Name: xenopus oocytes: optimized methods for microinjection, removal of follicular cell layers, and fast solution changes in electrophysiological experiments
Uploaded: 2016-12-31T15:00:00.000+00:00
Duration: 7 min 24 s
Description: Watch this Scientific Journal Video about Xenopus Oocytes: Optimized Methods for Microinjection, Removal of Follicular Cell Layers, and Fast Solution Changes in Electrophysiological Experiments at JoV

This work was supported by the Swiss National Science Foundation grant 315230_156929/1. M.C.M. is a recipient of a fellowship (Beca Chile Postdoctorado from CONICYT, Ministerio de Educacion, Chile).

동물 실험은 광저우 베른 Kantonstierarzt의 지역위원회, Kantonaler Veterinärdienst 베른 (BE85 / 15)에 의해 승인되었습니다. Xenopus의 난 모세포 1. 준비 <ol><li> 엄격하게 20 ° C로 유지되는 물에 12 시간 / 12 시간의 광 / 암주기 개구리 (Xenopus의의 laevis의)을 유지한다. </li><li> 여성 개구리 (9)에서 난소의 로브를 제거합니다. 참고 : 로브의 제거는 재생을위한 자극, 그리고 종종 난자의 질이 수술이 향상됩니다. 난자는 난황 막, 모낭 세포의 층과, 혈관 (21)이 포함하는 결합 조직으로 덮여있다. 전체 구조는 &quot;난포.&quot;불린다 </li><li> 밀도가 페니실린과 스트렙토 마이신 (88 mM의 염화나트륨, 1 ㎜의 KCl, 2.4 mM의 보충 멸균 바스의 식염수 7 (MBS)의 난 모세포를 포함하는 여포로 포장 난소의 로브를 배치의 NaHCO3, 0.82 mM의 황산, 0.41 mM의 CaCl2를 0.33 mM의 칼슘 (NO 3) 2, 10 mM의 4- (2- 히드 록시 에틸) -1- 피페 라진 에탄 술폰산; HEPES (pH가 7.5, 수산화 나트륨), 100 μg의 / ㎖ 페니실린, 100 μg의 / ㎖ 스트렙토 마이신). </li><li> 단일 아웃 스테이지 V-VI (21) 난포. 집게로 난소 로브를 잡습니다. 모공을 통해 백금 (그림 1)을 낮추고 부드럽게 난소 조직에서 뿌리를 포함하는 결합 조직을 방해하는 루프를 철회. 참고 : 스테이지 V-VI (21) 모공이 크기을 특징으로한다 (1.0 - 직경 1.2 mm)와 어두운 색소 동물 반구와 황색 식물 반구 사이의 좋은 반면. </li><li> 플라스틱 파스퇴르 피펫 (1.5 mm의 공경 뒤로 절단)에 의해 35 mm 직경의 페트리 접시 만 건강적인 모낭 (즉, 완벽한 구형 그대로 색소로)를 옮긴다. </li></ol> 참고 : 여기에 설명 된 미세 주입 시스템이 Kressmann과 Birnstiel (22)에 의해보고 그에서 파생됩니다. <ol><li> 시험 관내 전사에 의해 수용체, 폴리 (A +) 꼬리 및 RNA 정량의 첨가를 겔 전기 영동 (23)에 의해 α 4 β 2 δ GABA의 서브 유닛을 코딩하는 cDNA에서 각각 된 cRNA를 준비한다. </li><li> 마이크로 피펫 풀러를 사용하여 붕규산 유리 모세관 (1.0 mm 외경 (OD), 0.58 mm 내경 (ID), 100mm 길이)에서 미세 주입 피펫을 준비합니다. 경 사진 끝 15㎛ 인 - (12)의 선단부 직경을 만들기 위해 미세 조작기를 사용하고 미사 현미경 유리 모세관의 선단을 끊어. </li><li> 10 ML의 주사기를 사용하여 파라핀 오일과 미세 주입 피펫을 백필, 다음 및 자작 MICR 상에 미세 주입 피펫을 탑재oinjection 장치 (그림 2). 주 : 및 자작 유압 사출 장치는 발 페달 스위치에 의해 제어되는 그릴 모터로 구성되어 있습니다. 추가적인 스위치는 모터의 회전 모드를 변경한다. 이 모터는 전진 또는 10 μL 유리 주사기의 플런저를 후퇴 마이크로 미터 나사 (0.5 mm / 회전)를 구동한다. 주입 약 3 rpm의 속도이며, 0.5 회전은 모낭에 50 내셔널 리그 주입에 해당합니다. 주사기 팁 자체 타이곤 튜브의 짧은 부분으로 주사 바늘에 연결되어 두꺼운 폴리 테트라 플루오로 에틸렌 관에 연결된다. 주입 모세관은 미세 조작기에 의해 제어되는 드릴 척에 의해 유지된다. 전체 시스템은 파라핀 오일로 채워져있다. 그들은 주입 시스템을 저해하므로 기포는 피해야한다. 