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

이 작품 미국 생식 의학 협회와 찰스와 요한 나 부시 기념 기금 Rutgers에 연구 보조금에 의해 지원 되었다에 주립 대학 뉴저지 K.S. A.L.N.에 N.I.H. (F31 HD0989597)에서 교부 금에 의해 지원 되었다.

1. 분자 클로닝
<ol><li>관심과 전사 (pIVT) 벡터11 생체 외에서 플라스 미드의 유전자의 전체 길이 코딩 시퀀스를 가져옵니다. 참고: 전장 cDNA 클론 상업적으로 다양 한 공급 업체에서 제공 또는 반전 녹음 방송 연쇄 반응 (RT-PCR)을 통해 생성 될 수 있습니다. 생물 공학 정보 (NCBI) 온라인 리소스에 대 한 국립 센터는 뉴클레오티드에서 유전자에 대 한 사본을 시퀀스를 제공 합...

<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>

질병과 관련 된 인간의 유전 이체의 신속 하 고 증가 식별, 때문에 그들의 생물 학적 중요성을 평가 하기 위해 시스템 설립은 필수적 이다. 때문에 인간의 oocytes 소중한 희귀 하 고 인간의 정자는 의무가 유전자 조작 인간 감수 분열 포즈 특정 과제에서 단백질 기능을 이해. 마우스 oocytes는 인간의 meiotic 유전자 기능 10,23를 평가 하기 위한 귀중 한 포유류 ...

불 임은 재생산 나이 1에서 거의 절반 추구 치료 2인간의 인구의 10-15%에 영향을 미치는 조건이 다. 많은 경우 multifactorial 및 불 임 증의 병 인은 다양 한 인간에서 가장 흔한 유전 이상이 배아 이수성 3입니다. 이수성은 염색체 셀에 올바른 수의 편차 (이득 또는 손실)로 정의 됩니다. 인간 태아의 이수성의 현상 일반적 이며 진보 된 임산부 나이 4,5증가. 이 전략 증가 주입 속도, 낮은 유산 률과에 대 한 짧은 시간에 결과 때문에 자 궁 전송만 chromosomally 정상 (euploid) 배아 선택의 혜택을 강조 하는 4 개의 무작위 통제 실험 임신 6,7,,89를 달성. 따라서, 도움된을 재생산에 중요 한 의미를 가질 수 있습니다 인간의 이수성의 병 인을 이해.
Aneuploidies에 대 한 사전 주입 유전자 검사 하는 것이 불 임 치료에 도움이 있지만 aneuploidies 시작 하는 방법에 대 한 철저 한 이해는 여전히 부족 한. 그러나 Meiotic aneuploidies (생식 체 생산 동안 유래)와 산 모 나이,, 일부 여성 현재 배아 이수성 속도 그들의 주어진된 나이 4에 대 한 평균 속도에서 이탈 하는 긍정적인 상관관계가 있다는 것을 널리 받아들여진다. 이러한 경우는 혼자 나이 항상 aneuploid 배아 생성의 위험 예측 하지 것이 좋습니다. 다른 요인 배아 이수성, 유전자 변형 등의 위험 증가에 역할을 재생할 수 있습니다.
빠르게 meiotic 유전자 기능을 평가 하는 시스템 설계 조사 oocyte 감수 분열 동안 이수성을 유전자 이체의 잠재적인 기여의 핵심 요소가입니다. 윤리적 제약 및 제한 된 액세스, 인간의 계란을 사용 하 여이 실험을 수행 하는 실용적입니다. 마우스 oocytes를 사용 하 여 이러한 문제를 피할 수 있으며 여기 감수 분열 동안 인간 유전자 기능을 평가 하기 위해 분석 실험의 시리즈 나 설명. 관심사의 유전자 변형에 대 한 코딩 하는 메신저 RNA (mRNA) microinjecting에 의해 마우스 달걀에서 인간 단백질의 지역화 시각 고 수 경우는 야생 형 및 돌연변이 인간 단백질의 소성 식에서 결과 확인 하는 데 사용 이수성 이어질 수 있는 phenotypic 변경 Kinetochore 그리고 중요 한 것은 감수 분열 나의 분열에서 염색체 맞춤 지원 못하는 동생에 게 부적 절 한에 연결 하는 microtubules에 증가 포함 하는이 고기, 이득 및 기능의 상실을 조사 하이 프로토콜을 사용할 수 있습니다. 도전 oocyte 감수 분열의 키 이벤트와 같은 특정 실험 조건 설정 하 여 유전 이체는 건물 및 염색체 정렬 10스핀 들.

