이 작품은 샤론 E. 강사에 게 수 여 하는 NIH 그랜트 GM59354에 의해 지원 되었다. Confocal 현미경 검사 법에 대 한 앤 Lavanway와 기술 지원에 대 한 제이 에밀리 리드 형광 팔 프로브를 생성 하기 위한 프로토콜을 개발에 도움에 감사 위이 Q. 응웬 하 고. 우리는 또한 유용한 토론 및 조언을 위한 초파리 커뮤니티에서 수많은 동료를 감사합니다.

<ol>
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저자 아무 경쟁 금융 관심사를 선언합니다.

물고기를 사용 하 여 나 고 분열 prometaphase 팔 응집력의 상태를 평가 하기 위해 프로브 나 초파리 oocytes 초파리 감수 분열의 분야에서 중요 한 발전 이다. 역사적으로, 초파리 연구 성숙 oocytes11,,1819팔 응집력의 조 손실 유추를 유전자 검사로 제한 되었습니다. 자, 여기에 제시 된 방법으로 팔 단결력의 상태 ?...

유사 분열, 감수 분열 동안 염색체의 적절 한 분리 필요 그 자매 chromatid 단결력, 유지, 설립 될와 조정된 패션1,2에 발표. 단결력 S 단계 설정 되 고 복잡 한, 자매는 실제 관계를 형성 하는 cohesin에 의해 중재 chromatids 함께. 감수 분열에서 결합 한 크로스 오버를 원심 또한 함께 재조합 homologs를 기능과이 실제 협회를 통해 올바른 방향이는 분열에의 나는 (그림 1)3,4, 스핀 들 5. 팔의 릴리스 anaphase에 응집력 나 반대 스핀 들 극을 분리 하는 homologs 수 있습니다. 그러나, 팔 단결력은 경우 중간, 손실 재조합 homologs 그들의 물리적 연결을 잃게 되며 임의로, 분리는 aneuploid gametes (그림 1)에서 발생할 수 있습니다.
인간의 oocytes에서 염색체 분리에 오류 유산 및 기형 아 출산, 다운 증후군6, 등의 주요 원인이 되며 그들의 발생 산 모 나이7기 하 급수적으로 증가. 자매 chromatid 단결력 태아 oocytes에서 설정 되 고 meiotic 재결합 출생 하기 전에 완료 됩니다. Oocytes 다음 체포 중간 의향에 나 배 란까지 고이 체포 하는 동안, 재조합 homologs의 지속적인된 물리적 협회 의존 자매 chromatid 단결력. 따라서, 감수 분열과 정상 임신 결과 정확한 분리 결합 최대 5 년간 그대로 유지 해야 합니다.
인간의 oocytes의 장기간된 meiotic 체포 중 응집 조 손실 모성 연령 효과에 기여할 제안 되었습니다와 증거의 여러 줄이 가설8,9를 지원 합니다. 그러나,이 현상의 우리의 이해의 대부분 모델 생물5,10,,1112, 를 사용 하 여 의존 인간의 oocytes meiotic 응집력의 과제를 감안할 때, 13,,1415.
초파리 melanogaster oocytes meiotic 응집과 염색체 분리의 연구에 대 한 수많은 이점을 제공합니다. 간단한 유전자 분석 결과 수 aneuploid gametes에서 자손을 복구 하 고 X의 충실도 측정-대규모11,,1617에 염색체 분리. 또한, 하나 또한 나 팔 단결력11,18, 의 조 손실에 일치 하는 표현 형 감수 분열 시 재조합 homologs missegregate 때문에 염색체 분리 오류 발생 여부를 결정할 수 있습니다 19. 초파리 oocytes meiotic 응집력의 상태 관찰은 또한 형광 제자리에서 교 잡 (물고기)를 사용 하 여 직접. 비록 반복적인 위성 시퀀스에 교배 하는 형광 oligonucleotides 사용 되었습니다 10 년 이상 모니터링 성숙 초파리 oocytes4,20, 팔의 분석에서에서 pericentromeric 결합 하 단결력은 더욱 어려운 되었습니다. 팔 단결력의 상태 시각화 단일 복사본의 큰 영역에 걸쳐 프로브 시퀀스 이며 충분히 밝은 결과 개별 여동생에 대 한 표시 신호 chromatids 팔 단결력은 결 석 하는 경우 필요 합니다. 또한, oocyte 고정 조건 및 레이블이 지정 된 DNAs의 크기 큰 성숙한 초파리 oocyte (200 µ m 넓은 500 µ m 길이 의해)에 침투21 를 촉진 해야 합니다. 최근, 한 팔 조사는 성공적으로 활용, 하지만 저자 anaphase 동안 초파리 oocyte chromatids를 시각화 하기 위해 명시 된 그들은 prometaphase에 신호를 감지 하지 못했습니다 또는 분열 oocytes22를 체포. 여기 우리는 X의 생성에 대 한 자세한 프로토콜 제공-염색체 팔 물고기 조사 조건과 oocyte 준비 나 하 고 분열 chromatid 응집력 prometaphase 언 니의 조기 상실에 대 한 분석 결과 허용 난 oocytes. Meiotic 단결력의 유지 보수에 필요한 유전자 제품을 사용, 이러한 기술을 동생에 대 한 분석 결과를 다른 수 chromatid 단결력 다른 genotypes의 성숙한 초파리 oocytes에 결함.

