We would like to acknowledge Dr. Bradley Schulte for comments on this protocol. This work was supported by National Institutes of Health grant R00 (5R00DC010220). This project was performed in a renovated laboratory space supported by Grant C06RR014516.

ملاحظة: جميع البروتوكولات باستخدام الحيوانات الحية ويجب مراجعتها والموافقة عليها من قبل لجنة رعاية واستخدام المؤسسية الحيوان (IACUC) ويجب أن تتبع وافقت رسميا الطرق لرعاية واستخدام الحيوانات المختبرية. يجب أن يتم تنفيذ كافة تشريح باستخدام تقنية معقمة على مقاعد البدلاء نظيفة تدفق الصفحي. قفازات وقناع، إذا رغبت في ذلك، يجب أن ترتديه أثناء هذا الإجراء. 1. تشريح الأذن الداخلية الجنينية الماوس <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> الإعداد للهيئة في الثقافات يزدرع كورتي: <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تعقيم تدفق الصفحي غطاء نسيج الثقافة عن طريق تشغيل ضوء الأشعة فوق البنفسجية لمدة 30 دقيقة وتطهير السطح مع الايثانول 70٪ قبل استخدامها. وبالإضافة إلى ذلك، وتعقيم جميع الأدوات تشريح بما في ذلك أدوات تشريح الجميلة وأطباق Sylgard المغلفة للجنين عن طريق تشريح الأوتوكلاف أو عن طريق نقع في الايثانول 70٪ لمدة 20 دقيقة على الأقل قبل الاستخدام. تخلصي الايثانول 70٪ والسماح للأطباق وأدوات في الهواء الجاف في الصفحي نظيفة تدفق جزءا لا يتجزأ منن مقاعد البدلاء قبل استخدامها. </li><li style=";text-align:right;direction:rtl"> تحضير محلول الملح المتوازن معقم هانك (HBSS) / (4- (2-هيدروكسي إيثيل) حامض -1-piperazineethanesulfonic) (HEPES) خليط من خلال إضافة 10٪ من 10X HBSS و 0.5٪ HEPES وضبط درجة الحموضة إلى ما يقرب من 7.2 وفلتر تعقيم الحل النهائي وتخزينها في 4 ° C. تنفيذ جميع التشريح في برود حل HBSS / HEPES للحفاظ على أفضل الأنسجة في جميع أنحاء الداخلي. </li><li style=";text-align:right;direction:rtl"> أطباق أسفل الزجاج معطف مع مصفوفة الغشاء القاعدي عن طريق إضافة 5 مل من البرد (4 ° C) العقيمة Dulbecco لتعديل النسر المتوسطة (DMEM) إلى قسامة 300 ميكرولتر من المصفوفة الغشاء القاعدي في العقيمة 15 مل أنبوب البولي بروبلين. خلط قبل vortexing محتويات وإضافة 150 ميكرولتر من الغشاء القاعدي مصفوفة DMEM خليط من وسط كل صحن الثقافة بحيث يغطي ثقافة كاملة الحالية بشكل جيد في وسط الطبق. تغطية الأطباق وتخزينها في الحاضنة 37 درجة مئوية لمدة 45 دقيقة على الأقل قبل الاستخدام. </li><li style=";text-align:right;direction:rtl"> صب 4-5 مل المبردة HBSS إلى عشرري أطباق بتري معقمة البوليسترين وترك مغلقة في غطاء محرك السيارة. </li><li style=";text-align:right;direction:rtl"> إعداد مستنبت في أنبوب معقم 15 مل البولي بروبلين بإضافة 9 مل من DMEM، و 100 ميكرولتر من المكملات N2، 10 غرام / مل سيبرو و 1 مل من FBS. </li></ol></li><li style=";text-align:right;direction:rtl"> عزل الأجنة: ملاحظة: في هذا الإجراء لزراعة قوقعة إإكسبلنتس من الفئران الجنينية، الأذن الداخلية الحصاد من يوم الجنينية (E) 13 العظام الزمنية مع اليوم بعد الإخصاب التي تعتبر E1 9. ويمكن أيضا أن تستخدم بروتوكول مع الماوس الجنينية الأذن الداخلية ابتداء من E12 E18 ل. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> الموت ببطء ماوس توقيت الحوامل مع CO 2 والتضحية الحيوانية خلع عنق الرحم أو البروتوكولات المعتمدة المناسبة. تنفيذ euthanization بعيدا عن تدفق الصفحي غطاء نسيج الثقافة للحفاظ على عقم في غرفة زراعة الأنسجة. </li><li style=";text-align:right;direction:rtl"> ضع الماوس الموت الرحيم على منشفة ورقية مع البطن مواجهة وتطهير البطن مع الايثانول 70٪. </li><li style=";text-align:right;direction:rtl"> فتح تجويف البطن عن طريق الجلد الاستيلاءمع ملقط المنحنية مع يد واحدة وقطع البشرة والعضلات على طول خط الوسط مع مقص باستخدام جهة أخرى. جعل اثنين من التخفيضات عمودي على كلا الجانبين، وسحب الرحم بعناية من قبل رافعين قرون الرحم الثنائية مع ملقط وسلخه من تحت النسيج الضام باستخدام مقص. </li><li style=";text-align:right;direction:rtl"> وضع سلسلة الجنينية في طبق بتري تحتوي على حل المبردة تشريح (HBSS / HEPES خليط) ونقلها إلى الصفحي تدفق مقاعد البدلاء النظيفة. </li><li style=";text-align:right;direction:rtl"> إزالة الأجنة بعناية من المشيمة ووضعها في واحدة من أطباق بتري معقمة تحتوي على حل تشريح. إذا كان هذا الحل يتحول الدموي، نقلها إلى طبق بتري جديد. ملاحظة: عند هذه النقطة والأجنة يمكن نظموا باستخدام دليل انطلاق تايلر. </li><li