설정은 차가운 광원으로 점등됩니다. 실체 현미경은 광학 제어가 필요합니다. 여포는 stereomicr와 차가운 빛 아래 시각화oscope (40X 배율). 사출 설치의 성능은 Xenopus의 난자에 방사성 추적자의 주입에 의해 테스트됩니다. 45의 볼륨 - 55시 NL 주사 당 제공해야합니다. </li><li> 멸균 플라스틱 팁 피펫을 사용하여 내 습성 열가소성 (2 × 2cm)의 깨끗한 내부에 무균의 액체 방울을 배치하여 설치를 테스트한다. 이 물방울에 주입 피펫의 끝을 담그고, 다음 플런저 (주입 피펫 내부에 음압을) 철회 할 수있는 모터를 켭니다. 참고 : 물 피펫을 입력하고 기름 보이는 인터페이스를 형성한다. </li><li> 액적으로부터 피펫 팁을 후퇴 주입 피펫의 내부에 양압을 적용한다. 물방울이 분사 피펫의 선단에 형성한다. </li><li> 페디큐어의 바닥에 붙어 다음 식물 극 나일론 메쉬의 간극 (0.8 mm G)에서 위쪽으로 가리키는 그들을 일렬로하여 모낭의 주입을위한 준비MBS 덮여 트리 접시 (60mm). </li><li> 열가소성의 깨끗한 내부 상 멸균 플라스틱 팁 피펫을 사용하여 mRNA의 2 μL를 분배. 주입 피펫의 내부에 부압을인가에 의해 주입 피펫으로 mRNA를보십시오. </li><li> 미세 조작기를 사용하여 개별 모낭을 통해 주입 피펫을 배치합니다. </li><li> 식물성 극의 중심에 주사 바늘을 삽입하고 피펫의 내부에 정압을인가하여 0.6 μL / 분의 유속으로 50 mRNA의 거리 nL 주입. mRNA의 탈출을 방지하기 위해 난포에서 주입 피펫 팁을 제거하기 전에 10 초 - 5 기다립니다. 참고 : mRNA를 동물 반구에있는 핵에 주입을 방지하기 위해 식물 (황색) 극에 주입한다. </li><li> MBS의 2 mL로 가득 찬 새로운 배양 접시 (35mm)에 주입 된 모공을 전송합니다. 18 ° C로 설정 와인 쿨러에 접시를 놓습니다. 1 주입 된 모공을 품어 -7 일 이내 기록하기 전에, 새롭게 발현 된 단백질의 신원에 따라. </li></ol> 3. 모공의 스트리핑 (그림 3) 주 :으로 알려진, 상기 언급 된 혈관 (24)을 포함하는 난황 층 여포 세포 및 결합 조직에 의해 덮여 난자 같이 &quot;모낭.&quot; 세포 표면에 용액의 접근을 방해하지 않고, 기계적 안정성을 제공 난황 층을 제외한 모든 층들은 전기 생리 학적 실험을하기 전에 제거되어야한다. 주변 세포 층이없는 여포는 이전에 &quot;무 결함&quot;난자 (25)라고하고있다. 이 단계는 일반적으로 전기 생리 학적 실험과 동일한 날에 수행된다. <ol><li> 열 전달은 MBS 0.5 ㎖, 1 ㎎ / ㎖의 콜라게나 제, 및 0.1 ㎎ / ㎖의 트립신 억제제를 함유하는 보로 실리케이트 유리 튜브에 모낭 주입. 36 ° C로 유지 된 수욕 튜브 빠져. 품어20 분 동안 여포와 가끔 튜브를 흔들. </li><li> RT에서 MBS 1 ㎖를 함유하는 제 2 튜브로 전송하여 모낭을 씻어. 약 10 초 동안 튜브를 남겨주세요. </li><li> 4 mM의 에틸렌 글리콜 비스 (2- aminoethylether)를 포함하는 이중 농축 MBS 0.5ml를 함유 제 튜브 모낭 이동 - N은, N, N &#39;, N&#39;- 테트라 아세트산 (EGTA) 및 4 분으로 그들을 부화 RT. 때때로 튜브를 흔들어. 주 : 고장 성 용액 (배로 농축 MBS)를하여 난자로부터 모낭 세포 층을 분리 모낭 세포 내의 난자 층의 수축을 유도하기 위해 사용된다; EGTA은 콜라게나 제 활성을 억제하기 위해 첨가된다. </li><li> RT에서 MBS 1 ㎖를 함유하는 제 2 튜브로 전송하여 모낭을 씻어. 약 10 초 동안 튜브를 남겨주세요. </li><li> MBS 2 용액을 함유하는 페트리 접시 (직경 35mm)에 난자를 옮긴다. 실체 현미경 아래에서 모낭에서 외측 봉투 구분단순히 백금 루프를 사용하여 멀리 벌거 벗은 난자를 밀어. 주 : 난자의 외부 엔벨로프가 배양 접시에 단단히 달라 붙어 용이 난자로부터 분리 될 수있다. 일부 난자는 자발적으로 자신의 외부 봉투를 잃게됩니다 및 무 결함 난자로 복구됩니다. 이 절차는 난자의 구형 모양을 유지하는 상대적으로 안정적인 준비가 발생합니다. </li></ol> 난자의 주위에 4. 빠른 해결 방법 변경 <ol><li> 9,26 바와 같이, 전압 클램프 실험을 수행한다. </li><li> 실험에 의해 요구되는 관류 스위치를 켜서 중력식 관류 시스템 솔루션을 변경한다. <ol><li> 난자 주위 작용제 농도의 빠른 변화를 허용하도록, 난자의 표면으로부터 약 0.4 mm에 배치되는 입 어느 1.35 mm의 내경과 함께 유리 모세관을 6 mL / 분의 관류 액을 적용한다. 참고 : 변화의 속도는 이전에 추정하고있다0.5 미만의 27에서 70 % D 등. </li></ol></li></ol>

