-1331 (2013).</li><li>Boivin, J., Bunting, L., Collins, J. A., Nygren, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=International+estimates+of+infertility+prevalence+and+treatment-seeking:+potential+need+and+demand+for+infertility+medical+care.">International estimates of infertility prevalence and treatment-seeking: potential need and demand for infertility medical care.</a> Human Reproduction. 22 (6), 1506-1512 (2007).</li><li>Treff, N. R., Zimmerman, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+Preimplantation+Genetic+Testing+for+Monogenic+Disease+and+Aneuploidy.">Advances in Preimplantation Genetic Testing for Monogenic Disease and Aneuploidy.</a> Annu Rev Genomics Hum Genet. , (2017).</li><li>Franasiak, 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=The+nature+of+aneuploidy+with+increasing+age+of+the+female+partner:+a+review+of+15,169+consecutive+trophectoderm+biopsies+evaluated+with+comprehensive+chromosomal+screening.">The nature of aneuploidy with increasing age of the female partner: a review of 15,169 consecutive trophectoderm biopsies evaluated with comprehensive chromosomal screening.</a> Fertil Steril. 101 (3), 656-663 (2014).</li><li>Hassold, T., Hunt, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=To+err+(meiotically)+is+human:+the+genesis+of+human+aneuploidy.">To err (meiotically) is human: the genesis of human aneuploidy.</a> Nat Rev Genet. 2 (4), 280-291 (2001).</li><li>Scott, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blastocyst+biopsy+with+comprehensive+chromosome+screening+and+fresh+embryo+transfer+significantly+increases+in+vitro+fertilization+implantation+and+delivery+rates:+a+randomized+controlled+trial.">Blastocyst biopsy with comprehensive chromosome screening and fresh embryo transfer significantly increases in vitro fertilization implantation and delivery rates: a randomized controlled trial.</a> Fertil Steril. 100 (3), 697-703 (2013).</li><li>Scott, R. T., Upham, K. M., Forman, E. J., Zhao, T., Treff, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cleavage-stage+biopsy+significantly+impairs+human+embryonic+implantation+potential+while+blastocyst+biopsy+does+not:+a+randomized+and+paired+clinical+trial.">Cleavage-stage biopsy significantly impairs human embryonic implantation potential while blastocyst biopsy does not: a randomized and paired clinical trial.</a> Fertil Steril. 100 (3), 624-630 (2013).</li><li>Yang, Z., 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=Selection+of+single+blastocysts+for+fresh+transfer+via+standard+morphology+assessment+alone+and+with+array+CGH+for+good+prognosis+IVF+patients:+results+from+a+randomized+pilot+study.">Selection of single blastocysts for fresh transfer via standard morphology assessment alone and with array CGH for good prognosis IVF patients: results from a randomized pilot study.</a> Mol Cytogenet. 5 (1), 24 (2012).</li><li>Rubio, C., 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=In+vitro+fertilization+with+preimplantation+genetic+diagnosis+for+aneuploidies+in+advanced+maternal+age:+a+randomized,+controlled+study.">In vitro fertilization with preimplantation genetic diagnosis for aneuploidies in advanced maternal age: a randomized, controlled study.</a> Fertil Steril. 107 (5), 1122-1129 (2017).</li><li>Nguyen, A. L., 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=Identification+and+characterization+of+Aurora+Kinase+B+and+C+variants+associated+with+maternal+aneuploidy.">Identification and characterization of Aurora Kinase B and C variants associated with maternal aneuploidy.</a> Mol Hum Reprod. , (2017).</li><li>Igarashi, H., Knott, J. G., Schultz, R. M., Williams, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alterations+of+PLCbeta1+in+mouse+eggs+change+calcium+oscillatory+behavior+following+fertilization.">Alterations of PLCbeta1 in mouse eggs change calcium oscillatory behavior following fertilization.</a> Dev Biol. 312 (1), 321-330 (2007).</li><li>Database, J. S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+Methods+in+Cellular+and+Molecular+Biology.+Molecular+Cloning.">Basic Methods in Cellular and Molecular Biology. Molecular Cloning.</a> JoVE. , (2017).</li><li>Carey, M. F., Peterson, C. L., Smale, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PCR-mediated+site-directed+mutagenesis.">PCR-mediated site-directed mutagenesis.</a> Cold Spring Harb Protoc. 2013 (8), 738-742 (2013).</li><li>Armstrong, J. A., Schulz, J. 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> Current Protocols Essential Laboratory Techniques. , (2008).</li><li>Stein, P., Schindler, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+oocyte+microinjection,+maturation+and+ploidy+assessment.">Mouse oocyte microinjection, maturation and ploidy assessment.</a> J Vis Exp. (53), (2011).</li><li>Watanabe, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Geometry+and+force+behind+kinetochore+orientation:+lessons+from+meiosis.">Geometry and force behind kinetochore orientation: lessons from meiosis.</a> Nat Rev Mol Cell Biol. 13 (6), 370-382 (2012).</li><li>Shuda, K., Schindler, K., Ma, J., Schultz, R. M., Donovan, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aurora+kinase+B+modulates+chromosome+alignment+in+mouse+oocytes.">Aurora kinase B modulates chromosome alignment in mouse oocytes.</a> Mol Reprod Dev. 76 (11), 1094-1105 (2009).</li><li>Lane, S. I., Yun, Y., Jones, K. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Timing+of+anaphase-promoting+complex+activation+in+mouse+oocytes+is+predicted+by+microtubule-kinetochore+attachment+but+not+by+bivalent+alignment+or+tension.">Timing of anaphase-promoting complex activation in mouse oocytes is predicted by microtubule-kinetochore attachment but not by bivalent alignment or tension.</a> Development. 139 (11), 1947-1955 (2012).</li><li>Nguyen, A. L., 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=Phosphorylation+of+threonine+3+on+histone+H3+by+haspin+kinase+is+required+for+meiosis+I+in+mouse+oocytes.">Phosphorylation of threonine 3 on histone H3 by haspin kinase is required for meiosis I in mouse oocytes.</a> J Cell Sci. 127 (Pt 23), 5066-5078 (2014).</li><li>Tsafriri, A., Chun, S. Y., Zhang, R., Hsueh, A. J., Conti, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oocyte+maturation+involves+compartmentalization+and+opposing+changes+of+cAMP+levels+in+follicular+somatic+and+germ+cells:+studies+using+selective+phosphodiesterase+inhibitors.">Oocyte maturation involves compartmentalization and opposing changes of cAMP levels in follicular somatic and germ cells: studies using selective phosphodiesterase inhibitors.</a> Dev Biol. 178 (2), 393-402 (1996).</li><li>Kapoor, T. M., Mayer, T. U., Coughlin, M. L., Mitchison, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+spindle+assembly+mechanisms+with+monastrol,+a+small+molecule+inhibitor+of+the+mitotic+kinesin,+Eg5.">Probing spindle assembly mechanisms with monastrol, a small molecule inhibitor of the mitotic kinesin, Eg5.</a> Eg5. J Cell Biol. 150 (5), 975-988 (2000).</li><li>Jones, K. T., Lane, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+causes+of+aneuploidy+in+mammalian+eggs.">Molecular causes of aneuploidy in mammalian eggs.</a> Development. 140 (18), 3719-3730 (2013).</li><li>Fellmeth, J. E., 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=Expression+and+characterization+of+three+Aurora+kinase+C+splice+variants+found+in+human+oocytes.">Expression and characterization of three Aurora kinase C splice variants found in human oocytes.</a> Mol Hum Reprod. 21 (8), 633-644 (2015).</li><li>Rieder, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structure+of+the+cold-stable+kinetochore+fiber+in+metaphase+PtK1+cells.">The structure of the cold-stable kinetochore fiber in metaphase PtK1 cells.</a> Chromosoma. 84 (1), 145-158 (1981).</li><li>Brunet, 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=Kinetochore+fibers+are+not+involved+in+the+formation+of+the+first+meiotic+spindle+in+mouse+oocytes,+but+control+the+exit+from+the+first+meiotic+M+phase.">Kinetochore fibers are not involved in the formation of the first meiotic spindle in mouse oocytes, but control the exit from the first meiotic M phase.</a> J Cell Biol. 146 (1), 1-12 (1999).</li><li>Joung, 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=Genome-scale+CRISPR-Cas9+knockout+and+transcriptional+activation+screening.">Genome-scale CRISPR-Cas9 knockout and transcriptional activation screening.</a> Nat Protoc. 12 (4), 828-863 (2017).</li><li>Cong, L., 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=Multiplex+genome+engineering+using+CRISPR/Cas+systems.">Multiplex genome engineering using CRISPR/Cas systems.</a> Science. 339 (6121), 819-823 (2013).</li></ol>