<table><tbody><tr><td>&lt;strong&gt;Kits&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Midi Prep kit</td><td>Qiagen</td><td>12143</td><td>Prep BAC clone DNA</td></tr><tr><td>GenomePlex Complete Whole Genome Amplification (WGA) Kit</td><td>Sigma</td><td>WGA2</td><td>Amplify BAC clone DNA</td></tr><tr><td>ARES Alexa Fluor 647 DNA labeling kit</td><td>Invitrogen</td><td>A21676</td><td>Label BAC clone DNA</td></tr><tr><td>PCR purification kit</td><td>Qiagen</td><td>28104</td><td>Remove non-conjugated dye following labeling of BAC clone DNA</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Chemicals &amp;amp; Solutions&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Note: &lt;/strong&gt;All solutions are prepared using sterile ultrapure water and should be sterilized either by autoclave or filter sterilization.</td><td></td><td></td><td></td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Fisher Scientific</td><td>BP1600-100</td><td>Prepare 10% stock&lt;br/&gt; Freeze aliquots</td></tr><tr><td>Calcium chloride</td><td>Fisher Scientific</td><td>C75-500</td><td></td></tr><tr><td>DAPI (4&amp;rsquo;, 6-Diamidino-2-Phenylindole, Dihydrochloride)</td><td>Invitrogen</td><td>D1306</td><td>&lt;strong&gt;Toxic:&lt;/strong&gt; wear appropriate protection. Prepare 100&amp;micro;g/ml stock in 100% ethanol, store in aliquots at -20 &amp;deg;C. Prepare 1 &amp;micro;g/ml solution in 2X SSCT before use.</td></tr><tr><td>Dextran sulfate</td><td>Sigma</td><td>D-8906</td><td></td></tr><tr><td>Drierite</td><td>Drierite Company</td><td>23001</td><td></td></tr><tr><td>Dithiothreitol (DTT)</td><td>Invitrogen</td><td>15508-013</td><td>Prepare 10 mM stock</td></tr><tr><td>dTTP (10 &amp;micro;mol, 100 &amp;micro;l)</td><td>Boehringer Mannheim</td><td>1277049</td><td>Prepare 1 mM stock</td></tr><tr><td>EDTA (Disodium ethylenediamine tetraacetic acid)</td><td>Fisher Scientific</td><td>S311-500</td><td>Prepare 250 mM stock</td></tr><tr><td>100% ethanol (molecular grade, 200 proof)</td><td>Decon Laboratories</td><td>2716</td><td></td></tr><tr><td>Tris (Ultra Pure)</td><td>Invitrogen</td><td>15504-020</td><td></td></tr><tr><td>EGTA (Ethylenebis(oxyethylenenitrilo)tetraacetic acid)</td><td>Sigma</td><td>E-3889</td><td></td></tr><tr><td>16% formaldehyde</td><td>Ted Pella, Inc.</td><td>18505</td><td>&lt;strong&gt;Toxic:&lt;/strong&gt; wear appropriate protection</td></tr><tr><td>Formamide</td><td>Invitrogen</td><td>AM9342</td><td>&lt;strong&gt;Toxic: &lt;/strong&gt;wear appropriate protection</td></tr><tr><td>Glucose</td><td>Fisher Scientific</td><td>D16-1</td><td></td></tr><tr><td>Glycogen</td><td>Roche</td><td>901393</td><td></td></tr><tr><td>HEPES</td><td>Boehringer Mannheim</td><td>737-151</td><td></td></tr><tr><td>Heptane</td><td>Fisher Scientific</td><td>H350-4</td><td>&lt;strong&gt;Toxic:&lt;/strong&gt; wear appropriate protection.</td></tr><tr><td>Hydrochloric acid</td><td>Millipore</td><td>HX0603-4</td><td>&lt;strong&gt;Toxic:&lt;/strong&gt; wear appropriate protection.</td></tr><tr><td>Hydroxylamine</td><td>Sigma</td><td>438227</td><td>Prepare 3 M stock</td></tr><tr><td>4.9 M Magnesium chloride</td><td>Sigma</td><td>104-20</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4 &lt;/sub&gt;&amp;Yuml; 7H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Fisher Scientific</td><td>S373-500</td><td></td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4 &lt;/sub&gt;&amp;Yuml; 2H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Fisher Scientific</td><td>S369-500</td><td></td></tr><tr><td>Poly-L-lysine (0.1mg/ml)</td><td>Sigma</td><td>P8920-100</td><td></td></tr><tr><td>Potassium acetate</td><td>Fisher Scientific</td><td>BP364-500</td><td></td></tr><tr><td>Sodium acetate</td><td>Fisher Scientific</td><td>S209-500</td><td>Prepare 3M stock</td></tr><tr><td>Sodium cacodylate</td><td>Polysciences, Inc.</td><td>1131</td><td>&lt;strong&gt;Toxic: &lt;/strong&gt;wear appropriate protection. Prepare 400mM stock</td></tr><tr><td>Sodium citrate</td><td>Fisher Scientific</td><td>BP327-1</td><td></td></tr><tr><td>Sodium chloride</td><td>Fisher Scientific</td><td>S271-3</td><td>Sodium chloride</td></tr><tr><td>Sucrose</td><td>Fisher Scientific</td><td>S5-500</td><td></td></tr><tr><td>10% Tween 20</td><td>Thermo Scientific</td><td>28320</td><td>Surfact-Amps</td></tr><tr><td>10% Triton X-100</td><td>Thermo Scientific</td><td>28314</td><td>Surfact-Amps</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Solutions&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Note: &lt;/strong&gt;All solutions are prepared using sterile ultrapure water and should be sterilized either by autoclave or filter sterilization.</td><td></td><td></td><td></td></tr><tr><td>TE buffer</td><td></td><td></td><td>10 mM Tris, 1 mM EDTA, pH = 8.0</td></tr><tr><td>20X SSC (Saline Sodium Citrate)</td><td></td><td></td><td>3 M NaCl, 300 mM sodium citrate</td></tr><tr><td>2X cacodylate fix solution</td><td></td><td></td><td>&lt;strong&gt;Toxic:&lt;/strong&gt; wear appropriate protection. 200 mM sodium cacodylate, 200 mM sucrose, 80 mM sodium acetate, 20 mM EGTA</td></tr><tr><td>1.1X Hybridization buffer</td><td></td><td></td><td>3.3X SSC, 55% formamide, 11% dextran sulfate</td></tr><tr><td>Fix solution</td><td></td><td></td><td>&lt;strong&gt;Toxic: &lt;/strong&gt;wear appropriate protection. 