style=";text-align:right;direction:rtl"> قطع رأس الأجنة عن طريق معسر في منطقة الرقبة مع زوج من ملقط نظيفة أو المقص ووضع رؤساء في طبق بيتري الطازجة التي تحتوي على حل تشريح المبردة. </li></ol></li><li style=";text-align:right;direction:rtl"> تشريح الأذن الداخلية: <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> مكان سرئيس الجنين شمال شرق في طبق sylgard العقيمة التي تحتوي على تشريح البارد </li><li style=";text-align:right;direction:rtl"> الحل. العمل تحت المجهر تشريح في التكبير من ~ 1.6X، لشل حركة رأس الجنين عن طريق وضع دبابيس Minutien حول أو أقرب إلى منطقة العين (الشكل 1A). </li><li style=";text-align:right;direction:rtl"> باستخدام اثنين من أزواج من ملقط معقم إزالة بعناية الجلد وفتح الجمجمة على الجانب الظهري على طول خط الوسط (الشكل 1B). إزالة الدماغ من يمكن التعرف على تجويف الجمجمة والأذن الداخلية التي تقع داخل العظام الزمنية في هذه المرحلة (الشكل 1C، D). بطانة الأوعية الدموية، التي عادة ما تكون موجودة في جميع أنحاء الأذن الداخلية، ويمكن استخدامها كمعلم لتحديد الأذن الداخلية في هذه المرحلة (الشكل 1D). </li><li style=";text-align:right;direction:rtl"> تشريح الأذن الداخلية من العظام الزمنية عن طريق وضع ملقط تحت الأنسجة وعزل الأذن الداخلية من قاعدة الجمجمة ونقل إلى طبق جديد يحتوي الحل تشريح الباردة (الشكل 1E، F). </li></ol></li></ol> 2. جيل من الجهاز الثقافات كورتي يزدرع ملاحظة: في هذه المرحلة من التنمية، والأنسجة الغضروفية هي ويمكن تشريح بسهولة باستخدام ملقط. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> الشرق الأذن الداخلية بحيث الجانب البطني يكون مواجها لها (الشكل 2A) وتحقيق الاستقرار في الأذن الداخلية عن طريق إدراج بلطف على نهاية حادة من الدبابيس Minutien من خلال الجزء الدهليزي في الأذن الداخلية (الشكل 2B). </li><li style=";text-align:right;direction:rtl"> قطع متناهية الصغر باستخدام ملقط فتح الغضروف المغطي عن طريق شق باستخدام واحدة من نهاية ملقط بالقرب من النافذة البيضاوي وبعناية إزالة الغضروف من القوقعة (الشكل 2C). ملاحظة: من المهم للتأكد من أن ملقط لا تدرج كثيرا في الغضروف كما الغضروف المغطي تنصهر في بعض الأحيان مع قناة القوقعية وفي هذه الحالة فإنه من المفيد استخدام ملقط مغلقة لاطلاق سراح الغضروف من قناة القوقعية الأساسي عن طريق كشط تحت الصورةurface من الغضروف. في هذه المرحلة من التنمية، ودوامة القوقعة هي فقط ثلاثة أرباع منعطفا في الطول. </li><li style=";text-align:right;direction:rtl"> وبعد ذلك، فضح ظهارة الحسية عن طريق وضع ملقط في المنطقة المفضل للقناة القوقعية، إما القاعدة أو في ذروة جدا، وسحب بلطف من سطح القوقعة (الشكل 2D). ملاحظة: سقف القوقعة يجب إزالة تماما كما يمكن أن تحجب الظهارة الحسية التي تحول دون تحليل. ملاحظة: يجب أن يتم هذا الإجراء بلطف لأنه من السهل المسيل للدموع الظهارة الحسية. </li><li style=";text-align:right;direction:rtl"> وكخطوة الماضية، وإزالة بعناية النسيج الضام الكامنة من الظهارة الحسية تتعرض بحيث قاعدة يزدرع قوقعة فارغة بشكل موحد. </li><li style=";text-align:right;direction:rtl"> عزل القوقعة تشريح من الجزء الدهليزي في الأذن الداخلية باستخدام ملقط. هذا لا يمكن أن يؤديها قبل أو بعد الخطوة 2.4 (الشكل 2E). </li><li style=";text-align:right;direction:rtl"> نقل الظهارة الحسية قوقعة تشريح في الاغشيه الطابق السفليشمال شرق الثقافة جيدا باستخدام 1.5 ملم بالمغرفة معقمة (الشكل 2F) المغلفة المصفوفة. </li><li style=";text-align:right;direction:rtl"> الشرق ازدراع القوقعة مع سطح lumenal من ظهارة تواجه صعودا ونضح الغشاء القاعدي مصفوفة DMEM حل لشد بعناية النسيج واستبدالها مع 150 ميكرولتر من مستنبت الطازجة عن طريق إضافة بلطف إلى الطبق. ينبغي توخي الحذر للتأكد من أن كل يزدرع تلتزم بشكل جيد للساترة الزجاج المطلي وليس يعوم في مستنبت. ضمان غيض من ملقط وأشار يصل بينما الالصاق إلى البئر الزجاج لتجنب إتلاف يزدرع القوقعة. </li><li style=";text-align:right;direction:rtl"> بلطف نقل الطبق الثقافة في العقيمة 150 مم طبق ثقافة ومكان في حاضنة زراعة الأنسجة عند 37 درجة مئوية مع CO 2 5٪ للفترة من الوقت المطلوب، وعادة 3-6 أيام في المختبر (DIV). بعد 1 DIV، ودراسة الثقافات تحت المجهر تشريح للتأكد من أن يزدرع القوقعة التزمت كذلك إلى الطبق الثقافة. </li><li style=";text-align:right;direction:rtl"> بعد فيcubation في المختبر لفترات من الوقت المطلوب، تتم معالجة الثقافات لالمناعية. ملاحظة: الثقافات يزدرع