Vanwege de snelle en toenemende identificatie van menselijke genetische varianten ziekte is gekoppeld, is het noodzakelijk dat systemen voor de evaluatie van hun biologische betekenis worden vastgesteld. Begrip eiwitfunctie in menselijke meiose poses bijzondere uitdagingen omdat menselijke eicellen kostbare zijn en zeldzame en menselijk sperma niet vatbaar voor genetische manipulatie zijn. Muis eicellen zijn een waardevol voor de evaluatie van menselijk Meiotische gen functie 10,<sup cl...

Onvruchtbaarheid is een aandoening die 10-15% van de menselijke bevolking van reproductieve leeftijd 1, waaruit bijna de helft medische behandeling 2 zoekenbeïnvloedt. Hoewel de etiologie van onvruchtbaarheid divers is en in veel gevallen multifactoriële, de meest voorkomende genetische afwijking bij de mens embryonale aneuploïdie 3 is. Aneuploïdie wordt gedefinieerd als de afwijking (winst of verlies) van het juiste aantal chromosomen in een cel. Het fenomeen van aneuploïdie in menselijke embryo's is gemeenschappelijk en verhoogt met geavanceerde maternale leeftijd 4,5. Vier gerandomiseerde gecontroleerde onderzoeken gebleken dat het voordeel van de selectie van embryo's voor alleen inplanten normale-(euploïde) voor de overdracht van de baarmoeder, omdat deze strategie resulteerde in toegenomen implantatie tarieven, lagere tarieven van de miskraam en een kortere tijd voor bereiken van zwangerschap 6,,7,,8,9. Begrip van de etiologie van menselijke aneuploïdie kan dus grote gevolgen hebben in geassisteerde reproductie.
Hoewel de pre-implantatie genetische tests voor aneuploidies is gunstig in behandelingen van onvruchtbaarheid, een grondige kennis van hoe aneuploidies afkomstig zijn ontbreekt nog. Het is algemeen aanvaard dat er een positieve correlatie van Meiotische aneuploidies (ontstaan tijdens gameet productie) en maternale leeftijd, echter sommige vrouwen aanwezig embryonale aneuploïdie tarieven die van het gemiddelde tarief voor hun bepaalde leeftijd 4 afwijkenis. Deze gevallen suggereren dat leeftijd alleen niet altijd voorspellende van het risico van het genereren van een aneuploid embryo. Andere factoren kunnen een rol spelen bij waardoor het risico van embryonale aneuploïdie, zoals gene varianten.
Een belangrijk aspect van het onderzoek naar de potentiële bijdrage van een gen variant aneuploïdie tijdens oöcyt meiose is het ontwerpen van een systeem voor het snel evalueren Meiotische genfunctie. Als gevolg van de ethische beperkingen en een beperkte toegang is het onpraktisch voor het uitvoeren van deze experimenten met behulp van menselijke eicellen. Deze kwesties kunnen worden omzeild door het gebruik van de muis eicellen, en hier een serie van tests te beoordelen van menselijke genfunctie tijdens de meiose I zijn beschreven. Door microinjecting de boodschapper-RNA (mRNA) codering voor de gen variant van belang, kan de lokalisatie van de menselijke eiwitten in het ei van de muis worden gevisualiseerd en gebruikt om te bepalen als de ectopische expressie van het wild-type en gemuteerde menselijke eiwit in een resulteert fenotypische veranderingen die tot aneuploïdie leiden kunnen. Deze fenotypen omvatten een verhoging in microtubuli die aan het ongepast om zuster hechten kinetochoor en het onvermogen om ondersteuning van chromosoom uitlijning op de metafase van meiose I. belangrijker, dit protocol kan worden gebruikt voor het onderzoeken van zowel winst en verlies van functie genetische varianten door de oprichting van specifieke experimentele omstandigheden aan te vechten van belangrijke gebeurtenissen in oöcyt meiose zoals spindel gebouw en chromosoom uitlijning 10.