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B., Wilt, F. H., Wessels, N. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=American+clawed+frogs.">American clawed frogs.</a> Methods in developmental biology. , 75-84 (1967).</li><li>Elsner, H. A., Honck, H. H., Willmann, F., Kreienkamp, H. J., Iglauer, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poor+quality+of+oocytes+from+Xenopus+laevis+used+in+laboratory+experiments:+prevention+by+use+of+antiseptic+surgical+technique+and+antibiotic+supplementation.">Poor quality of oocytes from Xenopus laevis used in laboratory experiments: prevention by use of antiseptic surgical technique and antibiotic supplementation.</a> Comp Med. 50 (2), 206-211 (2000).</li><li>Green, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+affecting+oogenesis+in+the+South+African+clawed+frog+(Xenopus+laevis).">Factors affecting oogenesis in the South African clawed frog (Xenopus laevis).</a> Comp Med. 52 (4), 307-312 (2002).</li><li>Dumont, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oogenesis+in+Xenopus+laevis+(Daudin).+I.+Stages+of+oocyte+development+in+laboratory+maintained+animals.">Oogenesis in Xenopus laevis (Daudin). I. Stages of oocyte development in laboratory maintained animals.</a> J Morphol. 136 (2), 153-179 (1972).</li><li>Kressmann, A., Celis, J. E., Grassmann, A., Loyter, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Birnstiel+M.L.+Surrogate+genetics.">Birnstiel M.L. Surrogate genetics.</a> Transfer of Cell Constituents into Eucaryotic Cells. , 388-407 (1980).</li><li>Middendorp, S. J., Maldifassi, M. C., Baur, R., Sigel, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Positive+modulation+of+synaptic+and+extrasynaptic+GABAA+receptors+by+an+antagonist+of+the+high+affinity+benzodiazepine+binding+site.">Positive modulation of synaptic and extrasynaptic GABAA receptors by an antagonist of the high affinity benzodiazepine binding site.</a> Neuropharmacology. 95, 459-467 (2015).</li><li>Dumont, J. N., Brummett, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oogenesis+in+Xenopus+laevis+(Daudin).+V.+Relationships+between+developing+oocytes+and+their+investing+follicular+tissues.">Oogenesis in Xenopus laevis (Daudin). V. Relationships between developing oocytes and their investing follicular tissues.</a> J Morphol. 155 (1), 73-97 (1978).</li><li>Dascal, N., Landau, E. M., Lass, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenopus+oocyte+resting+potential,+muscarinic+responses+and+the+role+of+calcium+and+guanosine+3',5'-cyclic+monophosphate.">Xenopus oocyte resting potential, muscarinic responses and the role of calcium and guanosine 3',5'-cyclic monophosphate.</a> J Physiol. 352, 551-574 (1984).</li><li>Buckingham, S. D., Pym, L., Sattelle, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oocytes+as+an+expression+system+for+studying+receptor/channel+targets+of+drugs+and+pesticides.">Oocytes as an expression system for studying receptor/channel targets of drugs and pesticides.</a> Methods Mol Biol. 322, 331-345 (2006).</li><li>Sigel, E., Baur, R., Trube, G., M&#246;hler, H., Malherbe, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+subunit+combination+of+rat+brain+GABAA+receptors+on+channel+function.">The effect of subunit combination of rat brain GABAA receptors on channel function.</a> Neuron. 5, 703-711 (1990).</li></ol>

The authors have nothing to disclose.

이 문서에서 설명하는 방법은 전통적으로 7-9 사용되는 일탈. 1 내지 2 시간 콜라게나 제 처리 8 난소 로브를 노출하는 표준이다; 손상되지 않은, 무 결함 난 모세포를 분리; mRNA를 상업적 분사 장치를 사용하여 그것들을 주입. 난자게나 고농도로 긴 노출에 의해 손상 될 가능성이있다 1)이 고전 절차는 다음과 같은 단점을 갖는다. 2) 불안정 무 결함 난자가 실험까지 저장해야합니다. ...