عادة ما يتم تحضين لمدة 6 DIV وهو ما يعادل المرحلة التنموية، P0. بعد مرور فترة الحضانة في المختبر والثقافات يمكن معالجتها لمزيد من التطبيقات المصب مثل المناعية (الشكل 3B)، RT-PCR، التهجين في الموقع، و / أو لطخة غربية في. </li></ol> 3. بوساطة Electroporation للنقل الجينات <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إنشاء ل electroporation: <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تعقيم واحدة 100 ملم صحن الزجاج Sylgard المغلفة من قبل التعقيم أو تمرغ في الايثانول 70٪ لمدة 20 دقيقة والسماح للهواء الجاف في مقاعد البدلاء النظيفة قبل استخدامها. </li><li style=";text-align:right;direction:rtl"> إعداد DNA من متجه التعبير عن خيار استخدام maxi- أو ميدي الإعدادية مجموعات المناسبة. ناقلات التعبير المستخدمة في هذا البروتوكول هي pIRES2-Atoh1.EGFP وpCLIG-NeuroD1.EGFP. يجب أن يكون تركيز النهائي من الحمض النووي لا يقل عن 1 ميكروغرام / ميكرولتر في الحمض النووي معقم /خالية من ريبونوكلياز المياه. </li></ol></li><li style=";text-align:right;direction:rtl"> Electroporating الحمض النووي في إإكسبلنتس قوقعة الجنينية: <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إضافة 10 ميكرولتر من محلول الحمض النووي البلازميد إلى 100 ملم Sylgard المغلفة طبق الطازجة ونقل يزدرع القوقعة تشريح بالكامل (تم الحصول عليها من الخطوة 2.5) في حل DNA. </li><li style=";text-align:right;direction:rtl"> مع سطح lumenal من ظهارة مواجهة، إمالة القوقعة قليلا بحيث انها عمودي على الطائرة من الطبق. </li><li style=";text-align:right;direction:rtl"> ضع المجاذيف الكهربائي على جانبي القوقعة مع مجداف سلبي (الكاثود) نحو الظهارة الحسية ومجداف الإيجابي (الأنود) يقع بالقرب من قاعدة القوقعة. ملاحظة: بما أن اتهم DNA سلبا، فإنه يهاجر من السلبية إلى القطب الموجب وعن طريق وضع نهاية القطب السالب لجنة التحقيق المتاخمة للظهارة حسية. فإن DNA يذهب معظمها من خلال سطح الظهارة الحسية. </li><li style=";text-align:right;direction:rtl"> باستخدام electroporator، وتقديم 9-10 نبضات من 24 فولت، 30 ميللي ثانية مدة النبضة مع التبديل دواسة القدم على رانه electroporator، ثم قم بإضافة 100 ميكرولتر من المتوسط ​​الدافئة الثقافة إلى القوقعة electroporated. </li><li style=";text-align:right;direction:rtl"> كرر الخطوات من 3.2.1-4 لجميع إإكسبلنتس قوقعة وDNA من جميع الجينات في المصالح ونقل القوقعة electroporated في طبق المغلفة مصفوفة الغشاء القاعدي للتصفيح (الخطوة 2.7). ثقافة كل القوقعي electroporated في 37 ° C، 5٪ CO 2 حاضنة مرطب لفترات من الوقت المطلوب الذي سيتم بعد ذلك المجهزة للمناعية. ملاحظة: القوقعة من القمامة كاملة من CD1 الماوس (حوالي 8-12 الجراء) يمكن عزل، microdissected، electroporated ومطلي في &lt;4 ساعة. </li></ol></li></ol> 4. تحليل الثقافات القوقعة يزدرع ملاحظة: عادة ما يتم تحضين الثقافات لمدة 6 DIV وهو ما يعادل المرحلة التنموية، P0. بعد مرور فترة الحضانة في المختبر، يتم إصلاح الثقافات ومعالجتها للمناعية. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> بعد طول المرجوة من المؤتمر الوطني العراقيubation، وإزالة مستنبت وبسرعة شطف يزدرع القوقعة في الفوسفات مخزنة المالحة (PBS)، واحتضان في 4٪ بارافورمالدهيد (المعد في PBS) لمدة 15 دقيقة في درجة حرارة الغرفة. </li><li style=";text-align:right;direction:rtl"> غسل مثبت من قبل PBS إضافة وشطف في برنامج تلفزيوني ثلاث مرات لمدة 15 دقيقة. </li><li style=";text-align:right;direction:rtl"> Permeabilize باستخدام PBS-T (PBS + 0.5٪ توين) لمدة 30 دقيقة قبل حجب مع تحتوي على 10٪ مصل الماعز PBS-T. </li><li style=";text-align:right;direction:rtl"> احتضان في حل الأجسام المضادة الأولية التي تحتوي على الأجسام المضادة الأولية في برنامج تلفزيوني-T مع 1٪ مصل الماعز بين عشية وضحاها في 4 درجات مئوية. </li><li style=";text-align:right;direction:rtl"> غسل القوقعة ثلاث مرات في درجة حرارة الغرفة باستخدام PBS-T 15 دقيقة لكل منهما قبل أن يضيف الأجسام المضادة الثانوية اليكسا-مترافق المناسبة. </li><li style=";text-align:right;direction:rtl"> منذ يزدرع القوقعة هو مثقف على الزجاج ساترة يمكن التعامل معها بشكل فردي عن طريق عزل ساترة من الطبق وتصاعد على شريحة. ويمكن تحقيق ذلك عن طريق نقع الطبق الثقافة في OS30 مذيب لا يقل عن 1 ساعة في درجة حرارة الغرفة. باستخدام شفرة حلاقة معقمة، فصل ساترة من دإيش وجبل على شريحة وتصور تحت المجهر الفلورسنت. </li></ol>

