Research was partly supported by NIH research grants R01 AR062173 and SHC 250862 to IEA. ES is the recipient of NIH T32 CTSC predoctoral fellowship.

1. التسليم هيدرودينامي من sRANKL MC الحمض النووي <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> توصيل ماوس الهيدروديناميكية عبر الوريد الذيل <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> وزن الماوس قبل حقن الوريد الذيل. تمييع sRANKL أو بروتين الفلورية الخضراء (GFP) MC في حل رينغر (قبل دافئ عند 37 درجة مئوية) في إجمالي حجم ~ 10٪ من وزن جسم الفأر. </li><li style=";text-align:right;direction:rtl"> الاحماء الماوس في قفص لمدة 10 دقيقة قبل الحقن وذلك لتمدد الأوعية الدموية والأوردة جعل الجانبي (لفس) مرئية. رصد الماوس بعناية لتجنب الجفاف وارتفاع الحرارة. </li><li style=";text-align:right;direction:rtl"> حالما يتم المتوسعة لفس ومرئية، ونقل الماوس إلى كابح وتضاف المكونات بعيدا بما فيه الكفاية في برميل لكبح جماح الحركة. رصد الماوس للنشاط العادي مثل التنفس. ضبط المكونات من كابح إذا لزم الأمر. </li><li style=";text-align:right;direction:rtl"> تطهير منطقة الحقن 3/4 من غيض من الذيل مع الكحول مسح. استخدام إبرة G 27-30 للماوس حقن الوريد الذيل. عقد ذيل بحزم مع يد واحدة وادخال الإبرة فيإلى الوريد الذيل. الضغط على حقنة واستكمال الحقن داخل 5-7 ثانية. </li><li style=";text-align:right;direction:rtl"> إزالة الإبرة من الوريد الذيل والضغط مع كرة القطن على موقع الحقن لوقف النزيف. رصد الماوس ل15-30 دقيقة، ثم نقل الماوس مرة أخرى إلى الحظيرة مرة واحدة يتم خفض معدل التنفس إلى المعدل العادي. </li></ol></li><li style=";text-align:right;direction:rtl"> تأكيد الجهازية ماوس sRANKL التعبير والكمي لنقل ماوس سيروم TRACP5b آخر الجينات <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> الاحماء الماوس في قفص لمدة 5-10 دقيقة من أجل تمدد الأوعية الدموية وجعل لفس مرئية. رصد الماوس بعناية لتجنب الجفاف وارتفاع الحرارة. </li><li style=";text-align:right;direction:rtl"> إجراء شق صغير بشفرة إلى ذيل الماوس، في موقع واحد عليها من طرف الذيل. </li><li style=";text-align:right;direction:rtl"> جمع ما يقرب من 100 ميكرولتر من الدم في أنابيب فصل المصل. </li><li style=";text-align:right;direction:rtl"> احتضان أنابيب فصل المصل في درجة حرارة الغرفة لمدة 30 دقيقة. </li><li style=";text-align:right;direction:rtl"> عينات الطرد المركزي في 10،000 x ج لمدة 5 دقائق وجمع المصل. ستورمصل الإلكترونية في -80 درجة مئوية لمزيد من التحليل. </li><li style=";text-align:right;direction:rtl"> أداء sRANKL ELISA والماوس TRACP5b المصل فحص على عينات المصل باستخدام طقم التجارية المتاحة. </li></ol></li></ol> 2. في المختبر الجيل ناقضة العظم من ماوس BMMs <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> العزلة والتثقيف من BMMs <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> العزلة من الساق وعظم الفخذ العظام: الموت ببطء الماوس المانحة مع CO 2، وتطهير الساقين مع الايثانول 70٪. تشريح من عظم العانة حتى العظم العقبي، لإزالة عظم الفخذ والظنبوب عظام سليمة. </li><li style=";text-align:right;direction:rtl"> إزالة الجلد والعضلات بعناية دون الإضرار العظام. وضع عظم الفخذ والساق معالجة العظام في طبق بتري تحتوي على الفوسفات مخزنة المالحة (PBS). </li><li style=";text-align:right;direction:rtl"> إزالة نخاع العظم: قطع قبالة الطرف على جانب واحد من عظم الفخذ والساق وعظام طرد نخاع العظام في أنبوب 50 مل باستخدام حقنة 1ML مع 25 G إبرة، محملة α-MEM تحتوي على 1٪ البنسلين / الستربتوميسين (ناقضة العظم الثقافة متوسطة / OCM). </li><lط&gt; عملية نخاع العظم: مزيج ونقل نخاع العظام التعليق على الجديد 50 مل أنبوب بتمريرها من خلال 70 ميكرومتر الخلية النايلون مصفاة. الطرد المركزي الخلايا في 300 x ج وإعادة تعليق في α-MEM تحتوي على 1٪ البنسلين / الستربتوميسين و 10٪ FBS (ناقضة العظم الثقافة المتوسطة / OCM (+)). </li></ol></li><li style=";text-align:right;direction:rtl"> ثقافة الضامة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> عد الخلايا باستخدام عدادة الكريات أو خلية مكافحة التلقائي. </li><li style=";text-align:right;direction:rtl"> لوحة 1 × 10 6 خلايا لكل بئر لوحة 6 جيدا أو 3 × 10 5 خلايا إلى coverslips الزجاج (5 ملم) أو شرائح عاج وضعها في لوحة 96 جيدا واحتضان عند 37 درجة مئوية. </li><li style=";text-align:right;direction:rtl"> نضح كل وسائل الإعلام التي تحتوي على الخلايا غير ملتصقة واحتضان الخلايا عند 37 درجة مئوية مع OCM الطازجة (+) التي تحتوي على 25 نانوغرام / مل الماوس M-CSF لمدة 48 ساعة. </li><li style=";text-align:right;direction:rtl"> نضح جميع وسائل الإعلام واحتضان الخلايا عند 37 درجة مئوية مع OCM الطازجة (+) وسائل الإعلام التي تحتوي على 25 نانوغرام / مل الماوس M-CSF، و 30 الماوس نانوغرام / مل sRANKL لبدء osteoclastogenesis. تجديد وسائل الاعلام كل 3 أيام كما هو مطلوب. </li><li style=";text-align:right;direction:rtl"> تنمو الخلايالحوالي 6-8 أيام لتشكيل خلايا متعددة النووية العملاقة قادرة على resorbing العظام. حالما تظهر خلايا متعددة النوى، نفذ فخ (طرطرات الفوسفاتيز الحمضي مقاومة) تلطيخ استخدام عدة التجارية المتاحة، وعندما نضجت تماما، نفذ المقايسات الفنية. تصور حلقات F-الأكتين coverslips على استخدام وصمة عار phalloidin وصورة تعلق على الخلايا عاج شرائح بواسطة المجهر الإلكتروني (SEM) كما هو موضح سابقا 19. </li></ol></li></ol> 3. في المختبر الجيل ناقضة العظم من PBMCs الإنسان <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> عزل PBMCs الإنسان <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إضافة 10 مل من قبل تحسنت Histopaque-1077 في أنبوب 50 مل. </li><li style=";text-align:right;direction:rtl"> فارغة تصفية الكريات البيض التي تم الحصول عليها من بنك الدم مع برنامج تلفزيوني العقيمة في أنبوب 50 مل (نسبة 1:1 من الدم مع PBS). طبقة الدم المخفف على حل histopaque ببطء شديد، والتأكد من أن الدم لا يمتزج مع histopaque. </li><li style=";text-align:right;direction:rtl"> عينات الطرد المركزي في 1000 x ج لمدة 20 دقيقة في 18 درجة مئوية. تابعالطرد المركزي جي، عزل بعناية طبقة معطف الشهباء البيضاء التي تحتوي على خلايا الدم البيضاء ونقل إلى أنبوب جديد 50 مل. </li><li style=";text-align:right;direction:rtl"> تمييع الخلايا مع برنامج تلفزيوني وأجهزة الطرد المركزي مرة أخرى في 650 x ج لمدة 5 دقائق لجمع الخلايا. </li><li style=";text-align:right;direction:rtl"> إزالة طاف والخلايا resuspend في OCM (-) وسائل الإعلام وعدد الخلايا باستخدام عداد الخلية. </li></ol></li><li style=";text-align:right;direction:rtl"> الطلاء وزراعة PBMCs <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> resuspend الخلايا في OCM (+) وسائل الإعلام وصفيحة 8 × 10 6 خلية / مل لكل بئر في لوحة 6 جيدا أو 1 × 10 6 خلية / مل لكل بئر في لوحة 96 جيدا. خلايا لوحة على coverslips الزجاج (5 ملم) كما هو مطلوب للتصوير المناعي فضلا عن شرائح العظام مناسبة لفحوصات ارتشاف العظم كما هو موضح سابقا 20. </li><li style=";text-align:right;direction:rtl"> التغيير سائل الإعلام والثقافة، وتجديد الخلايا مع الإنسان M-CSF كل 2-3 أيام أو حسب الحاجة. ثم انتقل إلى الخطوة التمايز في يوم 5 عن طريق إضافة 30 نانوغرام / مل من sRANKL الإنسان جنبا إلى جنب مع 25 نانوغرام / مل الإنسان M-CSF في OCM (+) وسائل الإعلام لبدء osteoclastogenesهو. </li><li style=";text-align:right;direction:rtl"> تنمو الخلايا لحوالي 14 إلى 21 يوما لتشكيل خلايا متعددة النووية العملاقة. حالما تظهر خلايا متعددة النوى، ويمكن بعد ذلك أن هذه الخلايا مرقوم للشرك، وعندما نضجت تماما، المقايسات الفنية لا يمكن أن يؤديها. يمكن تصور حلقات F-الأكتين coverslips على استخدام phalloidin صمة عار. يمكن فحص مورفولوجيا الخلايا في الخلايا تعلق على عاج شرائح بواسطة SEM. </li></ol></li></ol>