제노 푸스 난 모세포 널리 (- 3, 참조 내부 참조 2) 발현 시스템으로서 사용된다. 이들은 적절히 조합 및 세포막에 기능적 활성 multisubunit 단백질을 포함 할 수있다. 이 시스템을 사용하면, 기능적으로, 단독으로 또는 다른 단백질과 결합 막 단백질을 조사 돌연변이 키메라 또는 연접 단백질의 성질을 연구하기 위해, 잠재적 약물을 스크리닝 할 수있다. 다른 이종 발현 시스템을 통해 난자를 사용 이점은 거대 세포의 간단한 처리 외국 유전 정보, 목욕 관류에 의해 난자의 환경의 간단한 제어를 발현하는 세포의 비율이 높은, 상기 막 전위의 제어를 포함 . 이러한 발현 시스템의 단점은 많은 실험실 17-20에서 관찰 계절 변동이다. 이러한 변화의 원인분명 거리가 멀다. 또한 난자의 품질이 종종 크게 변화하는 것으로 관찰된다. 전통적인 방법 7-9 난소 로브의 분리, 몇 시간 동안 콜라게나 제에 난소 로브의 노출, 무 결함 난자의 선택, 그리고 난자의 미세 주입을 포함했다. 여기서, 대안, 빠른 절차의 수는 우리가 난자의 질에 어떤 계절의 변화와 약간의 변화와 함께 30 년 이상이 발현 시스템에서 작동하도록 허용 한 것을보고됩니다. 난 모세포의 분리, cRNA를 미세 주사로하고, 모낭 세포 층의 제거를 위해 여기에 설명 된 수정 및 개선 방법은 제노 푸스 난 모세포에서 선택한 임의의 단백질의 발현을 위해 사용될 수있다. 난자 주위 매질의 빠른 변화 용액의 아주 간단한 방법은 리간드 개폐 통로와 캐리어들의 연구에 적용될 수있다.

<table><tbody><tr><td>NaCl</td><td>Sigma</td><td>71380</td><td></td></tr><tr><td>KCl</td><td>Sigma</td><td>P-9541</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma</td><td>S6014</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;&amp;nbsp;</td><td>Sigma</td><td>M-1880</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;&amp;nbsp;</td><td>Sigma</td><td>223560</td><td></td></tr><tr><td>Ca(NO&lt;sub&gt;3&lt;/sub&gt;)&lt;sub&gt;2&lt;/sub&gt;&amp;nbsp;</td><td>Sigma</td><td>C1396</td><td></td></tr><tr><td>HEPES</td><td>Sigma</td><td>H3375</td><td></td></tr><tr><td>Penicilin/streptomycin</td><td>Gibco</td><td>15140-148</td><td>100 &amp;mu;g penicillin/mL and 100 &amp;mu;g streptomycin/mL</td></tr><tr><td>Platinum wire loop</td><td>home-made</td><td></td><td></td></tr><tr><td>Micropipette puller</td><td>Zeitz-Instruments GmBH</td><td>DMZ</td><td></td></tr><tr><td>Hamilton syringe&amp;nbsp;</td><td>Hamilton</td><td>80300</td><td>10 &amp;mu;L, Type 701N</td></tr><tr><td>Thick walled polytetrafluoroethylene tubing</td><td>Labmarket GmBH</td><td></td><td>1.0 mm OD&amp;nbsp;</td></tr><tr><td>Paraffin oil</td><td>Sigma</td><td>18512</td><td></td></tr><tr><td>Nylon net, G: 0.8 mm&amp;nbsp;</td><td>ZBF Z&amp;uuml;richer Beuteltuchfabrik AG</td><td></td><td></td></tr><tr><td>Borosilicate glass tube&amp;nbsp;</td><td>Corning</td><td>99445-12</td><td>PYREX</td></tr><tr><td>Collagenase NB Standard Grade</td><td>SERVA</td><td>17454</td><td></td></tr><tr><td>Trypsin inhibitor type I-S</td><td>Sigma</td><td>T-9003</td><td></td></tr><tr><td>EGTA</td><td>Sigma</td><td>E3389</td><td></td></tr><tr><td>Glass capillary</td><td>Jencons (Scientific ) LTD.</td><td>H15/10</td><td>1.35 ID mm (for perfusion), alternative company: Harvard Apparatus Limited</td></tr><tr><td>Borosilicate glass capillary</td><td>Harvard Apparatus Limited</td><td>30-0019</td><td>1.0 OD x&amp;nbsp; 0.58 ID x 100 Length mm (for microinjection)&amp;nbsp;</td></tr><tr><td>Borosilicate glass capillary&amp;nbsp;</td><td>Harvard Apparatus Limited</td><td>30-0044</td><td>1.2 OD x 0.69 ID x 100 Length mm (for two-electrode voltage clamp)</td></tr><tr><td>&amp;gamma;-Aminobutyric acid (GABA)</td><td>Sigma</td><td>A2129</td><td></td></tr><tr><td>3&amp;alpha;,21-Dihydroxy-5&amp;alpha;-pregnan-20-one (THDOC)</td><td>Sigma</td><td>P2016</td><td></td></tr><tr><td>grill motor</td><td>Faulhaber&amp;nbsp;</td><td></td><td>DC micromotor Type 2230 with gear Type 22/2</td></tr><tr><td>micrometer screw</td><td>Kiener-Wittlin</td><td>10400</td><td>TESA, AR 02.11201</td></tr><tr><td>Sterile plastic transfer pipettes</td><td>Saint-Amand&amp;nbsp; Mfg.</td><td>222-20S</td><td></td></tr></tbody></table>

xenopus oocytes: optimized methods for microinjection, removal of follicular cell layers, and fast solution changes in electrophysiological experiments