<table>
	<tbody>
		<tr>
			<td>0.2 mL Seal-Rite PCR tube</td>
			<td>USA Scientific</td>
			<td>1602-4300</td>
			<td></td>
		</tr>
		<tr>
			<td>1 kb DNA Ladder</td>
			<td>Thermo Scientific</td>
			<td>SM0313</td>
			<td></td>
		</tr>
		<tr>
			<td>100 bp DNA Ladder</td>
			<td>Thermo Scientific</td>
			<td>SM0243</td>
			<td></td>
		</tr>
		<tr>
			<td>6X DNA loading Dye</td>
			<td>Thermo Scientific</td>
			<td>R0611</td>
			<td></td>
		</tr>
		<tr>
			<td>9-well glass spot plate</td>
			<td>Thomas Scientific</td>
			<td>7812G17</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Sigma Aldrich</td>
			<td>A9539</td>
			<td></td>
		</tr>
		<tr>
			<td>Albumin from bovine serum&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>A3294&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa-fluor-568 conjugated anti-mouse IgG</td>
			<td>Thermo Scientific</td>
			<td>A21050</td>
			<td>1:200 dilution</td>
		</tr>
		<tr>
			<td>Alexa-fluor-633 conjugated anti-human IgG</td>
			<td>Thermo Scientific</td>
			<td>A21091</td>
			<td>1:200 dilution</td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>VWR</td>
			<td>AA0356</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-vibration table</td>
			<td>Technical Manufacturing Corp</td>
			<td></td>
			<td>any standard model</td>
		</tr>
		<tr>
			<td>Anti-Acetylated Tubulin antibody</td>
			<td>Sigma Aldrich</td>
			<td>T7451</td>
			<td>1:100 diution</td>
		</tr>
		<tr>
			<td>Anti-centromeric (CREST) antibody</td>
			<td>Antibodies Incorportated</td>
			<td>15-234</td>
			<td>1:30 dilution&#160;</td>
		</tr>
		<tr>
			<td>Barrier (Filter) Pipette tips</td>
			<td>Thermo Scientific</td>
			<td>AM12635</td>
			<td>Make sure compatable with your brand of pipettors. These are compatible with Eppendorf brand pipettors.&#160;</td>
		</tr>
		<tr>
			<td>BD Difco Dehydrated Culture Media: LB Agar, Miller (Luria Bertani)</td>
			<td>Fisher Scientific</td>
			<td>DF0445-07-6</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Difco Dehydrated Culture Media: LB Broth, Miller (Luria Bertani)</td>
			<td>Fisher Scientific</td>
			<td>DF0446-07-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Capillary tubing</td>
			<td>Sutter</td>
			<td>B100-75-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Center Well organ culture dish</td>
			<td>VWR</td>
			<td>25381-141</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 tank</td>
			<td></td>
			<td></td>
			<td>For incubator</td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td>any standard model</td>
		</tr>
		<tr>
			<td>Centrifuge (With cooling ability)</td>
			<td>Thomas Scientific</td>
			<td></td>
			<td>any standard model&#160;</td>
		</tr>
		<tr>
			<td>Cover Glass 11 x 22 mm</td>
			<td>Thomas Scientific</td>
			<td>6663F10</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslips</td>
			<td>Thomas Scientific</td>
			<td>6663-F10</td>
			<td>thickness will vary for particular microscopes</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma Aldrich</td>
			<td>D9542</td>
			<td></td>
		</tr>
		<tr>
			<td>DEPC H20</td>
			<td>Life Technologies</td>
			<td>AM9922</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital Dry Bath</td>
			<td>Thermo Scientific</td>
			<td>888700001</td>
			<td></td>
		</tr>
		<tr>
			<td>Easy A high fidelity cloning enzyme</td>
			<td>Agilent</td>
			<td>600400</td>
			<td>For DNA cloning&#160;</td>
		</tr>
		<tr>
			<td>Enzymes for linearizing pIVT</td>
			<td>New England Biolabs</td>
			<td></td>
			<td>NdeI or KasI can be used</td>
		</tr>
		<tr>
			<td>Ethidium Bromide</td>
			<td>Thermo Scientific</td>
			<td>155585011</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent Microscope&#160;</td>
			<td></td>
			<td></td>
			<td>Any Fluorescent microscope may be used</td>
		</tr>
		<tr>
			<td>Formaldehyde (37%)</td>