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+osteoclast+differentiation.">Modulation of osteoclast differentiation.</a> Endocrine reviews. 13, 66-80 (1992).</li><li>Takahashi, N., 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=Osteoblastic+cells+are+involved+in+osteoclast+formation.">Osteoblastic cells are involved in osteoclast formation.</a> Endocrinology. 123, 2600-2602 (1988).</li><li>Bradley, E. W., Oursler, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteoclast+culture+and+resorption+assays.">Osteoclast culture and resorption assays.</a> Methods Mol Biol. 455, 19-35 (2008).</li><li>Arai, F., 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=Commitment+and+differentiation+of+osteoclast+precursor+cells+by+the+sequential+expression+of+c-Fms+and+receptor+activator+of+nuclear+factor+kappaB+(RANK)+receptors.">Commitment and differentiation of osteoclast precursor cells by the sequential expression of c-Fms and receptor activator of nuclear factor kappaB (RANK) receptors.</a> The Journal of experimental medicine. 190, 1741-1754 (1999).</li><li>Fuller, K., 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=Macrophage+colony-stimulating+factor+stimulates+survival+and+chemotactic+behavior+in+isolated+osteoclasts.">Macrophage colony-stimulating factor stimulates survival and chemotactic behavior in isolated osteoclasts.</a> The Journal of experimental medicin. 178, 1733-1744 (1993).</li><li>Edwards, J. R., Mundy, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+osteoclast+biology:+old+findings+and+new+insights+from+mouse+models.">Advances in osteoclast biology: old findings and new insights from mouse models.</a> Nature reviews. Rheumatology. 7, 235-243 (2011).</li><li>Weinstein, R. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Promotion+of+osteoclast+survival+and+antagonism+of+bisphosphonate-induced+osteoclast+apoptosis+by+glucocorticoids.">Promotion of osteoclast survival and antagonism of bisphosphonate-induced osteoclast apoptosis by glucocorticoids.</a> The Journal of clinical investigation. 109, 1041-1048 (2002).</li></ol>

The authors have nothing to disclose.

ظروف العضلات والعظام تقود أسباب الاعتلال والعجز وتتألف من أكثر من 150 من الأمراض والمتلازمات؛ التي تؤثر على ما يقرب من 90 مليون أميركي اليوم. التهاب المفاصل وتدمير العظام هي السمات الغالبة من الحالات العضلية الهيكلية، بما في ذلك التهاب المفاصل وهشاشة العظام. هشاشة الع...