단백질 이종 발현 시스템으로 제노 푸스 난 모세포는 제 Gurdon 동부 등에 의해 설명되었다. 1 널리 발견부터 사용되었다 (참고 문헌 2-3 및 참조 내부). 외국 채널 식의 난자가 매력적인 만드는 특징은 내생 이온 채널 4의 가난한 풍부하다. 이 발현 시스템은 리간드 게이트 이온 채널 그들 가운데 많은 단백질의 특성에 유용 입증되었습니다. GABA의 아프리카 손톱 개구리의 난모 세포 수용체와 기능적 특성의 발현은 난자, cRNA를, 여포 세포 층의 제거 및 전기 생리학 실험에서 빠른 솔루션 변경 microinjections의 분리를 포함하여, 여기에 설명되어 있습니다. 절차는이 실험 5,6에 최적화되어 있으며 정기적으로 7-9을 사용하는 것 이탈했다. 전통적으로, 무 결함 난자 제조RT에서 난소 로브의 연장 콜라게나 제 처리, 이러한 무 결함 난자와의 mRNA와 미세 주입된다. 최적화 된 방법을 사용하여 다양한 막 단백질은 발현되고 그러한 재조합 GABA A는 수용체 10-12 인간 재조합 클로라이드 채널 (13), 트리파노소마 칼륨 채널 (14), 및 미오의 -inositol 전달체 (15, 16),이 시스템을 연구 하였다. 여기에 설명 된 방법은 한 아프리카 손톱 개구리의 난모 세포의 선택 중의 단백질의 발현에인가 될 수 있고, 신속한 용액 변경은 다른 리간드 관문 이온 채널을 연구하는데 사용될 수있다.

아프리카 손톱 개구리의 난모 세포의 기계적 백금 루프 (도 1)를 이용하여 선발 하였다. 0.5 : 난자는 mRNA의 2, δ, 0.5 β, 수용체의 서브 유닛 4 α GABA 코딩에 미세 주입 된 2.5 fmol / 난 모세포 (그림 2). 4 D 후, 모낭 세포 층 (도 3)을 제거 하였다. 난 모세포 전압 -80 MV에 클램프 1 μM 3α, 21- 히드 록시 5α-레그나 20...

Watch this Scientific Journal Video about Xenopus Oocytes: Optimized Methods for Microinjection, Removal of Follicular Cell Layers, and Fast Solution Changes in Electrophysiological Experiments at JoV

Xenopus Oocytes: Optimized Methods for Microinjection, Removal of Follicular Cell Layers, and Fast Solution Changes in Electrophysiological Experiments

Optimized procedures for the isolation of single follicles, cytoplasmic RNA microinjections, the removal of surrounding cell layers, and protein expression in Xenopus oocytes are described. In addition, a simple method for fast solution changes in electrophysiological experiments with ligand-gated ion channels is presented.

Xenopus의 난 모세포 : 미세 주입을위한 최적화 방법, 여포 세포 레이어의 제거 및 전기 생리 실험에서 빠른 해결 방법 변경

The Xenopus oocyte as a heterologous expression system for proteins, was first described by Gurdon et al.1 and has been widely used since its discovery ...

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Xenopus Oocytes: Optimized Methods for Microinjection, Removal of Follicular Cell Layers, and Fast Solution Changes in Electrophysiological Experiments

20.1K 조회수.  University of Bern. Optimized procedures for the isolation of single follicles, cytoplasmic RNA microinjections, the removal of surrounding cell layers, and protein expression in Xenopus oocytes are described. In addition, a simple method for fast solution changes in electrophysiological experiments with ligand-gated ion channels is presented. 

비디오: Xenopus Oocytes: Optimized Methods for Microinjection, Removal of Follicular Cell Layers, and Fast Solution Changes in Electrophysiological Experiments

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

Amphibian eggs have been widely used to study embryonic development. Early embryonic development is driven by maternally stored factors accumulated during oogenesis. In order to study roles of such maternal factors in early embryonic development, it is desirable to manipulate their functions from the very beginning of embryonic development. Conventional ways of gene interference are achieved by injection of antisense oligonucleotides (oligos) or mRNA into fertilized eggs, enabling under- or over-expression of specific proteins, respectively. However, these methods normally require more than several hours until protein expression is affected, and, hence, the interference of gene functions is not effective during early embryonic stages. Here, we introduce an experimental system in which expression levels of maternal proteins can be altered before fertilization. Xenopus laevis oocytes obtained from ovaries are defolliculated by incubating with enzymes. Antisense oligos or mRNAs are injected into defolliculated oocytes at the germinal vesicle (GV) stage. These oocytes are in vitro matured to eggs at the metaphase II (MII) stage, followed by intracytoplasmic sperm injection (ICSI). By this way, up to 10% of ICSI embryos can reach the swimming tadpole stage, thus allowing functional tests of specific gene knockdown or overexpression. This approach can be a useful way to study roles of maternally stored factors in early embryonic development.