4% formaldehyde, 1X cacodylate fix solution</td></tr><tr><td>PBSBTx</td><td></td><td></td><td>1X PBS, 0.5% BSA, 0.1% Trition X-100</td></tr><tr><td>PBSTx</td><td></td><td></td><td>1X PBS, 1% Trition X-100</td></tr><tr><td>Extraction buffer (PBSTx + Rnase)</td><td></td><td></td><td>1X PBS, 1% Trition X-100, 100 &amp;micro;g/mL RNase</td></tr><tr><td>2X SSCT</td><td></td><td></td><td>2X SSC, 0.1% Tween 20</td></tr><tr><td>2X SSCT + 20% formamide</td><td></td><td></td><td>&lt;strong&gt;Toxic:&lt;/strong&gt; wear appropriate protection. 2X SSC, 0.1% Tween 20, 20% formamide</td></tr><tr><td>2X SSCT + 40% formamide</td><td></td><td></td><td>&lt;strong&gt;Toxic:&lt;/strong&gt; wear appropriate protection. 2X SSC, 0.1% Tween 20, 40% formamide</td></tr><tr><td>2X SSCT + 50% formamide</td><td></td><td></td><td>&lt;strong&gt;Toxic:&lt;/strong&gt; wear appropriate protection. 2X SSC, 0.1% Tween 20, 50% formamide</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Enzymes&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>AluI</td><td>New England Biolabs</td><td>R0137S</td><td></td></tr><tr><td>HaeIII</td><td>New England Biolabs</td><td>R0108S</td><td></td></tr><tr><td>MseI</td><td>New England Biolabs</td><td>R0525S</td><td></td></tr><tr><td>MspI</td><td>New England Biolabs</td><td>R0106S</td><td></td></tr><tr><td>RsaI</td><td>New England Biolabs</td><td>R0167S</td><td></td></tr><tr><td>BfuCI</td><td>New England Biolabs</td><td>R0636S</td><td></td></tr><tr><td>100X BSA</td><td>New England Biolabs</td><td></td><td>Comes with NEB enzymes</td></tr><tr><td>10X NEB buffer #2</td><td>New England Biolabs</td><td></td><td>Restriction enzyme digestion buffer. Comes with NEB enzymes</td></tr><tr><td>Terminal deoxynucleotidyl transferase (TdT) 400 U/&amp;micro;l</td><td>Roche/Sigma</td><td>3333566001</td><td></td></tr><tr><td>TdT buffer</td><td>Roche/Sigma</td><td></td><td>Comes with TdT enzyme</td></tr><tr><td>Cobalt chloride</td><td>Roche/Sigma</td><td></td><td>&lt;strong&gt;Toxic: &lt;/strong&gt;wear appropriate protection. Comes with TdT enzyme</td></tr><tr><td>RNase A (10 mg/mL)</td><td>Thermo-Scientific</td><td>EN0531</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Cytology Tools etc.&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Forceps</td><td>Dumont</td><td></td><td>#5 INOX, Biologie</td></tr><tr><td>9&amp;rdquo; Disposable glass Pasteur pipettes</td><td>Fisher</td><td>13-678-20C</td><td>Autoclave to sterilize</td></tr><tr><td>Shallow glass dissecting dish</td><td></td><td></td><td>Custom made</td></tr><tr><td>Deep well dish (3 wells)</td><td>Pyrex</td><td>7223-34</td><td></td></tr><tr><td>Fisherfinest Premium microscope slides</td><td>Fisher Scientific</td><td>22-038-104</td><td>Used to cover deep well dishes</td></tr><tr><td>Frosted glass slides, 25 x 75 mm</td><td>VWR Scientific</td><td>48312-002</td><td></td></tr><tr><td>Glass slides, 3 x 1 in, 1 mm thick</td><td>Thermo-Scientific</td><td>3051</td><td></td></tr><tr><td>Coverslips, 10 x 10 mm, No. 1.5</td><td>Thermo-Scientific</td><td>3405</td><td></td></tr><tr><td>Tungsten needle</td><td></td><td></td><td>homemade</td></tr><tr><td>Prolong GOLD mounting media</td><td>Molecular Probes</td><td>P36930</td><td></td></tr><tr><td>Compressed-air in can</td><td>Various</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>PCR machine</td><td>Various</td><td></td><td></td></tr><tr><td>Nanodrop 2000, spectrophotometer</td><td>Thermo-Scientific</td><td></td><td>microvolume spectrophotometer</td></tr><tr><td>Vortexer</td><td>Various</td><td></td><td></td></tr><tr><td>Table top microfuge at room temperature</td><td>Various</td><td></td><td></td></tr><tr><td>Table top microfuge at 4 &amp;deg;C</td><td>Various</td><td></td><td></td></tr><tr><td>Heat block</td><td>Various</td><td></td><td></td></tr><tr><td>Hybridization oven or incubator with rotator</td><td>Various</td><td></td><td></td></tr><tr><td>Nutator</td><td>Various</td><td></td><td></td></tr><tr><td>A1RSi laser scanning confoal</td><td>Nikon</td><td></td><td>40X oil Plan Fluor DIC (NA 1.3)</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Consumables etc.&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>50 mL conical tubes</td><td>Various</td><td></td><td></td></tr><tr><td>15 mL conical tubes</td><td>Various</td><td></td><td></td></tr><tr><td>1.5 mL microfuge tubes</td><td>Various</td><td></td><td></td></tr><tr><td>500 &amp;micro;l microfuge tubes</td><td>Various</td><td></td><td></td></tr><tr><td>200 &amp;micro;l PCR tubes</td><td>Various</td><td></td><td></td></tr><tr><td>Plastic container with tight fitting lid</td><td>Various</td><td></td><td>To hold Drierite</td></tr><tr><td>Kimwipes</td><td>Various</td><td></td><td>disposable wipes</td></tr><tr><td>Parafilm</td><td>Various</td><td></td><td>paraffin film</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Other&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>HPLC purified 5&#039;-labeled oligonucleotides</td><td>Integrated DNA Technologies</td><td></td><td>Cy3-labeled probes that recognize the 359 bp satellite repeat of the &lt;em&gt;X&lt;/em&gt; chromosome</td></tr><tr><td>Volocity 3D Image Analysis Software</td><td>PerkinElmer</td><td></td><td>Version 6.3</td></tr></tbody></table>