أمراض العضلات والعظام تؤثر على الملايين من الناس في الولايات المتحدة وعواقب وخيمة الحاضر على النظم الصحية الوطنية والمحلية 1. وتتميز هذه الاضطرابات بسبب فقدان العظام وظيفة مشتركة التي تتطلب معالجة مكثفة وفترات طويلة من الانتعاش. عادة، زيادة نسبية في عدد و / أو نشاط الخلايا الآكلة، وخلايا متخصصة ليرتشف العظام، في ترقق العظام والتهاب المفاصل لوحظ 2. في ظل الظروف الفسيولوجية وينظم عدد ونشاط الخلايا الآكلة بواسطة مستقبلات عامل منشط النووية κ-B يجند (RANKL)، الذي تنتجه الخلايا بانية العظم. Osteoprotegerin (OPG)، وينتج مستقبلات شرك لRANKL أيضا بانيات 3 في الجسم الحي النماذج الحيوانية التي تنطوي من overexpression النظامية من sRANKL، أو حذف OPG هي قيمة للغاية في مجال البحوث هشاشة العظام.؛ ومع ذلك، وهذه الأساليب تتطلب جيل من الفئران المعدلة وراثيا 4،5. هنا، بديل الروايةيوصف أسلوب overexpressing sRANKL لدراسة الاضطرابات المرتبطة العضلات والعظام. على وجه التحديد، واستخدمت minicircle (MC) تكنولوجيا الحمض النووي وطرق التسليم الهيدروديناميكية لتحقيق نقل الجينات من sRANKL في الجسم الحي وoverexpress الماوس sRANKL جهازيا 6. هذا الأسلوب هو أيضا مكملة للأخرى في الجسم الحي نماذج من ترقق العظام، مثل التشكيل الهرمونية من الآكلة التالية استئصال المبيض 7 والتدخل الغذائي من خلال اتباع نظام غذائي منخفض الكالسيوم 8. هذه النماذج هي مفيدة جدا لدراسة جوانب مختلفة من الاضطرابات العضلية الهيكلية ذات الصلة إلا أنها تتطلب عمليات جراحية ويمكن أن يستغرق فترة تصل الى عدة أشهر، بتكلفة كبيرة 9. مستأصلة المبيض (وفك) نموذج القوارض هو نموذج على حيوانات التجارب حيث إزالة المبايض يؤدي إلى نقص هرمون الاستروجين بعد سن اليأس وهشاشة العظام وبالتالي محاكاة الإنسان 10. الإنسان بهشاشة العظام بعد انقطاع الطمث، شرط فيها هرمون الاستروجين deficiency يؤدي إلى زيادة خطر كسور العظام وهشاشة العظام يؤثر على ما يقرب من ثمانية ملايين امرأة في الولايات المتحدة وحدها. على الرغم من أن نموذج وفك مفيد لهشاشة العظام بعد انقطاع الطمث أنه يوفر مزايا محدودة في دراسة هشاشة العظام بشكل عام. الاستروجين يمنع فقدان العظام، عن طريق حفز ناقضة العظم وتثبيط بناء العظم موت الخلايا المبرمج، وبالتالي في غيابها لوحظ زيادة النشاط ناقضة العظم 10-12. ويلاحظ وجود RANKL-OPG نسبة الخلل الذي يفضل ارتشاف العظام أيضا 13. ومع ذلك، ويرافق نقص هرمون الاستروجين في الجسم الحي أيضا من انخفاض مستويات عامل النمو تحويل β (TGF β)، وزيادة انترلوكين 7 (IL-7) وTNF، IL-1 و IL-6 14،15. لأن هذه السيتوكينات قد عرفت وظائف تغييري إعادة تشكيل العظام مستقلة عن المسار RANKL، فإنه من المستحيل أن ينسب أي تفعيل ناقضة العظم فقط إلى المحور RANKL رتبة. نموذج صفها في هذه الورقة تمكن الباحثون للدراسة في فيفس محور RANKL رتبة في osteoclastogenesis وفقدان العظام دون السيتوكينات الموالية للالتهابات مقارنة نماذج القوارض وفك. بالإضافة إلى ذلك، في المختبر تقنيات osteoclastogenesis هي أدوات أساسية لدراسة تفعيل ناقضة العظم لعلاج العلاجية المحتملة للأمراض العضلات والعظام. وقد أظهرت الدراسات السابقة أيضا أن زراعة نخاع العظم الضامة الماوس المستمدة (BMMs) مع الماوس بلعم مستعمرة عامل تحفيز (M-CSF) والماوس sRANKL يمكن أن تؤدي إلى التمايز ناقضة العظم 3،16،17. هنا، يتم وصف البروتوكولات لتوليد الخلايا ناقضة العظم مثل الماوس متعددة النوى من نخاع العظم وكذلك من الخلايا المحيطية وحيدات النوى الإنسان الدم (PBMCs) في المختبر 18. كما وصفها بإيجاز المقايسات خلية مقرها المطلوبة لتحديد ناقضة العظم متباينة عضال وتعمل بكامل طاقتها ناضجة. هذه في المختبر تقنيات تكمل الرواية في الجسم الحي والنهج معا بمثابة عأدوات التحقيق owerful لدراسة التمايز ناقضة العظم والتنشيط. باستخدام هذه الأنظمة، والعلماء قادرون على توليد الخلايا الآكلة في الجسم الحي في المختبر وتحديد المحفزات والإشارات اللازمة لانتشارها وتفعيل وكذلك اختبار فعالية مثبطات الدوائية والبيولوجية.