			<td>Thermo Fisher Scientifc</td>
			<td>9311</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde RNA loading dye</td>
			<td>Ambion</td>
			<td>8552</td>
			<td></td>
		</tr>
		<tr>
			<td>Frosted Microscope Slides (Uncharged) 25X75 mm</td>
			<td>Fisher Scientific</td>
			<td>12-544-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Full Length cDNA Clones</td>
			<td></td>
			<td></td>
			<td>Can be obtained from any vendor that supplies open reading frame clones</td>
		</tr>
		<tr>
			<td>Gel electrophoresis apparatus</td>
			<td>Bio-Rad</td>
			<td></td>
			<td>any standard model</td>
		</tr>
		<tr>
			<td>Glass Pasteur Pipets</td>
			<td>Fisher Scientific</td>
			<td>13-678-200</td>
			<td></td>
		</tr>
		<tr>
			<td>Globin Forward Primer for pIVT Construct</td>
			<td></td>
			<td></td>
			<td>5&#39;- GAA GCT CAG AAT AAA CGC -3&#39;.&#160; Can be purchased from any company that generates custom oligonucelotides</td>
		</tr>
		<tr>
			<td>Globin Reverse Primer for pIVT Construct</td>
			<td></td>
			<td></td>
			<td>5&#39;- ATT CGG GTG TTC TTG AGG CTG G -3&#39; Can be purchased from any company that generates custom oligonucelotides</td>
		</tr>
		<tr>
			<td>Holding pipettes</td>
			<td>Eppendorf</td>
			<td>930001015</td>
			<td>Vacutip</td>
		</tr>
		<tr>
			<td>Humidified Chamber</td>
			<td></td>
			<td></td>
			<td>Tupperware can be used</td>
		</tr>
		<tr>
			<td>Illustra Ready-To-Go RT-PCR beads</td>
			<td>GE Life Sciences</td>
			<td>27925901</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td></td>
			<td></td>
			<td>any standard model with CO2 and water jacketed technology</td>
		</tr>
		<tr>
			<td>Inverted Microscope</td>
			<td>Nikon instruments</td>
			<td></td>
			<td>Any Standard model</td>
		</tr>
		<tr>
			<td>Image J (NIH) Software</td>
			<td>NIH</td>
			<td></td>
			<td>Image Analysis software</td>
		</tr>
		<tr>
			<td>Lid of 96 well plate</td>
			<td>Nalgene Nunc International</td>
			<td>263339</td>
			<td></td>
		</tr>
		<tr>
			<td>Low Adhesion 0.5 mL microcentrifgue tube</td>
			<td>USA Scientific</td>
			<td>1405-2600</td>
			<td></td>
		</tr>
		<tr>
			<td>MacVector&#160;</td>
			<td>MacVector</td>
			<td></td>
			<td>Sequence analysis software</td>
		</tr>
		<tr>
			<td>MG132</td>
			<td>Selleckchem</td>
			<td>S2619</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope slides</td>
			<td>Fisher Scientific</td>
			<td>12-544-3&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Millenium RNA Markers-Formaldehyde&#160;</td>
			<td>Ambion</td>
			<td>AM7151</td>
			<td></td>
		</tr>
		<tr>
			<td>Milrinone</td>
			<td>Sigma-Aldrich</td>
			<td>M4659</td>
			<td>Resuspend in DMSO at 2.5mM</td>
		</tr>
		<tr>
			<td>Mineral Oil</td>
			<td>Sigma-Aldrich</td>
			<td>M5310</td>
			<td>Only used embryo-tested, sterile-filtered</td>
		</tr>
		<tr>
			<td>Monastrol</td>
			<td>Sigma-Aldrich</td>
			<td>M8515</td>
			<td>Resuspend in DMSO at 100 mM</td>
		</tr>
		<tr>
			<td>Mouthpiece</td>
			<td>Biodiseno</td>
			<td>MP-001-Y</td>
			<td></td>
		</tr>
		<tr>
			<td>N2 tank</td>
			<td></td>
			<td></td>
			<td>for antivibration table</td>
		</tr>
		<tr>
			<td>Nail Polish; Clear</td>
			<td></td>
			<td></td>
			<td>Any clear nailpolish can be used</td>
		</tr>
		<tr>
			<td>NanoDrop Microvolume UV-Vis Spectrophotometer</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td>any standard model</td>
		</tr>
		<tr>
			<td>NorthernMax 10X Denaturing Gel Buffer</td>
			<td>Life Technologies</td>
			<td>AM8676</td>
			<td></td>
		</tr>
		<tr>
			<td>NorthernMax 10X Running buffer</td>
			<td>Life Technologies</td>
			<td>AM8671</td>
			<td></td>
		</tr>
		<tr>
			<td>NuPAGE MOPS SDS Running buffer</td>
			<td>Thermo Scnentific</td>
			<td>NP0001</td>
			<td></td>
		</tr>
		<tr>
			<td>Organ Culture Dish 60x15mm</td>