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

We describe methods of manipulating Xenopus laevis immature oocytes, in vitro maturation of oocytes to eggs, and intracytoplasmic sperm injection. This protocol allows degradation of some maternal proteins and overexpression of genes of interest at fertilization, and hence is valuable to study roles of specific factors in early embryonic development.

조작 및<em&gt; 체외</em의&gt; 성숙<em&gt; 아프리카 발톱 개구리 laevis의</em세포질 내 정자 주입에 이어&gt; 난 모세포, 배아 발달을 연구하는

Amphibian eggs have been widely used to study embryonic development. Early embryonic development is driven by maternally stored factors accumulated during ...

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발생학

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

This protocol describes a method to visualise ligands distributed across a field of cells. The ease of expressing exogenous proteins, together with the large size of their cells in early embryos, make Xenopus laevis a useful model for visualising GFP-tagged ligands. Synthetic mRNAs are efficiently translated after injection into early stage Xenopus embryos, and injections can be targeted to a single cell. When combined with a lineage tracer such as membrane tethered RFP, the injected cell (and its descendants) that are producing the overexpressed protein can easily be followed. This protocol describes a method for the production of fluorescently tagged Wnt and Shh ligands from injected mRNA. The methods involve the micro dissection of ectodermal explants (animal caps) and the analysis of ligand diffusion in multiple samples. By using confocal imaging, information about ligand secretion and diffusion over a field of cells can be obtained. Statistical analyses of confocal images provide quantitative data on the shape of ligand gradients. These methods may be useful to researchers who want to test the effects of factors that may regulate the shape of morphogen gradients.

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

The manuscript here provides a simple set of methods for analysing the secretion and diffusion of fluorescently tagged ligands in Xenopus. This provides a context for testing the ability of other proteins to modify ligand distribution and allowing experiments that may give insight into mechanisms regulating morphogen gradients.

공 촛점 분석을 사용하여<em&gt; 아프리카 발톱 개구리 laevis의</em&gt;의 Wnt 및 쉬 Morphogen 그라디언트의 변조기를 조사하기 위해

This protocol describes a method to visualise ligands distributed across a field of cells. The ease of expressing exogenous proteins, together with the ...

Voltage-clamp Fluorometry in Xenopus Oocytes Using Fluorescent Unnatural Amino Acids

Voltage-Clamp Fluorometry (VCF) has been the technique of choice to investigate the structure and function of electrogenic membrane proteins where real-time measurements of fluorescence and currents simultaneously report on local rearrangements and global function, respectively1. While high-resolution structural techniques such as cryo-electron microscopy or X-ray crystallography provide static images of the proteins of interest, VCF provides dynamic structural data that allows us to link the structural rearrangements (fluorescence) to dynamic functional data (electrophysiology). Until recently, the thiol-reactive chemistry used for site-directed fluorescent labeling of the proteins restricted the scope of the approach because all accessible cysteines, including endogenous ones, will be labeled. It was thus required to construct proteins free of endogenous cysteines. Labeling was also restricted to sites accessible from the extracellular side. This changed with the use of Fluorescent Unnatural Amino Acids (fUAA) to specifically incorporate a small fluorescent probe in response to stop codon suppression using an orthogonal tRNA and tRNA synthetase pair2. The VCF improvement only requires a two-step injection procedure of DNA injection (tRNA/synthetase pair) followed by RNA/fUAA co-injection. Now, labelling both intracellular and buried sites is possible, and the use of VCF has expanded significantly. The VCF technique thereby becomes attractive for studying a wide range of proteins and &#8211; more importantly &#8211; allows investigating numerous cytosolic regulatory mechanisms.

Voltage-clamp Fluorometry in Xenopus Oocytes Using Fluorescent Unnatural Amino Acids

This article describes an enhancement of conventional Voltage-Clamp Fluorometry (VCF) where Fluorescent Unnatural Amino Acids (fUAA) are used instead of maleimide dyes, to probe structural rearrangements in ion channels. The procedure includes Xenopus oocyte DNA injection, RNA/fUAA coinjection, and simultaneous current and fluorescence measurements.

전압 클램프 형광 측정법<em&gt; Xenopus</em&gt; 형광성 비 자연 아미노산을 이용한 난 모세포

Voltage-Clamp Fluorometry (VCF) has been the technique of choice to investigate the structure and function of electrogenic membrane proteins where ...

