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

Este trabalho foi apoiado por uma bolsa de pesquisa da sociedade americana de medicina reprodutiva e do Charles e Johanna Busch Memorial Fund na Rutgers, Universidade do estado de NJ para Konzen A.L.N. foi apoiada por uma concessão do N.I.H (F31 HD0989597).

1. molecular clonagem
<ol><li>Obter a sequência de código completo do gene de interesse e o plasmídeo em vitro transcrição (pIVT) vetor11. Nota: Clones de cDNA completos estão comercialmente disponíveis a partir de vários fornecedores ou podem ser gerados através da reação em cadeia da polimerase transcrição reversa (RT-PCR). Centro Nacional para recursos on-line Biotechnology Information (NCBI) fornece sequências de transcrição de genes do nucleotídeo e sequência e...

<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>

Devido a rápida e crescente identificação do humanas variantes genéticas associadas com a doença, é essencial que os sistemas são estabelecidos para avaliar seu significado biológico. Compreensão da função das proteínas na meiose humana coloca desafios específicos porque oócitos humanos são preciosos e raras e humana de esperma não é passíveis de manipulação genética. Oócitos mouse são um sistema de modelo mamíferos valioso para avaliar o gene humano meiótica função 10<s...

Infertilidade é uma condição que afeta 10-15% da população humana da idade reprodutiva 1, dos quais quase metade procura tratamento médico 2. Embora a etiologia da infertilidade é diversa e em muitos casos multifatoriais, a anomalia genética mais comum em seres humanos é embrionário aneuploidia 3. Aneuploidia é definida como o desvio (ganho ou perda) do número correto de cromossomos em uma célula. O fenômeno de aneuploidia em embriões humanos é comum e aumenta com a idade materna avançada, 4,5. Quatro ensaios controlados randomizados evidenciaram o benefício da seleção de embriões (euploid) apenas cromossomicamente normais para transferência uterina porque esta estratégia resultou na implantação de aumento de taxas, menores taxas de aborto e um menor tempo para alcançar a gravidez 6,7,8,9. Portanto, compreender a etiologia da aneuploidia humana pode ter implicações importantes na reprodução assistida.
Embora o teste genético pré-implantação para aneuploidies é benéfica em tratamentos de infertilidade, continua a faltar uma compreensão completa de como originou aneuploidies. É amplamente aceito que existe uma correlação positiva de aneuploidies meióticas (originou-se durante a produção de gametas) e a idade materna, no entanto, algumas taxas de aneuploidia embrionário presente mulheres que desviam a taxa média para sua idade determinada 4. Estes casos sugerem que idade sozinha nem sempre é preditiva do risco de gerar um embrião aneuploid. Outros fatores podem desempenhar um papel no aumento do risco de aneuploidia embrionária, tais como variantes do gene.
Um aspecto fundamental de investigar o potencial de contribuição de uma variante do gene de aneuploidia durante a meiose oócito é projetar um sistema para avaliar rapidamente a função do gene meiótica. Devido a restrições éticas e acesso limitado, é impraticável para realizar esses experimentos usando ovos humanos. Estas questões podem ser contornadas usando oócitos de rato, e aqui uma série de ensaios para avaliar a função do gene humano durante a meiose são descritos. Por microinjecting a codificação do RNA mensageiro (mRNA) para a variante do gene de interesse, a localização da proteína humana no ovo do mouse pode ser visualizada e usada para determinar se a expressão ectópica da proteína humana tipo selvagem e mutante resulta em qualquer alterações fenotípicas que poderiam levar à aneuploidia. Estes fenótipos incluem um aumento no microtúbulos que anexar para o inadequado à irmã cinetócoro e a incapacidade de apoiar o alinhamento de cromossomos em metáfase da meiose I. importante, este protocolo pode ser usado para investigar tanto ganho e perda de função variantes genéticas através do estabelecimento de condições experimentais específicas para desafiar os principais eventos na meiose oócito tais como eixo de alinhamento edifício e cromossomo 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

Aneuploidia embrionária é a genética das principais causas de infertilidade em seres humanos. A maioria destes eventos se originam durante a meiose feminina, e embora positivamente correlacionada com a idade materna, idade sozinha nem sempre é preditiva do risco de gerar um embrião aneuploid. Portanto, variantes do gene podem contabilizar segregação do cromossomo incorreta durante a ovogênese. Dado que o acesso aos oócitos humanos é limitado para fins de pesquisa, uma série de ensaios foram desenvolvidos para estudar a função do gene humano durante a meiose I usando oócitos de rato. Primeiro, o RNA mensageiro (mRNA) do gene e variante do gene de interesse são injetadas em oócitos de rato prendi prófase. Após permitir tempo para expressão, oócitos sincronicamente são liberados na maturação meiose para completar a meiose eu. Marcando o mRNA com uma sequência de um repórter fluorescente, tais como a proteína verde fluorescente (Gfp), a localização da proteína humana pode ser avaliada para além das alterações fenotípicas. Por exemplo, ganho ou perda de função pode ser investigada através do estabelecimento de condições experimentais que desafiam o produto do gene para corrigir erros meióticos. Embora este sistema é vantajoso em investigar a função da proteína humana durante a ovogênese, adequada interpretação dos resultados deve ser realizada, dado que a expressão da proteína não está em níveis endógenos e, se não for controlada por (ou seja batido para fora ou para baixo), homologs murino também estão presentes no sistema.

Após in vitro transcrição RNA de alta qualidade terão uma relação A260/A280 (1.8-2.2) e um ≥1.7 de relação A260/A230 quando medido utilizando um espectrofotômetro. A imagem na Figura 1 mostra a migração do in vitro-produzido RNA em um gel de agarose a desnaturação, após eletroforese. Uma banda que é manchada no padrão ou uma amostra que tem várias faixas de tamanhos pode indicar a contaminação ou degradação da amostra....

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

Como as variantes genéticas associadas a doenças humanas começam a tornar-se descoberto, está se tornando cada vez mais importante desenvolver sistemas com os quais rapidamente avaliar o significado biológico dessas variantes identificadas. Este protocolo descreve métodos para avaliar a função do gene humano durante a meiose feminina eu usando oócitos de rato.

Usando oócitos de camundongo para avaliar a função de genes humanos durante a 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.

Education

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JoVE Core

JoVE Journal

Cell Biology

Molecular Biology

Genetics

Unit 2

Unit 1

Meiosis

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