			<td>Life Technologies</td>
			<td>08-772-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Polysciences, Inc.&#160;</td>
			<td>577773</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR Thermal Cycler</td>
			<td>Thermo Fisher Scientific</td>
			<td>4484075</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri Dish 139 mm</td>
			<td>Thermo Fisher Scientifc</td>
			<td>501V</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish 35 mm</td>
			<td>Thermo Fisher Scientifc</td>
			<td>121V</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri Dish 60 mm</td>
			<td>Falcon BD</td>
			<td>351007</td>
			<td></td>
		</tr>
		<tr>
			<td>Picoinjector</td>
			<td>XenoWorks Digital Microinjector</td>
			<td></td>
			<td>any standard model</td>
		</tr>
		<tr>
			<td>Pipette puller</td>
			<td>Flaming-Brown Micropipette puller</td>
			<td>Model P-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>pIVT plasmid</td>
			<td>AddGene</td>
			<td>32374</td>
			<td>Empty vector suitable for oocyte expression.</td>
		</tr>
		<tr>
			<td>Pregnant Mare Serum Gonadotropin</td>
			<td>Lee BioSolutions</td>
			<td>493-10</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAprep Spin Miniprep Kit</td>
			<td>Qiagen</td>
			<td>27104</td>
			<td>purification of up to 20 uL of plasmid DNA</td>
		</tr>
		<tr>
			<td>QIAquick PCR purification kit</td>
			<td>Qiagen</td>
			<td>28104</td>
			<td></td>
		</tr>
		<tr>
			<td>Quikchange II site directed mutagenesis kit</td>
			<td>Agilent&#160;</td>
			<td>200523</td>
			<td>mutagenesis kit for insertions and deletions</td>
		</tr>
		<tr>
			<td>Quikchange lightning multi-site directed mutagenesis kit</td>
			<td>Agilent&#160;</td>
			<td>210512</td>
			<td>mutagenesis kit for single site changes</td>
		</tr>
		<tr>
			<td>Scissors (Fine point)</td>
			<td>Fine science tools</td>
			<td>14393</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors (Medium point)</td>
			<td>Fine science tools</td>
			<td>WP114225</td>
			<td></td>
		</tr>
		<tr>
			<td>Seal-Rite 1.5 mL microcentrifuge tube</td>
			<td>USA Scientific</td>
			<td>1615-5500</td>
			<td></td>
		</tr>
		<tr>
			<td>Slide Warmer</td>
			<td></td>
			<td></td>
			<td>any standard model</td>
		</tr>
		<tr>
			<td>Spectrophotometer (Nanodrop)</td>
			<td>Thermo Fisher Scientific</td>
			<td>ND-ONE-W</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td></td>
			<td></td>
			<td>any standard model</td>
		</tr>
		<tr>
			<td>Subcloning Efficiency DH5a Competent Cells</td>
			<td>Thermo Fisher Scientifc</td>
			<td>18265017</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe</td>
			<td>BD Bioscienes</td>
			<td>309623</td>
			<td>1 ml, 27G(1/2)</td>
		</tr>
		<tr>
			<td>T4 DNA Ligase</td>
			<td>New England Biolabs</td>
			<td>M0202L</td>
			<td></td>
		</tr>
		<tr>
			<td>T7 mMessage Machine high-yield capped RNA transcription kit</td>
			<td>Life Technologies</td>
			<td>AM1340</td>
			<td></td>
		</tr>
		<tr>
			<td>TritonX-100</td>
			<td>Sigma-Aldrich</td>
			<td>x-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Sigma-Aldrich</td>
			<td>274348</td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezer (Fine point- Size 5)</td>
			<td>Fine science tools</td>
			<td>SN.743.12.1</td>
			<td></td>
		</tr>
		<tr>
			<td>UltraPure Dnase/Rnase-Free Distilled Water</td>
			<td>Thermo Fisher Scientifc</td>
			<td>10977015</td>
			<td></td>
		</tr>
		<tr>
			<td>UltraPure Ethidium Bromide 10mg/mL</td>
			<td>Thermo Fisher Scientifc</td>
			<td>15585011</td>
			<td></td>
		</tr>
		<tr>
			<td>UVP UV/White lite transilluminator</td>
			<td>Fisher Scientific</td>
			<td>UV95041501</td>
			<td></td>
		</tr>
		<tr>
			<td>Vectashield Mounting Medium</td>
			<td>Vector Laboratories</td>
			<td>H-1000</td>
			<td></td>
		</tr>
	</tbody>
</table>