using fluorescence in situ hybridization (fish) to monitor the state of arm cohesion in prometaphase and metaphase i drosophila oocytes

인간에서는, oocytes에서 염색체 분리 오류 유산 및 기형 아 출산의 대부분에 대 한 책임이 있습니다. 또한, 여자 나이, 그들 마음속에 aneuploid 태아는 극적으로 증가 하 고이 현상의 위험 모성 연령 효과로 알려져 있다. Meiotic 분열 동안 정확한 염색체 분리에 대 한 요구 사항 중 하나는 자매의 유지 보수 oocytes 경험 확장된 의향 기간 chromatid 단결력. 인간 및 모형 유기 체에서 cytological 증거 meiotic 단결력 노화 과정 동안 악화 나왔다. 또한, 인간의 oocytes에서 분리 오류는 감수 분열 중 가장 널리 퍼진 나 팔 단결력의 조 손실와 일치. 모델 생물의 사용은 기초가 단결력의 연령에 따라 손실 메커니즘을 탈피 중요 합니다. 초파리 melanogaster oocytes meiotic 응집력의 규칙을 공부에 대 한 몇 가지 장점이 있습니다. 그러나, 최근까지, 유전 시험만 팔 응집력 oocytes 다른 genotypes 또는 다른 실험 조건에서의 손실에 대 한 분석 결과를 사용할 수 없었습니다. 여기, 상세한 프로토콜 형광에서 제자리에서 사용 하기 위한 교 잡 (물고기)을 직접 시각화 결함 prometaphase 팔 응집력에 나 고 분열 초파리 oocytes를 체포 하는 제공 됩니다. X 염색체의 원심 팔 hybridizes를 물고기 프로브를 생성 하 여 수집 하는 confocal Z 스택 연구원 수 3 차원에서 개별 물고기 신호의 수를 시각화 하 고 자매 여부 결정 chromatid 무기 분리. 설명한 팔 단결력 결함 초파리 oocytes의 수백에 계량을 가능 하 게. 따라서,이 방법은 단결력 유지 보수 뿐만 아니라 노화 과정의 죽음으로 이어질 요인에 기여 하는 메커니즘을 조사는 중요 한 도구를 제공 합니다.

그림 5 는 X 염색체에 cytological 지역 6E-7B에 hybridizes는 팔 조사로 얻은 이미지를 선물 한다. 공동의 DAPI, localizes 신호에서이 조사 결과 배경에서 쉽게 구별할 수 이며 팔 단결력 결함 다른 genotypes19에 척도를 성공적으로 사용 되었습니다. 단결력 결함의 부 량 제한 되었다 prometaphase에 어 분열 I 단계; 핵 붕괴 이전 oocytes 분석<sup cl...

Watch this Scientific Journal Video about Using Fluorescence In Situ Hybridization FISH to Monitor the State of Arm Cohesion in Prometaphase and Metaphase I Drosophila Oocytes at JoVE.com

Using Fluorescence In Situ Hybridization FISH to Monitor the State of Arm Cohesion in Prometaphase and Metaphase I Drosophila Oocytes

Prometaphase 팔 응집력과 분열의 상태를 모니터링 하려면 형광 제자리에서 교 잡 (물고기)를 사용 하 여 나 초파리 Oocytes

이 원고 X를 생성 하는 자세한 방법을 제공-염색체 팔 프로브 및 수행 형광 제자리에서 교 잡 (물고기) 자매의 상태를 검사 하 chromatid 응집력 prometaphase 및 를 체포 하는 분열 초파리 oocytes. 이 프로토콜은 meiotic 팔 단결력은 그대로 또는 다른 genotypes에 방해 여부 결정 하는 데 적합 합니다.

In humans, chromosome segregation errors in oocytes are responsible for the majority of miscarriages and birth defects. Moreover, as women age, their risk ...

