وأيد هذا العمل من خلال منحة من جديد مبتكر برنامج جائزة مدير المعاهد الوطنية للصحة في (ED). وأيد AA في جزء من الجوائز البكالوريوس من المعهد الوطني للسرطان ومؤسسة العلوم الوطنية.

1. جعل النواقل الفيروسية تسلسل الترميز لهذا الجين من الفائدة، فضلا عن الضوابط الإيجابية والسلبية، تندس في pBABEpuro بالطرق التقليدية الاستنساخ (PCR التضخيم، والهضم تقييد وربط). هناك أربعة مواقع قيود على ناقلات حيث يمكن إدخال DNA: إنزيم BamHI، SnaBI، EcoRI وسالي. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إعداد ترنسفكأيشن الصف بلازميد DNA من خلايا المختصة باستخدام QIAGEN بلازميد ميدي عدة. </li><li style=";text-align:right;direction:rtl"> قياس تركيز الحمض النووي باستخدام معمل NanoDrop2000. </li></ol> 2. الارتجاعي الإنتاج سيتم استخدام خط الخلية التعبئة والتغليف بلات-E لإنتاج الفيروس الارتجاعي ecotropic التي سيلقي [كدنا] التي تهم خلايا NIH 3T3. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إنشاء الثقافات بلات-E من خلايا مجمدة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ذوبان الجليد بسرعة خلايا بلات-E في 37 ° C حمام الماء ونقل إلى 15 مل أنبوب مخروطي الشكل. إضافة ببطء 9 مل من بلات-E المتوسطة (دوlbecco في التعديل النسر متوسطة (DMEM) + 10٪ مصل بقري جنيني (FBS) + البنسلين والستربتوميسين). </li><li style=";text-align:right;direction:rtl"> أجهزة الطرد المركزي الأنبوب في 180 × ز لمدة 5 دقائق وتجاهل طاف. </li><li style=";text-align:right;direction:rtl"> اعادة تعليق الخلايا مع 10 مل من بلات-E متوسطة ونقل إلى صحن ثقافة 10 سم. </li><li style=";text-align:right;direction:rtl"> احتضان الخلايا في 37 درجة مئوية، 5٪ CO 2 الحاضنة حتى تكون 80-90٪ متموجة (حوالي 2 أيام). </li></ol></li><li style=";text-align:right;direction:rtl"> تقسيم الخلايا <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> نضح المتوسطة ويغسل خلايا مرة واحدة مع برنامج تلفزيوني. </li><li style=";text-align:right;direction:rtl"> إضافة 2 مل من 0.05٪ التربسين-EDTA واحتضان لمدة 1 دقيقة في RT. </li><li style=";text-align:right;direction:rtl"> فصل الخلايا عن طريق الإصبع والتنصت، وإضافة 10 مل من بلات-E المتوسطة، ونقل تعليق خلية إلى أنبوب 15 مل. </li><li style=";text-align:right;direction:rtl"> أجهزة الطرد المركزي الأنبوب في 180 × ز لمدة 5 دقائق وتجاهل طاف. </li><li style=";text-align:right;direction:rtl"> resuspend الخلايا مع 10 مل من بلات-E المتوسطة والبذور منها في أطباق جديدة في 1: 4-1: 6 التخفيفات. </li></ol></li><li style=";text-align:right;direction:rtl"> بلات-E البذر وترنسفكأيشن <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> البذور 2 × 10 6خلايا لكل 10 سم صحن الثقافة باستخدام بلات-E وسيط وبدون أي المضادات الحيوية. </li><li style=";text-align:right;direction:rtl"> احتضان الخلايا O / N في 37 ° C، 5٪ CO 2 الحاضنة. </li><li style=";text-align:right;direction:rtl"> اليوم التالي، ونقل 300 ميكرولتر من غروب-MEM إلى 1.5 مل microfuge أنبوب. </li><li style=";text-align:right;direction:rtl"> إضافة 27 ميكرولتر من polyethylenimine (PEI)، وكاشف ترنسفكأيشن فعالة من حيث التكلفة 12، إلى أنبوب استعداد مع غروب-MEM. المزيج بلطف بواسطة الإصبع التنصت واحتضان لمدة 5 دقائق على RT. </li><li style=";text-align:right;direction:rtl"> إضافة 9 ميكروغرام من DNA البلازميد ترنسفكأيشن الصف في أنبوب غروب-MEM / PEI، مزيج من قبل vortexing بلطف، واحتضان لمدة 15 دقيقة في RT. </li><li style=";text-align:right;direction:rtl"> إضافة DNA / PEI قطرة من الحكمة