using mouse oocytes to assess human gene function during meiosis i

Embryonale aneuploïdie is de voornaamste genetische oorzaak van onvruchtbaarheid bij de mens. De meeste van deze evenementen zijn afkomstig tijdens vrouwelijke meiose, en zij positief gecorreleerd met de maternale leeftijd, leeftijd alleen is niet altijd voorspellende van het risico van het genereren van een aneuploid embryo. Varianten van de gen kunnen daarom goed voor onjuiste chromosoom segregatie tijdens de oögenese. Gezien het feit dat toegang tot menselijke eicellen is beperkt voor onderzoeksdoeleinden, een serie van tests werden ontwikkeld om de studie van menselijke genfunctie tijdens meiose ik met behulp van de muis eicellen. Ten eerste zijn boodschapper-RNA (mRNA) van het gen en gen variant van belang microinjected in de profase ik-gearresteerd muis eicellen. Na die tijd voor expressie, eicellen synchroon vrijkomen Meiotische rijping te voltooien meiose ik. Door tagging de mRNA met een opeenvolging van een fluorescerende verslaggever, zoals groen fluorescente proteïne (Gfp), kan de lokalisatie van het menselijke eiwit naast de fenotypische wijzigingen worden beoordeeld. Bijvoorbeeld, winst of verlies van functie kan worden onderzocht door middel van experimentele omstandigheden die uitdaging van het gen product Meiotische fouten te herstellen. Hoewel dit systeem gunstig is in het onderzoek naar menselijke eiwitfunctie tijdens de oögenese, moet voldoende interpretatie van de resultaten worden verricht, gezien het feit dat eiwit expressie niet op endogene niveaus is en, tenzij gecontroleerd voor (dat wil zeggen klopte uit of naar beneden) zijn lymfkliertest homologen ook aanwezig in het systeem.