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			<td height="21" style="height:21px;width:320px;">Tailveiner Restrainer for mice</td>
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			<td style="width:119px;">MTR00</td>
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			<td height="21" style="height:21px;">Acid Phosphatase, Leukocyte (TRAP) Kit</td>
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a novel in vivo gene transfer technique and in vitro cell based assays for the study of bone loss in musculoskeletal disorders

Differentiation and activation of osteoclasts play a key role in the development of musculoskeletal diseases as these cells are primarily involved in bone resorption. Osteoclasts can be generated in vitro from monocyte/macrophage precursor cells in the presence of certain cytokines, which promote survival and differentiation. Here, both in vivo and in vitro techniques are demonstrated, which allow scientists to study different cytokine contributions towards osteoclast differentiation, signaling, and activation. The minicircle DNA delivery gene transfer system provides an alternative method to establish an osteoporosis-related model is particularly useful to study the efficacy of various pharmacological inhibitors in vivo. Similarly, in vitro culturing protocols for producing osteoclasts from human precursor cells in the presence of specific cytokines enables scientists to study osteoclastogenesis in human cells for translational applications. Combined, these techniques have the potential to accelerate drug discovery efforts for osteoclast-specific targeted therapeutics, which may benefit millions of osteoporosis and arthritis patients worldwide.

هنا، توصف تقنية نقل الجينات الرواية لتمايز الخلايا الآكلة في الجسم الحي والبروتوكولات الثقافة خلية للتمييز بين الخلايا الآكلة في السلائف في المختبر كوسيلة لدراسة آثار السيتوكينات على osteoclastogenesis. في الشكل 1، وأظهرت نتائج ممثل نقل الجينات الناجح لGF...

Watch this Scientific Journal Video about A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders at JoVE.com

A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

Differentiation of precursor cells into osteoclasts is regulated by cytokines and growth factors. Here, a novel gene transfer technique for differentiation of osteoclasts in vivo and cell culture protocols for differentiating precursor cells into osteoclasts in vitro as a method to study the effects of cytokines on osteoclastogenesis are described.

رواية<em&gt; في الجسم الحي</em&gt; تقنيات نقل الجينات و<em&gt; في المختبر</em&gt; فحوصات وبناء خلية لدراسة فقدان العظام في اضطرابات العضلات والعظام

Differentiation and activation of osteoclasts play a key role in the development of musculoskeletal diseases as these cells are primarily involved in bone ...

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A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

11.6K Views.  University of California, Davis. Differentiation of precursor cells into osteoclasts is regulated by cytokines and growth factors. Here, a novel gene transfer technique for differentiation of osteoclasts in vivo and cell culture protocols for differentiating precursor cells into osteoclasts in vitro as a method to study the effects of cytokines on osteoclastogenesis are described. 

Video: A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

A Strategy to Identify de Novo Mutations in Common Disorders such as Autism and Schizophrenia

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, the de novo mutation rate in humans is relatively high, so new mutations are generated at a high frequency in the population. However, de novo mutations have not been reported in most common diseases. Mutations in genes leading to severe diseases where there is a strong negative selection against the phenotype, such as lethality in embryonic stages or reduced reproductive fitness, will not be transmitted to multiple family members, and therefore will not be detected by linkage gene mapping or association studies. The observation of very high concordance in monozygotic twins and very low concordance in dizygotic twins also strongly supports the hypothesis that a significant fraction of cases may result from new mutations. Such is the case for diseases such as autism and schizophrenia. Second, despite reduced reproductive fitness1 and extremely variable environmental factors, the incidence of some diseases is maintained worldwide at a relatively high and constant rate. This is the case for autism and schizophrenia, with an incidence of approximately 1% worldwide. Mutational load can be thought of as a balance between selection for or against a deleterious mutation and its production by de novo mutation. Lower rates of reproduction constitute a negative selection factor that should reduce the number of mutant alleles in the population, ultimately leading to decreased disease prevalence. These selective pressures tend to be of different intensity in different environments. Nonetheless, these severe mental disorders have been maintained at a constant relatively high prevalence in the worldwide population across a wide range of cultures and countries despite a strong negative selection against them2. This is not what one would predict in diseases with reduced reproductive fitness, unless there was a high new mutation rate. Finally, the effects of paternal age: there is a significantly increased risk of the disease with increasing paternal age, which could result from the age related increase in paternal de novo mutations. This is the case for autism and schizophrenia3. The male-to-female ratio of mutation rate is estimated at about 4&#x2013;6:1, presumably due to a higher number of germ-cell divisions with age in males. Therefore, one would predict that de novo mutations would more frequently come from males, particularly older males4. A high rate of new mutations may in part explain why genetic studies have so far failed to identify many genes predisposing to complexes diseases genes, such as autism and schizophrenia, and why diseases have been identified for a mere 3% of genes in the human genome. Identification for de novo mutations as a cause of a disease requires a targeted molecular approach, which includes studying parents and affected subjects. The process for determining if the genetic basis of a disease may result in part from de novo mutations and the molecular approach to establish this link will be illustrated, using autism and schizophrenia as examples.

Molecular genetic strategy for finding de novo mutations causing common disorders such as autism and schizophrenia.

استراتيجية لتحديد الطفرات دي نوفو في الاضطرابات الشائعة مثل التوحد والفصام

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, ...

Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited time. These cultured tumorspheres may exhibit markedly different cellular phenotypes when compared to the original tumors. Demonstration that cultured cells have the capability of forming new tumors is important to ensure that cultured cells model the biology of the original tumor. 
Here we present a protocol for propagating human retinoblastoma tumors in vivo using Rag2-/- immune deficient mice. Cultured human retinoblastoma tumorspheres of low passage or cells obtained from freshly harvested human retinoblastoma tumors injected directly into the vitreous cavity of murine eyes form tumors within 2-4 weeks. These tumors can be harvested and either further passaged into murine eyes in vivo or grown as tumorspheres in vitro. Propagation has been successfully carried out for at least three passages thus establishing a continuing source of human retinoblastoma tissue for further experimentation. 
Wesley S. Bond and Lalita Wadhwa are co-first authors.

A method is described to propagate human retinoblastoma tumors in mice. Tumor cells are directly injected into the eyes of immune deficient mice. Secondary tumors have been successfully established using both cells directly harvested from human tumors and cultured tumorspheres.

إنشاء والدعوة للأورام الشبكية الإنسان في الفئران أن نقص المناعة

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited ...

Longitudinal Evaluation of Mouse Hind Limb Bone Loss After Spinal Cord Injury using Novel, in vivo, Methodology

Spinal cord injury (SCI) is often accompanied by osteoporosis in the sublesional regions of the pelvis and lower extremities, leading to a higher frequency of fractures 1. As these fractures often occur in regions that have lost normal sensory function, the patient is at a greater risk of fracture-dependent pathologies, including death. SCI-dependent loss in both bone mineral density (BMD, grams/cm2) and bone mineral content (BMC, grams) has been attributed to mechanical disuse 2, aberrant neuronal signaling 3 and hormonal changes 4. The use of rodent models of SCI-induced osteoporosis can provide invaluable information regarding the mechanisms underlying the development of osteoporosis following SCI as well as a test environment for the generation of new therapies 5-7 (and reviewed in 8). Mouse models of SCI are of great interest as they permit a reductionist approach to mechanism-based assessment through the use of null and transgenic mice. While such models have provided important data, there is still a need for minimally-invasive, reliable, reproducible, and quantifiable methods in determining the extent of bone loss following SCI, particularly over time and within the same cohort of experimental animals, to improve diagnosis, treatment methods, and/or prevention of SCI-induced osteoporosis.
An ideal method for measuring bone density in rodents would allow multiple, sequential (over time) exposures to low-levels of X-ray radiation. This study describes the use of a new whole-animal scanner, the IVIS Lumina XR (Caliper Instruments) that can be used to provide low-energy (1-3 milligray (mGy)) high-resolution, high-magnification X-ray images of mouse hind limb bones over time following SCI. Significant bone density loss was seen in the tibiae of mice by 10 days post-spinal transection when compared to uninjured, age-matched control (na&iuml;ve) mice (13% decrease, p&lt;0.0005). Loss of bone density in the distal femur was also detectable by day 10 post-SCI, while a loss of density in the proximal femur was not detectable until 40 days post injury (7% decrease, p&lt;0.05). SCI-dependent loss of mouse femur density was confirmed post-mortem through the use of Dual-energy X-ray Absorptiometry (DXA), the current "gold standard" for bone density measurements. We detect a 12% loss of BMC in the femurs of mice at 40 days post-SCI using the IVIS Lumina XR. This compares favorably with a previously reported BMC loss of 13.5% by Picard and colleagues who used DXA analysis on mouse femurs post-mortem 30 days post-SCI 9. Our results suggest that the IVIS Lumina XR provides a novel, high-resolution/high-magnification method for performing long-term, longitudinal measurements of hind limb bone density in the mouse following SCI.

Longitudinal Evaluation of Mouse Hind Limb Bone Loss After Spinal Cord Injury using Novel, in vivo, Methodology

A longitudinal examination of bone loss in the femurs and tibiae of adult mice was performed following spinal cord injury using sequential low-dose X-ray scans. Tibia bone loss was detected throughout the study, while bone loss in the femur was not detected until 40 days post injury.

التقييم الطولي للهند ماوس الاطراف فقدان العظام بعد اصابة النخاع الشوكي به الرواية ، في الجسم الحي ، منهجية

Spinal cord injury (SCI) is often accompanied by osteoporosis in the sublesional regions of the pelvis and lower extremities, leading to a higher ...