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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibitors+of+differentiation+and+DNA+binding+(Ids)+regulate+Math1+and+hair+cell+formation+during+the+development+of+the+organ+of+Corti.">Inhibitors of differentiation and DNA binding (Ids) regulate Math1 and hair cell formation during the development of the organ of Corti.</a> J Neurosci. 26 (2), 550-558 (2006).</li><li>Dabdoub, A., 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=Sox2+signaling+in+prosensory+domain+specification+and+subsequent+hair+cell+differentiation+in+the+developing+cochlea.">Sox2 signaling in prosensory domain specification and subsequent hair cell differentiation in the developing cochlea.</a> Proc. Natl. Acad. Sci. U.S.A. 105 (47), 18396-18401 (2008).</li><li>Doetzlhofer, A., Basch, M. L., Ohyama, T., Gessler, M., Groves, A. K., Segil, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hey2+regulation+by+FGF+provides+a+Notch-independent+mechanism+of+maintaining+pillar+cell+fate+in+the+organ+of+Corti.">Hey2 regulation by FGF provides a Notch-independent mechanism of maintaining pillar cell fate in the organ of Corti.</a> Dev Cell. 16 (1), 58-69 (2009).</li><li>Puligilla, C., Dabdoub, A., Brenowitz, S. D., Kelley, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sox2+induces+neuronal+formation+in+the+developing+cochlea.">Sox2 induces neuronal formation in the developing cochlea.</a> J Neurosci. 20 (2), 714-722 (2010).</li><li>Kaufman, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Atlas of Mouse Development. , (1995).</li><li>Sher, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+embryonic+and+postnatal+development+of+the+inner+ear+of+the+mouse.">The embryonic and postnatal development of the inner ear of the mouse.</a> Acta Otolaryngol Suppl. 285, 1-77 (1971).</li><li>Torres, M., Giraldez, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+the+vertebrate+inner+ear.">The development of the vertebrate inner ear.</a> Mech Dev. 71 (1-2), 5-21 (1998).</li><li>Brown, S. T., Martin, K., Groves, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+basis+of+inner+ear+induction.">Molecular basis of inner ear induction.</a> Curr Top Dev Biol. 57, 115-119 (2003).</li><li>Puligilla, C., Kelley, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Building+the+world&#8217;s+best+hearing+aid;+regulation+of+cell+fate+in+the+cochlea.">Building the world&#8217;s best hearing aid; regulation of cell fate in the cochlea.</a> Curr Opin Genet Dev. 19 (4), 368-373 (2009).</li><li>Wu, D. K., Kelley, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+of+inner+ear+development.">Molecular mechanisms of inner ear development.</a> Cold Spring Harb Perspect Biol. 4 (8), a008409 (2012).</li><li>Holt, 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=Viral-mediated+gene+transfer+to+study+the+molecular+physiology+of+the+mammalian+inner+ear.">Viral-mediated gene transfer to study the molecular physiology of the mammalian inner ear.</a> Audiol Neurootol. 7 (3), 157-160 (2002).</li><li>Stone, I. M., Lurie, D. I., Kelley, M. W., Poulsen, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adeno-associated+virus-mediated+gene+transfer+to+hair+cells+and+support+cells+of+the+murine+cochlea.">Adeno-associated virus-mediated gene transfer to hair cells and support cells of the murine cochlea.</a> Mol Ther. 11 (6), 843-848 (2005).</li></ol>