Na in vitro hebben transcriptie hoogwaardige RNA een verhouding van A260/A280 voor (1.8-2.2) en een A260/A230 verhouding ≥1.7 als gemeten met een spectrofotometer. De afbeelding in Figuur 1 ziet u de migratie van in vitro-RNA geproduceerd op een denatureren agarose gel na elektroforese. Een band die is ingesmeerd in patroon of een monster met meerdere formaat banden kunt aangeven verontreiniging of aantasting van het monster. Als alternati...

Watch this Scientific Journal Video about Using Mouse Oocytes to Assess Human Gene Function During Meiosis I at JoVE.com

Using Mouse Oocytes to Assess Human Gene Function During Meiosis I

Als de genetische varianten die zijn gekoppeld aan ziekten bij de mens begint te worden blootgelegd, wordt het steeds belangrijker systemen waarmee snel evalueren de biologische betekenis van deze geïdentificeerde varianten te ontwikkelen. Dit protocol wordt beschreven methoden voor de evaluatie van menselijke genfunctie tijdens vrouwelijke meiose ik met behulp van de muis eicellen.

Met behulp van de muis eicellen te beoordelen van menselijke genfunctie tijdens de meiose I

Embryonic aneuploidy is the major genetic cause of infertility in humans. Most of these events originate during female meiosis, and albeit positively ...