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

المؤشرات الحيوية في نموذج حيواني لكشف الاقتران العصبية، الدموية، والسلوكية للPTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

A Novel Surgical Approach for Intratracheal Administration of Bioactive Agents in a Fetal Mouse Model

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and acquired diseases. Apart from congenital or inherited abnormalities with the requirement for long-term expression of the delivered gene, several non-inherited perinatal conditions, where short-term gene expression or pharmacological intervention is sufficient to achieve therapeutic effects, are considered as potential future indications for this kind of approach. Candidate diseases for the application of short-term prenatal therapy could be the transient neonatal deficiency of surfactant protein B causing neonatal respiratory distress syndrome1,2 or hyperoxic injuries of the neonatal lung3. Candidate diseases for permanent therapeutic correction are Cystic Fibrosis (CF)4, genetic variants of surfactant deficiencies5 and &alpha;1-antitrypsin deficiency6.
Generally, an important advantage of prenatal gene therapy is the ability to start therapeutic intervention early in development, at or even prior to clinical manifestations in the patient, thus preventing irreparable damage to the individual. In addition, fetal organs have an increased cell proliferation rate as compared to adult organs, which could allow a more efficient gene or stem cell transfer into the fetus. Furthermore, in utero gene delivery is performed when the individual's immune system is not completely mature. Therefore, transplantation of heterologous cells or supplementation of a non-functional or absent protein with a correct version should not cause immune sensitization to the cell, vector or transgene product, which has recently been proven to be the case with both cellular and genetic therapies7.
In the present study, we investigated the potential to directly target the fetal trachea in a mouse model. This procedure is in use in larger animal models such as rabbits and sheep8, and even in a clinical setting9, but has to date not been performed before in a mouse model. When studying the potential of fetal gene therapy for genetic diseases such as CF, the mouse model is very useful as a first proof-of-concept because of the wide availability of different transgenic mouse strains, the well documented embryogenesis and fetal development, less stringent ethical regulations, short gestation and the large litter size.
Different access routes have been described to target the fetal rodent lung, including intra-amniotic injection10-12, (ultrasound-guided) intrapulmonary injection13,14 and intravenous administration into the yolk sac vessels15,16 or umbilical vein17. Our novel surgical procedure enables researchers to inject the agent of choice directly into the fetal mouse trachea which allows for a more efficient delivery to the airways than existing techniques18.

We developed a novel surgical approach for intratracheal administration of bioactive agents into the mouse fetus. The delivery route is more efficient in targeting the fetal mouse lungs than the commonly used intra-amniotic injection. This procedure has to date not been described in a mouse model.

وسيلة جديدة لإدارة الجراحية داخل الرغامى وكلاء الحيويه في نموذج الفأر الجنينية

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and ...

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a novel platform that allows for direct long-term visualization of tissue structure changes intracranially. Imaging at a single cell resolution in a real-time fashion provides supplementary dynamic information beyond that provided by standard end-point histological analysis, which looks solely at 'snap-shot' cross sections of tissue.
Establishing this intravital imaging technique in fluorescent chimeric mice, we are able to image four fluorescent channels simultaneously. By incorporating fluorescently labeled cells, such as GFP+ bone marrow, it is possible to track the fate of these cells studying their long-term migration, integration and differentiation within tissue. Further integration of a secondary reporter cell, such as an mCherry glioma tumor line, allows for characterization of cell:cell interactions. Structural changes in the tissue microenvironment can be highlighted through the addition of intra-vital dyes and antibodies, for example CD31 tagged antibodies and Dextran molecules.
Moreover, we describe the combination of our ICW imaging model with a small animal micro-irradiator that provides stereotactic irradiation, creating a platform through which the dynamic tissue changes that occur following the administration of ionizing irradiation can be assessed.
Current limitations of our model include penetrance of the microscope, which is limited to a depth of up to 900 &mu;m from the sub cortical surface, limiting imaging to the dorsal axis of the brain. The presence of the skull bone makes the ICW a more challenging technical procedure, compared to the more established and utilized chamber models currently used to study mammary tissue and fat pads 5-7. In addition, the ICW provides many challenges when optimizing the imaging.

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We describe a novel in vivo imaging technique that couples fluorescent chimeric mice with intracranial windows and high-resolution 2-photon microscopy. This imaging platform aids studies of dynamic changes in brain tissue and microvasculature, at a single-cell level, following pathological insults and is adaptable to assess intracranial drug delivery and distribution.

رواية عالية الدقة<em&gt; في الجسم الحي</em&gt; تقنية التصوير لدراسة الاستجابة الديناميكية للمنشآت داخل القحف لنمو الورم والتداوي

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a ...