معقدة في طبق بلات-E، واحتضان O / N عند 37 درجة مئوية، و 5٪ CO 2. </li><li style=";text-align:right;direction:rtl"> اليوم التالي، نضح في المتوسط ​​تحتوي على كاشف ترنسفكأيشن وإضافة 10 مل من جديد بلات-E متوسطة (بدون المضادات الحيوية). </li><li style=";text-align:right;direction:rtl"> عودة إلى الخلايا الحاضنة. </li></ol></li></ol> 3. خلايا NIH 3T3 والعدوى <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إنشاء المعاهد الوطنية للصحة3T3 الثقافات والبذر <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ذوبان الجليد بسرعة خلايا NIH 3T3 في 37 ° C حمام الماء ونقل إلى أنبوب 15 مل. إضافة ببطء 9 مل من NIH 3T3 المتوسطة (DMEM + 10٪ FBS). </li><li style=";text-align:right;direction:rtl"> أجهزة الطرد المركزي الأنبوب في 180 × ز لمدة 5 دقائق وتجاهل طاف. </li><li style=";text-align:right;direction:rtl"> resuspend الخلايا مع 10 مل من NIH 3T3 المتوسطة ونقل إلى صحن ثقافة 10 سم. </li><li style=";text-align:right;direction:rtl"> احتضان الخلايا في 37 درجة مئوية، 5٪ CO 2 الحاضنة. من المهم جدا أن الخلايا NIH 3T3 يتم الاحتفاظ دائما underconfluent (50-60٪)، كما تردد التحول التلقائي (وبالتالي مستويات القاعدية من تشكيل بؤر) يزيد مرة واحدة تصل إلى ثقافة confluency. </li><li style=";text-align:right;direction:rtl"> البذور 3 × 10 5 خلايا لكل 10 سم طبق واحتضان O / N للعدوى في اليوم التالي. </li></ol></li><li style=";text-align:right;direction:rtl"> عدوى <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> حصاد طاف فيروسات من الطبق بلات-E باستخدام 10 مل حقنة واحدة، وتصفية عليه من خلال 0.45 ميكرون حجم المسام النايلون مرشح غشاء، وتحويلها إلى 15 ملأنبوب. </li><li style=";text-align:right;direction:rtl"> إضافة 10 مل من جديد بلات-E المتوسطة للخلايا (بدون المضادات الحيوية)، وإعادتها إلى الحاضنة. </li><li style=";text-align:right;direction:rtl"> نضح المتوسطة من الطبق خلية NIH 3T3 للإصابة، وإضافة 5 مل من العادية المتوسطة NIH 3T3 و 5 مل من التي تحتوي على فيروس تصفيتها طاف. </li><li style=";text-align:right;direction:rtl"> إضافة polybrene إلى الطبق بتركيز 6 ميكروغرام / مل. </li><li style=";text-align:right;direction:rtl"> احتضان الخلايا O / N عند 37 درجة مئوية، و 5٪ CO 2. </li><li style=";text-align:right;direction:rtl"> اليوم التالي، كرر الخطوات من 3.2.1 - 3.2.5 لجولة ثانية من العدوى. </li><li style=";text-align:right;direction:rtl"> استبدال المتوسطة التي تحتوي على الفيروس مع منتظم المتوسطة NIH 3T3. </li><li style=";text-align:right;direction:rtl"> السماح للخلايا NIH 3T3 لتنمو لمدة 2-3 أسابيع، لتحل محل المتوسطة حسب الضرورة. تحقق من التعبير عن بروتين الاهتمام من جانب التقليدي الكهربائي البروتين وطخة مناعية على لست] خلية كاملة من لوحات طبق الأصل. </li></ol></li></ol> 4. تلطيخ والكمي <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> الكريستال البنفسجي تلطيخ <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> نضح المتوسطة NIH 3T3 ووضع الأطباقعلى الجليد. </li><li style=";text-align:right;direction:rtl"> غسل الأطباق مرتين مع PBS الجليد الباردة. </li><li style=";text-align:right;direction:rtl"> إصلاح الخلايا مع الميثانول الجليد الباردة لمدة 10 دقيقة. </li><li style=";text-align:right;direction:rtl"> إزالة أطباق من الجليد، ونضح الميثانول، وإضافة 3 مل من 0.5٪ محلول الكريستال البنفسجي المحرز في 25٪ الميثانول (RT). </li><li style=";text-align:right;direction:rtl"> احتضان الأطباق لمدة 5 دقائق على RT. </li><li