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Using Fluorescence In Situ Hybridization (FISH) to Monitor the State of Arm Cohesion in Prometaphase and Metaphase I Drosophila Oocytes

Meiosis

SCIENTISTS IN ACTION

11.3K 조회수.  Dartmouth College. 이 원고 X를 생성 하는 자세한 방법을 제공-염색체 팔 프로브 및 수행 형광 제자리에서 교 잡 (물고기) 자매의 상태를 검사 하 chromatid 응집력 prometaphase 및 를 체포 하는 분열 초파리 oocytes. 이 프로토콜은 meiotic 팔 단결력은 그대로 또는 다른 genotypes에 방해 여부 결정 하는 데 적합 합니다.

비디오: Using Fluorescence In Situ Hybridization FISH to Monitor the State of Arm Cohesion in Prometaphase and Metaphase I Drosophila Oocytes

Using Mouse Oocytes to Assess Human Gene Function During Meiosis I

Embryonic aneuploidy is the major genetic cause of infertility in humans. Most of these events originate during female meiosis, and albeit positively correlated with maternal age, age alone is not always predictive of the risk of generating an aneuploid embryo. Therefore, gene variants might account for incorrect chromosome segregation during oogenesis. Given that access to human oocytes is limited for research purposes, a series of assays were developed to study human gene function during meiosis I using mouse oocytes. First, messenger RNA (mRNA) of the gene and gene variant of interest are microinjected into prophase I-arrested mouse oocytes. After allowing time for expression, oocytes are synchronously released into meiotic maturation to complete meiosis I. By tagging the mRNA with a sequence of a fluorescent reporter, such as green fluorescent protein (Gfp), the localization of the human protein can be assessed in addition to the phenotypic alterations. For example, gain or loss of function can be investigated by establishing experimental conditions that challenge the gene product to fix meiotic errors. Although this system is advantageous in investigating human protein function during oogenesis, adequate interpretation of results should be undertaken given that protein expression is not at endogenous levels and, unless controlled for (i.e. knocked out or down), murine homologs are also present in the system.

As the genetic variants associated with human disease begin to become uncovered, it is becoming increasingly important to develop systems with which to rapidly evaluate the biological significance of those identified variants. This protocol describes methods for evaluating human gene function during female meiosis I using mouse oocytes.

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 ...

연구

유전학

Use of Single Molecule Fluorescent In Situ Hybridization (SM-FISH) to Quantify and Localize mRNAs in Murine Oocytes

Current methods routinely used to quantify mRNA in oocytes and embryos include digital reverse-transcription polymerase chain reaction (dPCR), quantitative, real-time RT-PCR (RT-qPCR) and RNA sequencing. When these techniques are performed using a single oocyte or embryo, low-copy mRNAs are not reliably detected. To overcome this problem, oocytes or embryos can be pooled together for analysis; however, this often leads to high variability amongst samples. In this protocol, we describe the use of fluorescence in situ hybridization (FISH) using branched DNA chemistry. This technique identifies the spatial pattern of mRNAs in individual cells. When the technique is coupled with Spot Finding and Tracking&#160;computer software, the abundance of mRNAs in the cell can also be quantified. Using this technique, there is reduced variability within an experimental group and fewer oocytes and embryos are required to detect significant differences between experimental groups. Commercially available branched-DNA SM-FISH kits have been optimized to detect mRNAs in sectioned tissues or adherent cells on slides. However, oocytes do not effectively adhere to slides and some reagents in the kit were too harsh resulting in oocyte lysis. To prevent this lysis, several modifications were made to the FISH kit. Specifically, oocyte permeabilization and wash&#160;buffers designed for the immunofluorescence of oocytes and embryos replaced the proprietary buffers. The permeabilization, washes, and incubations with probes and amplifier were performed in 6-well plates and oocytes were placed on slides at the end of the protocol using mounting media. These modifications were able to overcome the limitations of the commercially available kit, in particular, the oocyte lysis. To accurately and reproducibly count the number of mRNAs in individual oocytes, computer software was used. Together, this protocol represents an alternative to PCR and sequencing to compare the expression of specific transcripts in single cells.

Use of Single Molecule Fluorescent In Situ Hybridization SM-FISH to Quantify and Localize mRNAs in Murine Oocytes

To reproducibly count the numbers of mRNAs in individual oocytes, single molecule RNA fluorescence in situ hybridization (RNA-FISH) was optimized for non-adherent cells. Oocytes were collected, hybridized with the transcript specific probes, and quantified using an image quantification software.

Murine Oocytes에 계량 및 방화 mRNAs 단일 분자 형광에서 제자리 교 잡 (SM-물고기)의 사용

Current methods routinely used to quantify mRNA in oocytes and embryos include digital reverse-transcription polymerase chain reaction (dPCR), ...

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

This protocol describes the use of the Drosophila melanogaster syncytial embryo for studying mitosis1. Drosophila has useful genetics with a sequenced genome, and it can be easily maintained and manipulated. Many mitotic mutants exist, and transgenic flies expressing functional fluorescently (e.g. GFP) - tagged mitotic proteins have been and are being generated. Targeted gene expression is possible using the GAL4/UAS system2. 