No conflicts of interest declared.

كل الخلايا داخل التيه الغشائي في الأذن الداخلية الماوس بما في ذلك الحسية، وnonsensory وتستمد جميع الخلايا العصبية دوامة من العقدة المستمدة placodally-الكيسة السمعية يقع بالقرب من الدماغ المؤخر من الأديم الظاهر، حول E8 10-14. في E11، المنطقة البطنية من الكيسة السمعية تمتد لت?...

The mammalian organ of Corti is comprised of a mosaic of specialized cell types, including two types of mechanosensory hair cells as well as at least four types of nonsensory supporting cells making it an ideal model system to study normal cellular processes like proliferation, fate specification, differentiation and patterning. In addition, the normal development of these different cell types is essential for normal hearing function. Hence, it is crucial to understand the factors, both molecular and cellular, that regulate their development. However, the small size of the mouse cochlea as well as its inaccessibility poses a particular challenge for gene expression studies. Moreover, most of the cell fate specification and patterning events occur during embryonic time periods and are mostly completed before birth. Therefore, identification and characterization of signaling events during embryonic time periods is essential to gain insight into the molecular basis of cochlear morphogenesis. 
Here, we demonstrate a method to culture intact cochlea in vitro from embryonic mouse inner ears. The rationale behind the use of this technique is that cultured cochleae maintain their molecular and morphological characteristics thereby providing a valuable model for investigating potential candidate genes and exploring the mechanisms involved in cochlear morphogenesis. Although transgenic mice can be used for gene expression studies, an in vitro system is often needed for monitoring specific gene functions. Moreover, cochlear cultures can be established from transgenic mouse embryos so that their in vitro development and response to various soluble factors and antagonists can be studied. Although embryos at day 13 (E13) are used in this protocol, cultures from E12 or E14 to early postnatal inner ears can give similar results. 
We also present a gene transfer technique in cultured embryonic cochlear explants using square wave electroporation. Following the isolation of the cochlear explants, electroporation can be used to express DNA plasmids of gene(s) of interest in individual cells within the cochlear duct. This technique serves as a complementary approach to studies utilizing transgenic mice to gain insight into the molecular pathways underlying cellular phenotype. Using this method of gene transfer, a variety of epithelial cell types within the embryonic cochlea are transfected, thereby enabling loss-and gain-of-function analyses at the single-cell level. In addition, electrophysiological studies can also be performed in cochlear explant cultures8. This method of in vitro electroporation is relatively simple and straightforward, combined with minimal damage to the tissue, has resulted in a rapid expansion of this technique.

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culture of embryonic mouse cochlear explants and gene transfer by electroporation

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair cells are aligned in one row of inner hair cells and three rows of outer hair cells that extend along the basal to apical axis of the cochlea. The explant culture technique described here provides an efficient method to isolate and maintain cochlear explants from the embryonic mouse inner ear. Also, the morphology and molecular characteristics of sensory hair cells and nonsensory supporting cells within the cochlear explant cultures resemble those observed in vivo and can be studied within its intrinsic cellular environment. The cochlear explants can serve as important experimental tools for the identification and characterization of molecular and genetic pathways that are involved in cellular specification and patterning. Although transgenic mouse models provide an effective approach for gene expression studies, a considerable number of mouse mutants die during embryonic development thereby hindering the analysis and interpretation of developmental phenotypes. The organ of Corti from mutant mice that die before birth can be cultured so that their in vitro development and responses to different factors can be analyzed. Additionally, we describe a technique for electroporating embryonic cochlear explants ex vivo which can be used to downregulate or overexpress specific gene(s) and analyze their potential endogenous function and test whether specific gene product is necessary or sufficient in a given context to influence mammalian cochlear development1-8.

We describe a method to isolate cochlea from embryonic inner ears and micro-dissect to expose the sensory epithelium. Once dissected, it may be plated and cultured as an intact cochlear duct (Figure 3) and analyzed by immunohistochemistry. The cultured cochlear explants provide a useful assay to examine the effect of variety of soluble factors and pharmacological drugs on cochlear development. Following dissection of cochlear explants, electroporation technique can be used to misexpress genes of interest...

Watch this Scientific Journal Video about Culture of Embryonic Mouse Cochlear Explants and Gene Transfer by Electroporation at JoVE.com

Culture of Embryonic Mouse Cochlear Explants and Gene Transfer by Electroporation

We present a method that describes isolation and culture of cochlear explants from embryonic mouse inner ear. We also demonstrate a method for gene transfer into cochlear explants via square-wave electroporation. The in vitro explant culture coupled with gene transfer technique enables researchers to study the effects of altering gene expression during development.

ثقافة الجنينية الماوس القوقعة إإكسبلنتس وجين نقل من قبل Electroporation لل

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair ...

culture-embryonic-mouse-cochlear-explants-gene-transfer

Research

JoVE Journal

Developmental Biology

12.8K Views.  Medical University of South Carolina, College of Medicine. We present a method that describes isolation and culture of cochlear explants from embryonic mouse inner ear. We also demonstrate a method for gene transfer into cochlear explants via square-wave electroporation. The in vitro explant culture coupled with gene transfer technique enables researchers to study the effects of altering gene expression during development. 

Video: Culture of Embryonic Mouse Cochlear Explants and Gene Transfer by Electroporation

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

The cone photoreceptor-enriched cultures derived from embryonic chick retinas have become an indispensable tool for researchers around the world studying the biology of retinal neurons, particularly photoreceptors. The applications of this system go beyond basic research, as they can easily be adapted to high throughput technologies for drug development. However, genetic manipulation of retinal photoreceptors in these cultures has proven to be very challenging, posing an important limitation to the usefulness of the system. We have recently developed and validated an ex&#160;ovo plasmid electroporation technique that increases the rate of transfection of retinal cells in these cultures by five-fold compared to other currently available protocols1. In this method embryonic chick eyes are enucleated at stage 27, the RPE is removed, and the retinal cup is placed in a plasmid-containing solution and electroporated using easily constructed custom-made electrodes. The retinas are then dissociated and cultured using standard procedures. This technique can be applied to overexpression studies as well as to the downregulation of gene expression, for example via the use of plasmid-driven RNAi technology, commonly achieving transgene expression in 25% of the photoreceptor population. The video format of the present publication will make this technology easily accessible to researchers in the field, enabling the study of gene function in primary retinal cultures. We have also included detailed explanations of the critical steps of this procedure for a successful outcome and reproducibility.