style=";text-align:right;direction:rtl"> نضح الحل الكريستال البنفسجي وشطف بعناية الطبق مع ميلي-Q H 2 O حتى يأتي لا لون ثماره في الشطف. </li><li style=";text-align:right;direction:rtl"> السماح للأطباق لتجف O / N على الفوق (كشف). </li></ol></li><li style=";text-align:right;direction:rtl"> بؤر الكمي <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> مرة واحدة الأطباق جافة، استخدام مسطرة لقياس مجموعات الملونة على نحو مظلم من الخلايا على طبق من ذهب. الاعتماد فقط تلك التي هي أكبر من 5 ملم في القطر بأنها &quot;بؤر&quot;. سجل عدد من البؤر وحساب المتوسطات وأهمية مقارنة مع الضوابط. </li></ol></li></ol>

<ol>
	<li>Clark, G. J., Cox, A. D., Graham, S. M., Der, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+assays+for+Ras+transformation.">Biological assays for Ras transformation.</a> Methods in enzymology. , 255-395 (1995).</li><li>Celis, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cell biology : a laboratory handbook. , 345-352 (2006).</li><li>Raptis, L., Vultur, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neoplastic+transformation+assays.">Neoplastic transformation assays.</a> Methods in molecular biology. 165, 151-164 (2001).</li><li>Johnson, P. J., Coussens, P. M., Danko, A. V., Shalloway, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overexpressed+pp60c-src+can+induce+focus+formation+without+complete+transformation+of+NIH+3T3+cells.">Overexpressed pp60c-src can induce focus formation without complete transformation of NIH 3T3 cells.</a> Molecular and cellular biology. 5, 1073-1083 (1985).</li><li>Bonner, T. I., Kerby, S. B., Sutrave, P., Gunnell, M. A., Mark, G., Rapp, U. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+biological+activity+of+human+homologs+of+the+raf/mil+oncogene.">Structure and biological activity of human homologs of the raf/mil oncogene.</a> IMolecular and cellular biology. 5, 71400-71407 (1985).</li><li>Yancopoulos, G. D., 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=N-myc+can+cooperate+with+ras+to+transform+normal+cells+in+culture.">N-myc can cooperate with ras to transform normal cells in culture.</a> Proceedings of the National Academy of Sciences of the United States of America. 82, 5455-5459 (1985).</li><li>Fujiwara, 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=Transforming+activity+of+purinergic+receptor+P2Y,+G+protein+coupled,+8+revealed+by+retroviral+expression+screening.">Transforming activity of purinergic receptor P2Y, G protein coupled, 8 revealed by retroviral expression screening.</a> Leukemia &amp; lymphoma. 48, 978-986 (2007).</li><li>Morita, S., Kojima, T., Kitamura, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plat-E:+an+efficient+and+stable+system+for+transient+packaging+of+retroviruses.">Plat-E: an efficient and stable system for transient packaging of retroviruses.</a> Gene. 7, 1063-1066 (2000).</li><li>Morgenstern, J. P., Land, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advanced+mammalian+gene+transfer:+high+titre+retroviral+vectors+with+multiple+drug+selection+markers+and+a+complementary+helper-free+packaging+cell+line.">Advanced mammalian gene transfer: high titre retroviral vectors with multiple drug selection markers and a complementary helper-free packaging cell line.