A Novel Application of Musculoskeletal Ultrasound Imaging

Ultrasound is an attractive modality for imaging muscle and tendon motion during dynamic tasks and can provide a complementary methodological approach for biomechanical studies in a clinical or laboratory setting. Towards this goal, methods for quantification of muscle kinematics from ultrasound imagery are being developed based on image processing. The temporal resolution of these methods is typically not sufficient for highly dynamic tasks, such as drop-landing. We propose a new approach that utilizes a Doppler method for quantifying muscle kinematics. We have developed a novel vector tissue Doppler imaging (vTDI) technique that can be used to measure musculoskeletal contraction velocity, strain and strain rate with sub-millisecond temporal resolution during dynamic activities using ultrasound. The goal of this preliminary study was to investigate the repeatability and potential applicability of the vTDI technique in measuring musculoskeletal velocities during a drop-landing task, in healthy subjects. The vTDI measurements can be performed concurrently with other biomechanical techniques, such as 3D motion capture for joint kinematics and kinetics, electromyography for timing of muscle activation and force plates for ground reaction force. Integration of these complementary techniques could lead to a better understanding of dynamic muscle function and dysfunction underlying the pathogenesis and pathophysiology of musculoskeletal disorders.

We describe a new ultrasound-based vector tissue Doppler imaging technique to measure muscle contraction velocity, strain and strain rate with sub-millisecond temporal resolution during dynamic activities. This approach provides complementary measurements of dynamic muscle function and could lead to a better understanding of mechanisms underlying musculoskeletal disorders.

تطبيق رواية من العظام والعضلات التصوير بالموجات فوق الصوتية

Ultrasound is an attractive modality for imaging muscle and tendon  motion during dynamic tasks and can provide a complementary methodological  approach ...

Cell-based Assay Protocol for the Prognostic Prediction of Idiopathic Scoliosis Using Cellular Dielectric Spectroscopy

This protocol details the experimental and analytical procedure for a cell-based assay developed in our laboratory as a functional test to predict the prognosis of idiopathic scoliosis in asymptomatic and affected children. The assay consists of the evaluation of the functional status of Gi and Gs proteins in peripheral blood mononuclear cells (PBMCs) by cellular dielectric spectroscopy (CDS), using an automated CDS-based instrument, and the classification of children into three functional groups (FG1, FG2, FG3) with respect to the profile of imbalance between the degree of response to Gi and Gs proteins stimulation. The classification is further confirmed by the differential effect of osteopontin (OPN) on response to Gi stimulation among groups and the severe progression of disease is referenced by FG2. Approximately, a volume of 10 ml of blood is required to extract PBMCs by Ficoll-gradient and cells are then stored in liquid nitrogen. The adequate number of PBMCs to perform the assay is obtained after two days of cell culture. Essentially, cells are first incubated with phytohemmaglutinin (PHA). After 24 hr incubation, medium is replaced by a PHA-free culture medium for an additional 24 hr prior to cell seeding and OPN treatment. Cells are then spectroscopically screened for their responses to somatostatin and isoproterenol, which respectively activate Gi and Gs proteins through their cognate receptors. Both somatostatin and isoproterenol are simultaneously injected with an integrated fluidics system and the cells&#39; responses are monitored for 15 min. The assay can be performed with fresh or frozen PBMCs and the procedure is completed within 4 days.

A structured protocol is presented for a cell-based assay as a functional test to predict the prognosis of idiopathic scoliosis using cellular dielectric spectroscopy (CDS). The assay can be performed with fresh or frozen peripheral blood mononuclear cells (PBMCs) and the procedure is completed within 4 days.

بروتوكول الفحص على أساس خلية للتنبؤ النذير من الجنف مجهول السبب عن طريق الخلوي عازل الطيفي

This protocol details the experimental and analytical procedure for a cell-based assay developed in our laboratory as a functional test to predict the ...

In vivo 19F MRI for Cell Tracking

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. Here, we describe a general protocol for cell labeling, imaging, and image processing. The technique is applicable to various cell types and animal models, although here we focus on a typical mouse model for tracking murine immune cells. The most important issues for cell labeling are described, as these are relevant to all models. Similarly, key imaging parameters are listed, although the details will vary depending on the MRI system and the individual setup. Finally, we include an image processing protocol for quantification. Variations for this, and other parts of the protocol, are assessed in the Discussion section. Based on the detailed procedure described here, the user will need to adapt the protocol for each specific cell type, cell label, animal model, and imaging setup. Note that the protocol can also be adapted for human use, as long as clinical restrictions are met.

In vivo 19F MRI for Cell Tracking

We describe a general protocol for in vivo cell tracking using MRI in a mouse model with ex vivo labeled cells. A typical protocol for cell labeling, image acquisition processing and quantification is included.

في الجسم الحي 19 F التصوير بالرنين المغناطيسي لتتبع الخليوي

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

الحقن الشريان السباتي لوأضافا الدوائية وتحليل Taxanes في الفئران

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

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