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

Embryonic chick retinal cell cultures constitute valuable tools for the study of photoreceptor biology. We have developed an efficient gene transfer technique based on ex&#160;ovo plasmid electroporation of the retinas prior to culture. This technique considerably increases transfection efficiencies over currently available protocols, making genetic manipulation feasible for this system.

كفاءة نقل الجينات في شبكية العين الفرخ عن الخلية الأولية دراسات الثقافة: ل<em&gt; السابقين البيضة</em&gt; Electroporation للمنهج

The cone photoreceptor-enriched cultures derived from embryonic chick retinas have become an indispensable tool for researchers around the world studying ...

Culture and Co-Culture of Mouse Ovaries and Ovarian Follicles

The mammalian ovary is composed of ovarian follicles, each follicle consisting of a single oocyte surrounded by somatic granulosa cells, enclosed together within a basement membrane. A finite pool of follicles is laid down during embryonic development, when oocytes in meiotic arrest form a close association with flattened granulosa cells, forming primordial follicles. By or shortly after birth, mammalian ovaries contain their lifetime&#8217;s supply of primordial follicles, from which point onwards there is a steady release of follicles into the growing follicular pool.
The ovary is particularly amenable to development in vitro, with follicles growing in a highly physiological manner in culture. This work describes the culture of whole neonatal ovaries containing primordial follicles, and the culture of individual ovarian follicles, a method which can support the development of follicles from an immature through to the preovulatory stage, after which their oocytes are able to undergo fertilization in vitro. The work outlined here uses culture systems to determine how the ovary is affected by exposure to external compounds. We also describe a co-culture system, which allows investigation of the interactions that occur between growing follicles and the non-growing pool of primordial follicles.

This protocol describes the primary culture/co-culture of mouse ovarian tissue, using ovaries from neonatal mice and individual ovarian follicles from prepubertal mice. The culture techniques support development in a highly physiological manner, allowing investigation of the effect of extrinsic agents on the ovary, and of interactions between ovarian follicles.

الثقافة والمشارك ثقافة ماوس المبايض وحويصلات المبيض

The mammalian ovary is composed of ovarian follicles, each follicle consisting of a single oocyte surrounded by somatic granulosa cells, enclosed together ...

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

Mouse genetics offers a powerful tool determining the role of specific genes during development. Analyzing the resulting phenotypes by immunohistochemical and molecular methods provides information of potential target genes and signaling pathways. To further elucidate specific regulatory mechanisms requires a system allowing the manipulation of only a small number of cells of a specific tissue by either overexpression, ablation or re-introduction of specific genes and follow their fate during development. To achieve this ex utero electroporation of hippocampal structures, especially the dentate gyrus, followed by organotypic slice culture provides such a tool. Using this system to generate mosaic deletions allows determining whether the gene of interest regulates cell-autonomously developmental processes like progenitor cell proliferation or neuronal differentiation. Furthermore it facilitates the rescue of phenotypes by re-introducing the deleted gene or its target genes. In contrast to in utero electroporation the ex utero approach improves the rate of successfully targeting deeper layers of the brain like the dentate gyrus. Overall ex utero electroporation and organotypic slice culture provide a potent tool to study regulatory mechanisms in a semi-native environment mirroring endogenous conditions.

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

Here we present a protocol providing a tool to examine regulatory mechanisms of specific genes during hippocampal development. Employing ex utero electroporation and organotypic slice culture allows the up- and down-regulation of the expression of genes of interest in single cells and follow their fate during development.

السابق الرحم Electroporation للوعضوي النمط من الثقافة شريحة ماوس الحصين الأنسجة

Mouse genetics offers a powerful tool determining the role of specific genes during development. Analyzing the resulting phenotypes by immunohistochemical ...

Neural Differentiation of Mouse Embryonic Stem Cells in Serum-free Monolayer Culture

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as well as the generation of mature neural cell types for further study. In this protocol we describe a method for the differentiation of ESC to neural progenitors using serum-free, monolayer culture. The method is scalable, efficient and results in production of ~70% neural progenitor cells within 4 - 6 days. It can be applied to ESC from various strains grown under a variety of conditions. Neural progenitors can be allowed to differentiate further into functional neurons and glia or analyzed by microscopy, flow cytometry or molecular techniques. The differentiation process is amenable to time-lapse microscopy and can be combined with the use of reporter lines to monitor the neural specification process. We provide detailed instructions on media preparation and cell density optimization to allow the process to be applied to most ESC lines and a variety of cell culture vessels.

This protocol describes in detail the method for generating neural progenitors from embryonic stem cells using a serum-free monolayer method. These progenitors can be used to derive mature neural cell types or to study the process of neural specification and is amenable to multiwell format scaling for compound screening.

التمايز العصبي للماوس الخلايا الجذعية الجنينية في المصل خالية أحادي الطبقة الثقافة

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as ...