</a> Nucleic acids research. 18, 3587-3596 (1990).</li><li>Kim, T. K., Eberwine, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammalian+cell+transfection:+the+present+and+the+future.">Mammalian cell transfection: the present and the future.</a> Analytical and bioanalytical chemistry. 397, 3173-3178 (2010).</li><li>Klaver, B., Berkhout, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+5'+and+3'+long+terminal+repeat+promoter+function+in+human+immunodeficiency+virus.">Comparison of 5' and 3' long terminal repeat promoter function in human immunodeficiency virus.</a> Journal of virology. 68, 3830-3840 (1994).</li><li>Fukumoto, Y., 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=Cost-effective+gene+transfection+by+DNA+compaction+at+pH+4.0+using+acidified,+long+shelf-life+polyethylenimine.">Cost-effective gene transfection by DNA compaction at pH 4.0 using acidified, long shelf-life polyethylenimine.</a> Cytotechnology. 62, 73-82 (2010).</li><li>Fox, E. J., Wright, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=S-phase-specific+expression+of+the+Mad3+gene+in+proliferating+and+differentiating+cells.">S-phase-specific expression of the Mad3 gene in proliferating and differentiating cells.</a> The Biochemical journal. 359, 361-367 (2001).</li><li>Yun, J. S., Rust, J. M., Ishimaru, T., Diaz, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+role+of+the+Mad+family+member+Mad3+in+cerebellar+granule+neuron+precursor+proliferation.">A novel role of the Mad family member Mad3 in cerebellar granule neuron precursor proliferation.</a> Molecular and cellular biology. 27, 8178-8189 (2007).</li><li>Gore, Y., Lantner, F., Hart, G., Shachar, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mad3+negatively+regulates+B+cell+differentiation+in+the+spleen+by+inducing+Id2+expression.">Mad3 negatively regulates B cell differentiation in the spleen by inducing Id2 expression.</a> Molecular biology of the cell. 21, 1864-1871 (2010).</li><li>Barisone, G. A., Yun, J. S., Diaz, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+cerebellar+proliferation+to+tumorigenesis:+new+insights+into+the+role+of+Mad3.">From cerebellar proliferation to tumorigenesis: new insights into the role of Mad3.</a> Cell cycle. 7, 423-427 (2008).</li><li>Barisone, G. 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=Role+of+MXD3+in+proliferation+of+DAOY+human+medulloblastoma+cells.">Role of MXD3 in proliferation of DAOY human medulloblastoma cells.</a> PloS One. 7, e38508 (2012).</li><li>Nair, S. K., Burley, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=X-ray+structures+of+Myc-Max+and+Mad-Max+recognizing+DNA.+Molecular+bases+of+regulation+by+proto-oncogenic+transcription+factors.">X-ray structures of Myc-Max and Mad-Max recognizing DNA. Molecular bases of regulation by proto-oncogenic transcription factors.</a> Cell. 112, 193-205 (2003).</li></ol>

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يوفر FFA طريقة سريعة وسهلة لتقييم التحول الخبيث في المختبر. فمن قابلة للفحص عدد كبير نسبيا من الجينات المرشحة، والمتطلبات التقنية المتواضعة جعلها فعالة من حيث التكلفة. وعلاوة على ذلك، اثنين أو أكثر من الجينات يمكن coexpressed (يشار إليها أحيانا باسم &quot;التعاون&quot; فحص)...