The Drosophila early embryo carries out multiple mitoses very rapidly (cell cycle duration, &asymp;10 min). It is well suited for imaging mitosis, because during cycles 10-13, nuclei divide rapidly and synchronously without intervening cytokinesis at the surface of the embryo in a single monolayer just underneath the cortex. These rapidly dividing nuclei probably use the same mitotic machinery as other cells, but they are optimized for speed; the checkpoint is generally believed to not be stringent, allowing the study of mitotic proteins whose absence would cause cell cycle arrest in cells with a strong checkpoint. Embryos expressing GFP labeled proteins or microinjected with fluorescently labeled proteins can be easily imaged to follow live dynamics (Fig. 1). In addition, embryos can be microinjected with function-blocking antibodies or inhibitors of specific proteins to study the effect of the loss or perturbation of their function3. These reagents can diffuse throughout the embryo, reaching many spindles to produce a gradient of concentration of inhibitor, which in turn results in a gradient of defects comparable to an allelic series of mutants. Ideally, if the target protein is fluorescently labeled, the gradient of inhibition can be directly visualized4. It is assumed that the strongest phenotype is comparable to the null phenotype, although it is hard to formally exclude the possibility that the antibodies may have dominant effects in rare instances, so rigorous controls and cautious interpretation must be applied. Further away from the injection site, protein function is only partially lost allowing other functions of the target protein to become evident.

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

This protocol describes the use of microinjection and high resolution imaging in the Drosophila melanogaster syncytial embryo to study mitosis.

의 유사 분열 유학을위한 Microinjection 기법 Drosophila melanogaster Syncytial 엠브료

This protocol describes the use of the Drosophila melanogaster syncytial embryo  for studying mitosis1.  Drosophila has useful genetics with a sequenced ...

생물학

Nuclear Migration in the Drosophila Oocyte

Live cell imaging is particularly necessary to understand the cellular and molecular mechanisms that regulate organelle movements, cytoskeleton rearrangements, or polarity patterning within the cells. When studying oocyte nucleus positioning, live-imaging techniques are essential to capture the dynamic events of this process. The Drosophila egg chamber is a multicellular structure and an excellent model system to study this phenomenon because of its large size and availability of numerous genetic tools. During Drosophila mid-oogenesis, the nucleus migrates from a central position within the oocyte to adopt an asymmetric position mediated by microtubule-generated forces. This migration and positioning of the nucleus are necessary to determine the polarity axes of the embryo and the subsequent adult fly. One characteristic of this migration is that it occurs in three dimensions (3D), creating a necessity for live imaging. Thus, to study the mechanisms that regulate nuclear migration, we have developed a protocol to culture the dissected egg chambers and perform live imaging for 12 h by time-lapse acquisitions using spinning-disk confocal microscopy. Overall, our conditions allow us to preserve Drosophila egg chambers alive for a long period of time, thereby enabling the completion of nuclear migration to be visualized in a large number of samples in 3D.

Nuclear Migration in the Drosophila Oocyte

In Drosophila, the oocyte nucleus undergoes microtubule-dependent migration during oogenesis. Here, we describe a protocol that was developed to follow the migration by performing live imaging on egg chambers ex-vivo. Our procedure maintains egg chambers alive for 12 h to acquire multi-position 3D time-lapse movies using spinning-disk confocal microscopy.

드로소필라 오키테의 핵 이주

Live cell imaging is particularly necessary to understand the cellular and molecular mechanisms that regulate organelle movements, cytoskeleton ...

발생학

Confocal and Super-Resolution Imaging of Polarized Intracellular Trafficking and Secretion of Basement Membrane Proteins During Drosophila Oogenesis

The basement membrane (BM) - a specialized sheet of extracellular matrix present at the basal side of epithelial cells - is critical for the establishment and maintenance of epithelial tissue morphology and organ morphogenesis. Moreover, the BM is essential for tissue modeling, serving as a signaling platform, and providing external forces to shape tissues and organs. Despite the many important roles that the BM plays during normal development and pathological conditions, the biological pathways controlling the intracellular trafficking of BM-containing vesicles and how basal secretion leads to the polarized deposition of BM proteins are poorly understood. The follicular epithelium of the Drosophila ovary is an excellent model system to study the basal deposition of BM membrane proteins, as it produces and secretes all major components of the BM. Confocal and super-resolution imaging combined with image processing in fixed tissues allows for the identification and characterization of cellular factors specifically involved in the intracellular trafficking and deposition of BM proteins. This article presents a detailed protocol for staining and imaging BM-containing vesicles and deposited BM using endogenously tagged proteins in the follicular epithelium of the Drosophila ovary. This protocol can be applied to address both qualitative and quantitative questions and it was developed to accommodate high-throughput screening, allowing for the rapid and efficient identification of factors involved in the polarized intracellular trafficking and secretion of vesicles during epithelial tissue development.

Confocal and Super-Resolution Imaging of Polarized Intracellular Trafficking and Secretion of Basement Membrane Proteins During Drosophila Oogenesis

The basement membrane is essential for tissue and organ morphogenesis during development. To better understand the mechanisms leading to proper placement of this structure, the protocol presented describes methods to visualize and characterize the intracellular trafficking and secretion of basement membrane proteins in epithelial cells using confocal and super-resolution microscopy.

초 파리 Oogenesis 동안 양극화 된 세포 내 밀매 및 기저막 단백질의 분비의 공초점 및 수퍼 해상도 이미징

The basement membrane (BM) - a specialized sheet of extracellular matrix present at the basal side of epithelial cells - is critical for the establishment ...