생화학

Microinjection of Xenopus Laevis Oocytes

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic transport. Because of its large nucleus and high density of nuclear pore complexes (NPCs), nuclear transport can be easily visualized in the Xenopus oocyte. Much insight into the mechanisms of nuclear import and export has been gained through use of this system (reviewed by Pant&eacute;, 2006). In addition, we have used microinjection of Xenopus oocytes to dissect the nuclear import pathways of several viruses that replicate in the host nucleus. 
Here we demonstrate the cytoplasmic microinjection of Xenopus oocytes with a nuclear import substrate. We also show preparation of the injected oocytes for visualization by thin-sectioning EM, including dissection, dehydration, and embedding of the oocytes into an epoxy embedding resin. Finally, we provide representative results for oocytes that have been microinjected with the capsid of the baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV) or the parvovirus Minute Virus of Mice (MVM), and discuss potential applications of the technique.

Here we demonstrate cytoplasmic microinjection of Xenopus laevis oocytes with a nuclear import substrate, as well as preparation of the injected oocytes for visualization by thin-sectioning electron microscopy.

Xenopus Laevis의 Oocytes의 Microinjection

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

생물학

Visualizing RNA Localization in Xenopus Oocytes

RNA localization is a conserved mechanism of establishing cell polarity. Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to spatially restrict gene expression of Vg1 protein. Tight control of Vg1 distribution in this manner is required for proper germ layer specification in the developing embryo. RNA sequence elements in the 3' UTR of the mRNA, the Vg1 localization element (VLE) are required and sufficient to direct transport. To study the recognition and transport of Vg1 mRNA in vivo, we have developed an imaging technique that allows extensive analysis of trans-factor directed transport mechanisms via a simple visual readout. 
To visualize RNA localization, we synthesize fluorescently labeled VLE RNA and microinject this transcript into individual oocytes. After oocyte culture to allow transport of the injected RNA, oocytes are fixed and dehydrated prior to imaging by confocal microscopy. Visualization of mRNA localization patterns provides a readout for monitoring the complete pathway of RNA transport and for identifying roles in directing RNA transport for cis-acting elements within the transcript and trans-acting factors that bind to the VLE (Lewis et al., 2008, Messitt et al., 2008). We have extended this technique through co-localization with additional RNAs and proteins (Gagnon and Mowry, 2009, Messitt et al., 2008), and in combination with disruption of motor proteins and the cytoskeleton (Messitt et al., 2008) to probe mechanisms underlying mRNA localization.

Visualizing RNA Localization in Xenopus Oocytes

Visualization of in vivo RNA transport is accomplished by microinjection of fluorescently labeled RNA transcripts into Xenopus oocytes, followed by confocal microscopy.

머릿속 RNA 현지화에서 Xenopus Oocytes

RNA localization is a conserved mechanism of establishing cell polarity.  Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to ...

Fertilization of Xenopus oocytes using the Host Transfer Method

Studying the contribution of maternally inherited molecules to vertebrate early development is often hampered by the time and expense necessary to generate maternal-effect mutant animals. Additionally, many of the techniques to overexpress or inhibit gene function in organisms such as Xenopus and zebrafish fail to sufficiently target critical maternal signaling pathways, such as Wnt signaling. In Xenopus, manipulating gene function in cultured oocytes and subsequently fertilizing them can ameliorate these problems to some extent. Oocytes are manually defolliculated from donor ovary tissue, injected or treated in culture as desired, and then stimulated with progesterone to induce maturation. Next, the oocytes are introduced into the body cavity of an ovulating host female frog, whereupon they will be translocated through the host's oviduct and acquire modifications and jelly coats necessary for fertilization. The resulting embryos can then be raised to the desired stage and analyzed for the effects of any experimental perturbations. This host-transfer method has been highly effective in uncovering basic mechanisms of early development and allows a wide range of experimental possibilities not available in any other vertebrate model organism.

Fertilization of Xenopus oocytes using the Host Transfer Method

Procedure for fertilizing Xenopus oocytes by the host transfer method.

의 수정 Xenopus의 oocytes 호스트 전송 방법을 사용하여

Studying the contribution of maternally inherited molecules to vertebrate early development is often hampered by the time and expense necessary to ...

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may also be used to study the structure-function relationship for other ion channels and neurotransmitter receptors1. BK channels are widely expressed in different tissues and have been implicated in many physiological functions, including regulation of smooth muscle contraction, frequency tuning of inner hair cells and regulation of neurotransmitter release2-6. BK channels are activated by membrane depolarization and by intracellular Ca2+ and Mg2+ 6-9. Therefore, the protocol is designed to control both the membrane voltage and the intracellular solution. In this protocol, messenger RNA of BK channels is injected into Xenopus laevis oocytes (stage V-VI) followed by 2-5 days of incubation at 18&deg;C10-13. Membrane patches that contain single or multiple BK channels are excised with the inside-out configuration using patch clamp techniques10-13. The intracellular side of the patch is perfused with desired solutions during recording so that the channel activation under different conditions can be examined. To summarize, the mRNA of BK channels is injected into Xenopus laevis oocytes to express channel proteins on the oocyte membrane; patch clamp techniques are used to record currents flowing through the channels under controlled voltage and intracellular solutions.