Whole Retinal Explants from Chicken Embryos for Electroporation and Chemical Reagent Treatments

The retina is a good model for the developing central nervous system. The large size of the eye and most importantly the accessibility for experimental manipulations in ovo/in vivo makes the chicken embryonic retina a versatile and very efficient experimental model. Although the chicken retina is easy to target in ovo by intraocular injections or electroporation, the effective and exact concentration of the reagents within the retina may be difficult to fully control. This may be due to variations of the exact injection site, leakage from the eye or uneven diffusion of the substances. Furthermore, the frequency of malformations and mortality after invasive manipulations such as electroporation is rather high.
This protocol describes an ex ovo technique for culturing whole retinal explants from chicken embryos and provides a method for controlled exposure of the retina to reagents. The protocol describes how to dissect, experimentally manipulate, and culture whole retinal explants from chicken embryos. The explants can be cultured for approximately 24 hr and be subjected to different manipulations such as electroporation. The major advantages are that the experiment is not dependent on the survival of the embryo and that the concentration of the introduced reagent can be varied and controlled in order to determine and optimize the effective concentration. Furthermore, the technique is rapid, cheap and together with its high experimental success rate, it ensures reproducible results. It should be emphasized that it serves as an excellent complement to experiments performed in ovo.

This protocol describes a method to dissect, experimentally manipulate and culture whole retinal explants from chicken embryos. The explant cultures are useful when high success rate, efficacy and reproducibility are needed to test the effects of plasmids for electroporation and/or reagent substances, i.e., enzymatic inhibitors.

إإكسبلنتس الشبكية كاملة من الدجاج الأجنة لعلاج Electroporation للوالكيميائية الكاشف

The retina is a good model for the developing central nervous system. The large size of the eye and most importantly the accessibility for experimental ...

Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation

Chicken embryos are ideal model systems for studying embryonic development as manipulations of gene function can be conducted with relative ease in ovo. The inner ear auditory sensory organ is critical for our ability to hear. It houses a highly specialized sensory epithelium that consists of mechano-transducing hair cells (HCs) and surrounding glial-like supporting cells (SCs). Despite structural differences in the auditory organs, molecular mechanisms regulating the development of the auditory organ are evolutionarily conserved between mammals and aves. In ovo electroporation is largely limited to early stages at E1 - E3. Due to the relative late development of the auditory organ at E5, manipulations of the auditory organ by in ovo electroporation past E3 are difficult due to the advanced development of the chicken embryo at later stages. The method presented here is a transient gene transfer method for targeting genes of interest at stage E4 - E4.5 in the developing chicken auditory sensory organ via in ovo micro-electroporation. This method is applicable for gain- and loss-of-functions with conventional plasmid DNA-based expression vectors and can be combined with in ovo cell proliferation assay by adding EdU (5-ethynyl-2&#180;-deoxyuridine) to the whole embryo at the time of electroporation. The use of green or red fluorescent protein (GFP or RFP) expression plasmids allows the experimenter to quickly determine whether the electroporation successfully targeted the auditory portion of the developing inner ear. In this method paper, representative examples of GFP electroporated specimens are illustrated; embryos were harvested 18 - 96 hr&#160;after electroporation and targeting of GFP to the pro-sensory area of the auditory organ was confirmed by RNA in situ hybridization. The method paper also provides an optimized protocol for the use of the thymidine analog EdU to analyze cell proliferation; an example of an EdU based cell proliferation assay that combines immuno-labeling and click EdU chemistry is provided.

Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation

The auditory organ mediates hearing. Here we present a modified in ovo micro-electroporation method optimized for studying auditory progenitor cell proliferation and differentiation in the developing chicken auditory organ.

نقل الجينات إلى داخل الجهاز الدجاج السمعية التي كتبها<em&gt; في البيضة</em&gt; الصغرى Electroporation لل

Chicken embryos are ideal model systems for studying embryonic development as manipulations of gene function can be conducted with relative ease in ovo. ...

Culture of Adult Transgenic Zebrafish Retinal Explants for Live-cell Imaging by Multiphoton Microscopy

An endogenous regeneration program is initiated by M&#252;ller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The M&#252;ller glia re-enter the cell cycle and produce neuronal progenitor cells that undergo subsequent rounds of cell divisions and differentiate into the lost neuronal cell types. Both M&#252;ller glia and neuronal progenitor cell nuclei replicate their DNA and undergo mitosis in distinct locations of the retina, i.e. they migrate between the basal Inner Nuclear Layer (INL) and the Outer Nuclear Layer (ONL), respectively, in a process described as Interkinetic Nuclear Migration (INM). INM has predominantly been studied in the developing retina. To examine the dynamics of INM in the adult regenerating zebrafish retina in detail, live-cell imaging of fluorescently-labeled M&#252;ller glia/neuronal progenitor cells is required. Here, we provide the conditions to isolate and culture dorsal retinas from Tg[gfap:nGFP]mi2004 zebrafish that were exposed to constant intense light for 35 h. We also show that these retinal cultures are viable to perform live-cell imaging experiments, continuously acquiring z-stack images throughout the thickness of the retinal explant for up to 8 h using multiphoton microscopy to monitor the migratory behavior of gfap:nGFP-positive cells. In addition, we describe the details to perform post-imaging analysis to determine the velocity of apical and basal INM. To summarize, we established conditions to study the dynamics of INM in an adult model of neuronal regeneration. This will advance our understanding of this crucial cellular process and allow us to determine the mechanisms that control INM.

Zebrafish retinal regeneration has mostly been studied using fixed retinas. However, dynamic processes such as interkinetic nuclear migration occur during the regenerative response and require live-cell imaging to investigate the underlying mechanisms. Here, we describe culture and imaging conditions to monitor Interkinetic Nuclear Migration (INM) in real-time using multiphoton microscopy.