Studying Mitotic Checkpoint by Illustrating Dynamic Kinetochore Protein Behavior and Chromosome Motion in Living Drosophila Syncytial Embryos

The spindle assembly checkpoint (SAC) mechanism is an active signal, which monitors the interaction between chromosome kinetochores and spindle microtubules to prevent anaphase onset until the chromosomes are properly connected. Cells use this mechanism to prevent aneuploidy or genomic instability, and hence cancers and other human diseases like birth defects and Alzheimer's1. A number of the SAC components such as Mad1, Mad2, Bub1, BubR1, Bub3, Mps1, Zw10, Rod and Aurora B kinase have been identified and they are all kinetochore dynamic proteins2. Evidence suggests that the kinetochore is where the SAC signal is initiated. The SAC prime regulatory target is Cdc20. Cdc20 is one of the essential APC/C (Anaphase Promoting Complex or Cyclosome) activators3 and is also a kinetochore dynamic protein4-6. When activated, the SAC inhibits the activity of the APC/C to prevent the destruction of two key substrates, cyclin B and securin, thereby preventing the metaphase to anaphase transition7,8. Exactly how the SAC signal is initiated and assembled on the kinetochores and relayed onto the APC/C to inhibit its function still remains elusive.
Drosophila is an extremely tractable experimental system; a much simpler and better-understood organism compared to the human but one that shares fundamental processes in common. It is, perhaps, one of the best organisms to use for bio-imaging studies in living cells, especially for visualization of the mitotic events in space and time, as the early embryo goes through 13 rapid nuclear division cycles synchronously (8-10 minutes for each cycle at 25 &deg;C) and gradually organizes the nuclei in a single monolayer just underneath the cortex9.
Here, I present a bio-imaging method using transgenic Drosophila expressing GFP (Green Fluorescent Protein) or its variant-targeted proteins of interest and a Leica TCS SP2 confocal laser scanning microscope system to study the SAC function in flies, by showing images of GFP fusion proteins of some of the SAC components, Cdc20 and Mad2, as the example.

Studying Mitotic Checkpoint by Illustrating Dynamic Kinetochore Protein Behavior and Chromosome Motion in Living Drosophila Syncytial Embryos

The kinetochore is where the SAC initiates its signal monitoring the mitotic segregation of the sister chromatids. A method is described to visualize the recruitment and turnover of one of the kinetochore proteins and its coordination with the chromosome motion in Drosophila embryos using a Leica laser scanning confocal system.

생활에서 보여주는 동적 Kinetochore 단백질 행동과 염색체 모션으로 Mitotic 검사 점 유학<em&gt; Drosophila</em&gt; Syncytial 태아

The spindle assembly checkpoint (SAC) mechanism is an active signal, which monitors the interaction between chromosome kinetochores and spindle ...

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

The Drosophila oocyte has been established as a versatile system for investigating fundamental questions such as cytoskeletal function, cell organization, and organelle structure and function. The availability of various GFP-tagged proteins means that many cellular processes can be monitored in living cells over the course of minutes or hours, and using this technique, processes such as RNP transport, epithelial morphogenesis, and tissue remodeling have been described in great detail in Drosophila oocytes1,2.
The ability to perform video imaging combined with a rich repertoire of mutants allows an enormous variety of genes and processes to be examined in incredible detail. One such example is the process of ooplasmic streaming, which initiates at mid-oogenesis3,4. This vigorous movement of cytoplasmic vesicles is microtubule and kinesin-dependent5 and provides a useful system for investigating cytoskeleton function at these stages.
Here I present a protocol for time lapse imaging of living oocytes using virtually any confocal microscopy setup.

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

A protocol for live imaging of GFP-tagged proteins or autofluorescent structures in individual Drosophila oocytes is described.

에 GFP-라벨 단백질의 영상을 라​​이브<em&gt; Drosophila</em&gt; Oocytes

The  Drosophila oocyte has been established as a versatile system for investigating  fundamental questions such as cytoskeletal function, cell ...

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Assessing the expression pattern of a gene, as well as the subcellular localization properties of its transcribed RNA, are key features for understanding its biological function during development. RNA in situ hybridization (RNA-ISH) is a powerful method used for visualizing RNA distribution properties, be it at the organismal, cellular or subcellular levels 1. RNA-ISH is based on the hybridization of a labeled nucleic acid probe (e.g. antisense RNA, oligonucleotides) complementary to the sequence of an mRNA or a non-coding RNA target of interest 2. As the procedure requires primary sequence information alone to generate sequence-specific probes, it can be universally applied to a broad range of organisms and tissue specimens 3. Indeed, a number of large-scale ISH studies have been implemented to document gene expression and RNA localization dynamics in various model organisms, which has led to the establishment of important community resources 4-11. While a variety of probe labeling and detection strategies have been developed over the years, the combined usage of fluorescently-labeled detection reagents and enzymatic signal amplification steps offer significant enhancements in the sensitivity and resolution of the procedure 12. Here, we describe an optimized fluorescent in situ hybridization method (FISH) employing tyramide signal amplification (TSA) to visualize RNA expression and localization dynamics in staged Drosophila embryos. The procedure is carried out in 96-well PCR plate format, which greatly facilitates the simultaneous processing of large numbers of samples.

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Here we describe a whole-mount fluorescent in situ hybridization (FISH) protocol for determining the expression and localization properties of RNAs expressed during embryogenesis in the fruit fly, Drosophila melanogaster.

전체 마운트 RNA 형광<em&gt; 현장에서</em의&gt; 하이브리드<em&gt; Drosophila</em&gt; 태아

Assessing  the expression pattern of a gene, as well as the subcellular localization  properties of its transcribed RNA, are key features for ...

Techniques for Imaging Prometaphase and Metaphase of Meiosis I in Fixed Drosophila Oocytes

Chromosome segregation in human oocytes is error prone, resulting in aneuploidy, which is the leading genetic cause of miscarriage and birth defects. The study of chromosome behavior in oocytes from model organisms holds much promise to uncover the molecular basis of the susceptibility of human oocytes to aneuploidy. Drosophila melanogaster is amenable to genetic manipulation, with over 100 years of research, community, and technique development. Visualizing chromosome behavior and spindle assembly in Drosophila oocytes has particular challenges, however, due primarily to the presence of membranes surrounding the oocyte that are impenetrable to antibodies. We describe here protocols for the collection, preparation, and imaging of meiosis I spindle assembly and chromosome behavior in Drosophila oocytes, which allow the molecular dissection of chromosome segregation in this important model organism.