<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

배아 이수성은 인간에서 불모의 주요 유전 원인입니다. 이러한 이벤트의 대부분 여성 감수 분열 동안 시작 하며 나이 혼자 항상 aneuploid 배아 생성의 위험 예측은 긍정적으로 산 모 나이 상관, 이기는 하지만. 따라서, 유전자 변형 oogenesis 동안 잘못 된 염색체 분리에 대 한 계정 수 있습니다. 인간의 oocytes에 대 한 액세스는 제한 된 연구 목적을 위해, 분석 실험의 시리즈 마우스 oocytes를 사용 하 여 내가 감수 분열 동안 인간 유전자 기능을 공부에 개발 되었다. 첫째, 유전자 및 유전자 변형 관심의 메신저 RNA (mRNA) 의향 나 체포 마우스 oocytes에 microinjected 있다. 후 식에 대 한 시간을 허용, oocytes 동기적으로 감수 분열을 완료 meiotic 성숙에 출시 되 나. 형광 기자, 녹색 형광 단백질 (Gfp) 등의 순서로 mRNA에 태그를 추가 하 여 인간의 단백질의 지역화 phenotypic 변경 뿐만 아니라 평가 수 있습니다. 예를 들어 이득 또는 기능의 손실 meiotic 오류를 해결 하기 위해 유전자 제품에 도전 하는 실험 조건을 설정 하 여 조사 수 있습니다. 이 시스템은 유리한 oogenesis 동안 인간의 단백질 기능을 조사, 결과의 적절 한 해석 이루어져야 한다 단백질 표정은 내 생 수준에서 그에 주어진 고 (즉, 절에 대 한 제어 하지 않는 한 밖으로 또는 아래로), murine homologs 시스템에 있습니다.

생체 외에서 후 전사 고품질 RNA (1.8-2.2) A260/A280 비율과 A260/A230 비율 ≥1.7은 분 광 광도 계를 사용 하 여 측정 하는 때 있을 것 이다. 그림 1 에서 그림 보여준다 생체 외에서의 마이그레이션-변성 agarose 젤 전기 이동 법 후 RNA 생성. 패턴 또는 여러 크기의 밴드는 샘플에 얼룩져 밴드 오염 또는 샘플의 저하를 나타낼 수 있습니다. 또는, 여러...

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

인간의 질병과 관련 된 유전자 변종 발견 될 하기 시작, 그것은 점점 더 빠르게 그 확인 된 이체의 생물학적 의미를 평가 하는 시스템을 개발 하는 것이 중요 되고있다. 이 프로토콜에는 인간 유전자 기능 여성 감수 분열 동안 마우스 oocytes를 사용 하 여 나로 평가 하는 방법을 설명 합니다.

감수 분열 동안 인간 유전자 기능을 평가 하기 위해 마우스 Oocytes를 사용 하 여 나

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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Unit 2

Unit 1

Meiosis

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멀티 플렉스 RT-qPCR에 사용 단일 세포 유전자 발현은 희귀 림프 인구의 이질성의 특성을

Gene expression heterogeneity is an interesting feature to investigate in lymphoid populations. Gene expression in these cells varies during cell ...

Retroviral Scanning: Mapping MLV Integration Sites to Define Cell-specific Regulatory Regions

레트로 바이러스 스캐닝 : MLV 통합 사이트 맵핑으로 세포 특정 규제 지역 정의

Moloney murine leukemia (MLV) virus-based retroviral vectors integrate predominantly in acetylated enhancers and promoters. For this reason, mLV ...

A Standard Methodology to Examine On-site Mutagenicity As a Function of Point Mutation Repair Catalyzed by CRISPR/Cas9 and SsODN in Human Cells

점 돌연변이 수리 CRISPR/Cas9 및 SsODN 인간 세포에 의해 촉매의 기능으로 현지 변이 검사 하는 표준 방법론

Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point ...

Highly Efficient Gene Disruption of Murine and Human Hematopoietic Progenitor Cells by CRISPR/Cas9

CRISPR/Cas9에 의해 Murine 인간과 조 혈 조상 세포의 고효율 유전자 장애

Advances in the hematopoietic stem cell (HSCs) field have been aided by methods to genetically engineer primary progenitor cells as well as animal models. ...

CRISPR/Cas9 Gene Editing to Make Conditional Mutants of Human Malaria Parasite P. falciparum

CRISPR/Cas9 Gene Editing to Make Conditional Mutants of Human Malaria Parasite P. falciparum

CRISPR/Cas9 유전자 인간 말라리아 기생충의 조건부 돌연변이 게 P. falciparum 에 편집

Malaria is a significant cause of morbidity and mortality worldwide. This disease, which primarily affects those living in tropical and subtropical ...

The Use of Mouse Splenocytes to Assess Pathogen-associated Molecular Pattern Influence on Clock Gene Expression

병원 체 관련 된 분자 패턴 시계 유전자 발현에 영향을 평가 하기 위해 마우스 Splenocytes 사용 하 여

From behavior to gene expression, circadian rhythms regulate nearly all aspects of physiology. Here, we present a methodology to challenge mouse ...

Manipulation of Gene Function in Mexican Cavefish

멕시코 Cavefish에서 유전자 기능 조작

Cave animals provide a compelling system for investigating the evolutionary mechanisms and genetic bases underlying changes in numerous complex traits, ...

Overexpressing Long Noncoding RNAs Using Gene-activating CRISPR

Overexpressing 긴 Noncoding RNAs 유전자 활성화 CRISPR를 사용 하 여

Long noncoding RNA (lncRNA) biology is a new and exciting field of research, with the number of publications from this field growing exponentially since ...

Lentiviral Mediated Gene Silencing in Human Pseudoislet Prepared in Low Attachment Plates

렌티 바이러스는 낮은 부착 플레이트에 준비 인간의 의사에 유전자 침묵 중재

Various genetic tools are available to modulate genes in pancreatic islets of rodents to dissect function of islet genes for diabetes research. However, ...