ثقافة الكبار المعدلة وراثيا الزرد الشبكية إإكسبلنتس التصوير خلية حية من قبل Multiphoton المجهر

An endogenous regeneration program is initiated by M&#252;ller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The ...

Dissection and Culture of Mouse Embryonic Kidney

The goal of this protocol is to describe a method for the dissection, isolation, and culture of mouse metanephric rudiments. 
During mammalian kidney development, the two progenitor tissues, the ureteric bud and the metanephric mesenchyme, communicate and reciprocally induce cellular mechanisms to eventually form the collecting system and the nephrons of the kidney. As mammalian embryos grow intrauterine and therefore are inaccessible to the observer, an organ culture has been developed. With this method, it is possible to study epithelial-mesenchymal interactions and cellular behavior during kidney organogenesis. Furthermore, the origin of congenital kidney and urogenital tract malformations can be investigated. After careful dissection, the metanephric rudiments are transferred onto a filter that floats on culture medium and can be kept in a cell culture incubator for several days. However, one must be aware that the conditions are artificial and could influence the metabolism in the tissue. Also, the penetration of test substances could be limited due to the extracellular matrix and basal membrane present in the explant.
One main advantage of organ culture is that the experimenter can gain direct access to the organ. This technology is cheap, simple, and allows a large number of modifications, such as the addition of biologically active substances, the study of genetic variants, and the application of advanced imaging techniques.

This protocol describes a method for isolating and culturing metanephric rudiments from mouse embryos.

تشريح وثقافة ماوس الجنينية الكلى

The goal of this protocol is to describe a method for the dissection, isolation, and culture of mouse metanephric rudiments. 
During mammalian kidney ...

CRISPR-mediated Loss of Function Analysis in Cerebellar Granule Cells Using In Utero Electroporation-based Gene Transfer

Brain malformation is often caused by genetic mutations. Deciphering the mutations in patient-derived tissues has identified potential causative factors of the diseases. To validate the contribution of a dysfunction of the mutated genes to disease development, the generation of animal models carrying the mutations is one obvious approach. While germline genetically engineered mouse models (GEMMs) are popular biological tools and exhibit reproducible results, it is restricted by time and costs. Meanwhile, non-germline GEMMs often enable exploring gene function in a more feasible manner. Since some brain diseases (e.g., brain tumors) appear to result from somatic but not germline mutations, non-germline chimeric mouse models, in which normal and abnormal cells coexist, could be helpful for disease-relevant analysis. In this study, we report a method for the induction of CRISPR-mediated somatic mutations in the cerebellum. Specifically, we utilized conditional knock-in mice, in which Cas9 and GFP are chronically activated by the CAG (CMV enhancer/chicken &#223;-actin) promoter after Cre-mediated recombination of the genome. The self-designed single-guide RNAs (sgRNAs) and the Cre recombinase sequence, both encoded in a single plasmid construct, were delivered into cerebellar stem/progenitor cells at an embryonic stage using in utero electroporation. Consequently, transfected cells and their daughter cells were labeled with green fluorescent protein (GFP), thus facilitating further phenotypic analyses. Hence, this method is not only showing electroporation-based gene delivery into embryonic cerebellar cells but also proposing a novel quantitative approach to assess CRISPR-mediated loss-of-function phenotypes.

CRISPR-mediated Loss of Function Analysis in Cerebellar Granule Cells Using In Utero Electroporation-based Gene Transfer

Conventional loss-of-function studies of genes using knockout animals have often been costly and time-consuming. Electroporation-based CRISPR-mediated somatic mutagenesis is a powerful tool to understand gene functions in vivo. Here, we report a method to analyze knockout phenotypes in proliferating cells of the cerebellum.

فقدان وظيفة تحليل في الحبيبية للدماغ كريسبر بوساطة خلايا تستخدم في الرحم على أساس انهانسر نقل الجينات

Brain malformation is often caused by genetic mutations. Deciphering the mutations in patient-derived tissues has identified potential causative factors ...

Isolation and Differentiation of Primary Myoblasts from Mouse Skeletal Muscle Explants

Primary myoblasts are undifferentiated proliferating precursors of skeletal muscle. They can be cultured and studied as muscle precursors or induced to differentiate into later stages of muscle development. The protocol provided here describes a robust method for the isolation and culture of a highly proliferative population of myoblast cells from young adult mouse skeletal muscle explants. These cells are useful for the study of the metabolic properties of skeletal muscle of different mouse models, as well as in other downstream applications such as transfection with exogenous DNA or transduction with viral expression vectors. The level of differentiation and metabolic profile of these cells depends on the length of exposure, and composition of the media used to induce myoblast differentiation. These methods provide a robust system for the study of mouse muscle cell metabolism ex vivo. Importantly, unlike in vivo models, the methods described here provide a cell population that can be expanded and studied with high levels of reproducibility.

Myoblasts are proliferating precursor cells that differentiate to form polynucleated myotubes and eventually skeletal muscle myofibers. Here, we present a protocol for efficient isolation and culture of primary myoblasts from young adult mouse skeletal muscles. The method enables molecular, genetic, and metabolic studies of muscle cells in culture.

العزلة والتمايز من المقاطع الاساسيه من الماوس الهيكل العظمي العضلات الشرح

Primary myoblasts are undifferentiated proliferating precursors of skeletal muscle. They can be cultured and studied as muscle precursors or induced to ...