Techniques for Imaging Prometaphase and Metaphase of Meiosis I in Fixed Drosophila Oocytes

We present protocols for the collection, preparation, and imaging of mature Drosophila oocytes. These methods allow the visualization of chromosome behavior and spindle assembly and function during meiosis.

고정의 이미지 Prometaphase 및 중기 감수 분열의 I위한 기술<em&gt; 초파리</em&gt; 난 모세포

Chromosome segregation in human oocytes is error prone, resulting in aneuploidy, which is the leading genetic cause of miscarriage and birth defects. The ...

Visualizing and Tracking Endogenous mRNAs in Live Drosophila melanogaster Egg Chambers

Fluorescence-based imaging techniques, in combination with developments in light microscopy, have revolutionized how cell biologists conduct live cell imaging studies. Methods for detecting RNAs have expanded greatly since seminal studies linked site-specific mRNA localization to gene expression regulation. Dynamic mRNA processes can now be visualized via approaches that detect mRNAs, coupled with microscopy set-ups that are fast enough to capture the dynamic range of molecular behavior. The molecular beacon technology is a hybridization-based approach capable of direct detection of endogenous transcripts in living cells. Molecular beacons are hairpin-shaped, internally quenched, single-nucleotide discriminating nucleic acid probes, which fluoresce only upon hybridization to a unique target sequence. When coupled with advanced fluorescence microscopy and high-resolution imaging, they enable one to perform spatial and temporal tracking of intracellular movement of mRNAs. Although this technology is the only method capable of detecting endogenous transcripts, cell biologists have not yet fully embraced this technology due to difficulties in designing such probes for live cell imaging. A new software application, PinMol, allows for enhanced and rapid design of probes best suited to efficiently hybridize to mRNA target regions within a living cell. In addition, high-resolution, real-time image acquisition and current, open source image analysis software allow for a refined data output, leading to a finer evaluation of the complexity underlying the dynamic processes involved in the mRNA&#39;s life cycle.
Here we present a comprehensive protocol for designing and delivering molecular beacons into Drosophila melanogaster egg chambers. Direct and highly specific detection and visualization of endogenous maternal mRNAs is performed via spinning disc confocal microscopy. Imaging data is processed and analyzed using object detection and tracking in Icy software to obtain details about the dynamic movement of mRNAs, which are transported and localized to specialized regions within the oocyte.

Visualizing and Tracking Endogenous mRNAs in Live Drosophila melanogaster Egg Chambers

Here, we present a protocol for the visualization, detection, analysis and tracking of endogenous mRNA trafficking in live Drosophila melanogaster egg chamber using molecular beacons, spinning disc confocal microscopy, and open-source analysis software.

라이브 드로소필라 멜라노가스터 에그 챔버에서 내인성 mRNA 를 시각화하고 추적

Fluorescence-based imaging techniques, in combination with developments in light microscopy, have revolutionized how cell biologists conduct live cell ...

Prometaphase 팔 응집력과 분열의 상태를 모니터링 하려면 형광 제자리에서 교 잡 (물고기)를 사용 하 여 나 초파리 Oocytes

요약

더 많은 비디오 탐색

이 비디오의 챕터

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

Murine Oocytes에 계량 및 방화 mRNAs 단일 분자 형광에서 제자리 교 잡 (SM-물고기)의 사용

의 유사 분열 유학을위한 Microinjection 기법 Drosophila melanogaster Syncytial 엠브료

드로소필라 오키테의 핵 이주

초 파리 Oogenesis 동안 양극화 된 세포 내 밀매 및 기저막 단백질의 분비의 공초점 및 수퍼 해상도 이미징

생활에서 보여주는 동적 Kinetochore 단백질 행동과 염색체 모션으로 Mitotic 검사 점 유학

에 GFP-라벨 단백질의 영상을 라이브

전체 마운트 RNA 형광

고정의 이미지 Prometaphase 및 중기 감수 분열의 I위한 기술

라이브 드로소필라 멜라노가스터 에그 챔버에서 내인성 mRNA 를 시각화하고 추적

Prometaphase 팔 응집력과 분열의 상태를 모니터링 하려면 형광 제자리에서 교 잡 (물고기)를 사용 하 여 나 초파리 Oocytes

요약

더 많은 비디오 탐색

이 비디오의 챕터

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

Murine Oocytes에 계량 및 방화 mRNAs 단일 분자 형광에서 제자리 교 잡 (SM-물고기)의 사용

의 유사 분열 유학을위한 Microinjection 기법 Drosophila melanogaster Syncytial 엠브료

드로소필라 오키테의 핵 이주

초 파리 Oogenesis 동안 양극화 된 세포 내 밀매 및 기저막 단백질의 분비의 공초점 및 수퍼 해상도 이미징

생활에서 보여주는 동적 Kinetochore 단백질 행동과 염색체 모션으로 Mitotic 검사 점 유학

에 GFP-라벨 단백질의 영상을 라​​이브

전체 마운트 RNA 형광

고정의 이미지 Prometaphase 및 중기 감수 분열의 I위한 기술

라이브 드로소필라 멜라노가스터 에그 챔버에서 내인성 mRNA 를 시각화하고 추적

에 GFP-라벨 단백질의 영상을 라이브