The overall goal of this series of assays is to investigate human gene function during Meiosis I using mouse oocytes. These methods can help answer key questions in the human reproductive field, such as whether there's a genetic predisposition to maternal aneuploidy. The main advantage of this technique is that it uses mouse oocytes as a model for mammalian meiosis, which is useful given the inaccessibility of human eggs for gene function studies.We first had the idea for this method when we sequenced the exomes of women who had undergone in vitro fertilization treatments and who's embryonic aneuploidy rates could not be explained by their maternal age. Visual demonstration of this method is critical, as the image analysis steps are difficult to learn and defining strict analysis parameters is key to rigor and reproducibility. Using a validated cloning strategy, insert the full-length coding sequence of the gene of interest into the multiple cloning region of the in vitro transcription vector.Mutagenize the DNA if generating single nucleotide polymorphisms or insertions and deletions, as described in the text protocol. Next, transform the mutagenized DNA into a bacterial host. Use a kit to isolate and purify the plasmid, following the manufacturer's instructions.Then, linearize the final products using single restriction enzyme digestion of the purified DNA. Purify the digested product using a validated DNA cleanup protocol or a kit. And elute in 30 microliters of RNase/DNase-free water.In vitro, transcribe DNA using T7 polymerase following the manufacturer's protocol. Finally, purify and elute RNA in 30 microliters of RNase/DNase-free water using a silica matrix spin-column based nucleic acid purification kit following the manufacturer's protocol. Divide oocytes into equal groups for microinjection.Microinject one group with experimental mRNA and the other group with the wild-type control NPBs or GFP mRNA. Using a hand or mouth-operated pipetting apparatus where the glass pipette opening is slightly larger than the diameter of an oocyte, transfer oocytes into a 100 microliter drop of milrinone-free culture medium in a Petri dish covered in embryo-quality mineral oil. Then, incubate the oocytes for seven hours at 37 degrees Celsius in 5%CO2 to allow sufficient maturation to metaphase of Meiosis I.After incubation, use the same pipette apparatus to transfer the oocytes into a center-well organ culture dish containing 700 microliters of pre-chilled MEM collection medium in the center well and 500 microliters of water in the outside ring.Place the dish on ice for seven minutes. Fix the oocytes by transferring them into a well of a clear glass spot plate containing 400 microliters of 2%paraformaldehyde in 1x PBS. Incubate for 20 minutes at room temperature.Following immunostaining, as described in the text protocol, image the oocytes using a 40 to 63x objective on a confocal microscope. Capture small steps in the Z-plane optimized for the working objective, and image the entire region of the metaphase spindle. To identify the kinetochore microtubule attachment status, first open the image by dragging the file into the ImageJ toolbar.In the opened Z-series, navigate to the image drop down menu, and under the Color sub tab, click Merge Channels to make all channels visible. It is critical that the analyst be familiar with what type of attachments to kinetochores are possible in mouse oocytes. The quality of the image will greatly influence your ability to properly analyze these data.Using the toggle at the bottom of the image, move through each Z slice and determine kinetochore microtubule attachment. Using the same pipette apparatus, transfer the oocytes into a 100 microliter drop of milrinone-free culture medium under oil. Incubate the oocytes for seven hours to allow sufficient maturation to metaphase of Meiosis I as before.Then, transfer the oocytes to a center-well organ culture dish containing 700 microliters of culture medium containing monastrol in the center well and 400 microliters of water in the surrounding ring. Incubate at 37 degrees Celsius for two hours. Rinse out the monastrol by transferring oocytes through three drops of 100 microliters of CZB culture medium.Then transfer the oocytes to a new center-well organ culture dish containing 700 microliters of culture medium plus proteasome inhibitor in the center ring. Add 400 microliters of water to the outside ring and incubate for three hours. Next, fix the oocytes by transferring them into a well of a clear glass spot plate containing 400 microliters of 2%paraformaldehyde in PBS for 20 minutes at room temperature.Image the oocytes using a 40 to 63x objective on a confocal microscope, capturing less than one micron steps in the Z-plane and making sure to image the entire region of the metaphase spindle. To identify chromosome alignment status, open the image files using imaging software. Then drag the image into the ImageJ control panel.In the open Z-series, use the point tool to mark points at the end of each spindle pole in their respective Z slice by placing the tool over the spindle pole in the image and clicking on the image. Add these points to the ROI Manager by simultaneously pressing the command button and the T button on the keyboard. Determine the specific coordinates for each of the set positions by highlighting the specific point in the ROI Manager and clicking the Measure button.This will provide a Results table containing X and Y coordinates for each point, which are the X1, Y1 and X1, Y2 points, respectively. Next, determine the true Z coordinate. This is done by multiplying the slice number of the specific Z slice in the Z stack in which the spindle pole was identified by the stack thickness.These will be the Z1 and Z2 points, respectively. Determine the spindle length using the Pythagorean theorem equation with the previously defined X, Y, and Z coordinates. Next, determine the spindle midzone.This is calculated as 1/2 the length of the spindle. Click the line icon in the ImageJ toolbar and draw a line that is from one spindle pole to the spindle midzone. The length will be displayed on the ImageJ toolbar.Defining strict chromosome alignment parameters allows for a robust quantitative analysis, important in data reproducibility. Determine the chromosome alignment status by assessing chromosome distance from the spindle midzone using the line measurement tool. Click the line icon in the ImageJ toolbar, and draw a line from the spindle midzone to the chromosome.The length will be displayed on the ImageJ toolbar. Chromosomes greater than four microns from the spindle midzone are considered unaligned. After the oocytes have undergone immunostaining and imaging, correct microtubule kinetochore attachments can be assessed.In a normal attachment, each sister kinetochore pair of the bivalent is attached to spindle microtubules from opposite poles. Up to three different types of abnormal attachments may be observed. One example occurs when there are no microtubules contacting the kinetochore.Alternatively, microtubules can bind incorrectly, producing syntelic or merotelic attachments. Syntelic attachments form with both pairs of sister kinetochores attached to microtubules emanating from a single pole. Merotelic attachments occur when the sister kinetochores in a pair are attached to opposite poles.After oocytes recover from the induced misalignment, the ability for chromosomes to realign is assessed by imaging. In this example, one chromosome bivalent is considered misaligned since it is located at more than four microns from the spindle midzone. When choosing which assay is appropriate for assessing gene variant function, it's important to remember that the human gene variants under study are being overexpressed and that the endogenous mouse homologs may need to be depleted at the same time.Following this procedure, other methods like CRISPR-Cas9 to create humanized mouse's strains can be performed to answer additional questions, such as understanding how the gene variant influences egg and embryo quality and if it impacts reproductive health of the individual. After its development, this technique paved the way for researchers in the field of reproductive genetics to explore other causative factors to maternal aneuploidy in humans. Gene identification could provide powerful diagnostics and a basis for studies at correcting aneuploidies.Thank you for watching. Good luck with your experiments.

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JoVE Core

JoVE Journal

Cell Biology

Molecular Biology

Genetics

Unit 2

Unit 1

Meiosis

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