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Ionic current of BK channels is recorded using patch clamp techniques. BK channels are expressed in Xenopus oocytes by injecting messenger RNA. The intracellular solution during patch clamp recordings is controlled by a perfusion system.

이온 채널 유학을위한 패치 클램프 및 재관류 기법으로 표현 Xenopus oocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...

Mouse Oocyte Microinjection, Maturation and Ploidy Assessment

Mistakes in chromosome segregation lead to aneuploid cells. In somatic cells, aneuploidy is associated with cancer but in gametes, aneuploidy leads to infertility, miscarriages or developmental disorders like Down syndrome. Haploid gametes form through species-specific developmental programs that are coupled to meiosis. The first meiotic division (MI) is unique to meiosis because sister chromatids remain attached while homologous chromosomes are segregated. For reasons not fully understood, this reductional division is prone to errors and is more commonly the source of aneuploidy than errors in meiosis II (MII) or than errors in male meiosis 1,2. 
In mammals, oocytes arrest at prophase of MI with a large, intact germinal vesicle (GV; nucleus) and only resume meiosis when they receive ovulatory cues. Once meiosis resumes, oocytes complete MI and undergo an asymmetric cell division, arresting again at metaphase of MII. Eggs will not complete MII until they are fertilized by sperm. Oocytes also can undergo meiotic maturation using established in vitro culture conditions 3. Because generation of transgenic and gene-targeted mouse mutants is costly and can take long periods of time, manipulation of female gametes in vitro is a more economical and time-saving strategy. 
Here, we describe methods to isolate prophase-arrested oocytes from mice and for microinjection. Any material of choice may be introduced into the oocyte, but because meiotically-competent oocytes are transcriptionally silent 4,5 cRNA, and not DNA, must be injected for ectopic expression studies. To assess ploidy, we describe our conditions for in vitro maturation of oocytes to MII eggs. Historically, chromosome-spreading techniques are used for counting chromosome number 6. This method is technically challenging and is limited to only identifying hyperploidies. Here, we describe a method to determine hypo-and hyperploidies using intact eggs 7-8. This method uses monastrol, a kinesin-5 inhibitor, that collapses the bipolar spindle into a monopolar spindle 9 thus separating chromosomes such that individual kinetochores can readily be detected and counted by using an anti-CREST autoimmune serum. Because this method is performed in intact eggs, chromosomes are not lost due to operator error.

Oocytes are prone to aneuploidy due to errors in chromosome segregation during meiotic maturation. Aneuploid eggs can cause infertility, miscarriages or developmental disorders like Down syndrome. Here, we describe methods to introduce materials of choice into oocytes and methods to study meiotic maturation and assess ploidy.

마우스 Oocyte의 Microinjection, 성숙과 Ploidy 평가

Mistakes in chromosome segregation lead to aneuploid cells.  In somatic cells, aneuploidy is associated with cancer but in gametes, aneuploidy leads to ...

Microtransplantation of Synaptic Membranes to Reactivate Human Synaptic Receptors for Functional Studies

Excitatory and inhibitory ionotropic receptors are the major gates of ion fluxes that determine the activity of synapses during physiological neuronal communication. Therefore, alterations in their abundance, function, and relationships with other synaptic elements have been observed as a major correlate of alterations in brain function and cognitive impairment in neurodegenerative diseases and mental disorders. Understanding how the function of excitatory and inhibitory synaptic receptors is altered by disease is of critical importance for the development of effective therapies. To gain disease-relevant information, it is important to record the electrical activity of neurotransmitter receptors that remain functional in the diseased human brain. So far this is the closest approach to assess pathological alterations in receptors' function. In this work, a methodology is presented to perform microtransplantation of synaptic membranes, which consists of reactivating synaptic membranes from snap frozen human brain tissue containing human receptors, by its injection and posterior fusion into the membrane of Xenopus laevis oocytes. The protocol also provides the methodological strategy to obtain consistent and reliable responses of &#945;-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and &#947;-aminobutyric acid (GABA) receptors, as well as novel detailed methods that are used for normalization and rigorous data analysis.

The protocol demonstrates that by performing microtransplantation of synaptic membranes into Xenopus laevis oocytes, it is possible to record consistent and reliable responses of &#945;-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and &#947;-aminobutyric acid receptors.

기능적 연구를 위한 인간 시냅틱 수용체를 재활성화하기 위한 시냅틱 멤브레인의 미세이식

Excitatory and inhibitory ionotropic receptors are the major gates of ion fluxes that determine the activity of synapses during physiological neuronal ...

신경과학

Xenopus laevis as a Model to Identify Translation Impairment

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

Xenopus laevis as a Model to Identify Translation Impairment

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

Xenopus의 번역 손상을 식별 할 수있는 모델로 laevis의

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

Xenopus의 난 모세포 : 미세 주입을위한 최적화 방법, 여포 세포 레이어의 제거 및 전기 생리 실험에서 빠른 해결 방법 변경

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