الخلايا السرطانية يمكن تمييزها عن نظيراتها العادية من قبل مجموعة واسعة من التعديلات، من أنماط التعبير الجيني لepigenomics إلى تغيرات شكلية والتكاثري. ومن بين هذه الأخيرة، وخفض الاعتماد على المصل، وفقدان الاتصال (الكثافة) تثبيط، واقتناء الانتشار مرسى مستقل، وفي نهاية المطاف، القدرة على تكوين الأورام عند حقنه في الحيوانات، مؤشرات قابلة للقياس مفيدة التحول الخبيث 3. عدة في المختبر والمقايسات فيفو تم وضعها من أجل التحول الخلوي. في المختبر فحوصات تهدف إلى تحديد وقياس التغيرات في التشكل الثقافة (التركيز تشكيل فحص)، وديناميات الثقافة (معدل النمو والكثافة التشبع)، وعامل النمو (النمو في انخفاض المصل) أو مرسى (النمو في أجار لينة) المتطلبات. لا يزال المعيار الذهبي لتحديد طبيعة الخبيثة من نوع الخلية تشكيل الورم (xenografts) في حيوانات التجارب. ومع ذلك، رانه يكلف وطول من الدراسات في الجسم الحي لا تجعل لهم دائما مبررا كخطوة أولى التحقق من صحة أو فحص الجينات المسرطنة مرشح. على الرغم من عدم الفحص في المختبر يوفر تقييم واضح للإمكانات أنكجنيك من الجين، فإنها توفر نظرة ثاقبة إمكانات أنكجنيك التي قد تضييق المستقبل في الدراسات المجراة. واحد من أكثر الأنظمة المستخدمة على نطاق واسع لتقييم إمكانات أنكجنيك في المختبر هو التركيز تشكيل الفحص 2. ويعتمد هذا الأسلوب على استخدام NIH 3T3 الخلايا الليفية الماوس، خط خلية غير حولت الذي يظهر تثبيط اتصال قوية. overexpression من على النتائج الجين الورمي في فقدان النمو التي تعتمد على كثافة. الخلايا تحولت يمكن بعد ذلك تنمو في طبقات متعددة، وتشكيل &quot;بؤر&quot;، تصور بسهولة ضد أحادي الطبقة الخلفية للخلايا غير متحول. تشكيل التركيز الفحص، ثم، يقيس قدرة الجين الورمي مرشح للحث على التحول الخبيث، كما يتضح من فقدان الاتصال inhibition باعتباره النمط الظاهري للقياس. وقد استخدم FFA لتقييم التحول من overexpression من تحركات البروتينات (على سبيل المثال، SRC 4، BRAF 5)، عوامل النسخ (على سبيل المثال، N-MYC 6) مستقبلات، إلى جانب G-البروتين (على سبيل المثال، P2RY8 7) وGTPases (على سبيل المثال ورأس 1)، وغيرها. السهولة النسبية لهذا الاختبار يجعلها خيارا جيدا من شأنها أن توفر إجابة واضحة وسريعة بصريا ما إذا كان overexpression من الجينات يكفي لتحويل NIH 3T3 خلايا فأر الليفية في المختبر. وFFA الموصوفة في هذا البروتوكول يستخدم بلات-E خط الخلية التعبئة والتغليف 8، والذي يوفر البروتينات الفيروسية التعبئة والتغليف، وفيروسات ناقلات pBABEpuro 9 (Addgene بلازميد 1764) لإنتاج الفيروس الارتجاعي. بعد ترنسفكأيشن مع بناء pBABEpuro التي تحتوي على الجينات في المصالح، فإن خط الخلية بلات-E تنتج الارتجاعي ecotropic التي يمكن استخدامها لتصيب خلايا NIH 3T3. تيطريقة الفيروسي له من توصيل الجينات هو أكثر فعالية من الأساليب ترنسفكأيشن الكيميائية التقليدية، وأنه يوفر وسيلة للتعبير عن هذا الجين على نحو مستدام 10. مرة واحدة دمجها في جينوم الخلايا NIH 3T3، والدافع overexpression من هذا الجين من الفائدة من جانب يكرر محطة الطويلة الفيروسية (LTR) المروج 11. ويمكن استخدام هذا التعبير المستمر لتحديد ما إذا كانت الجينات في المصالح والنشاط أنكجنيك، مقاسا تشكيل بؤر، على خلايا NIH 3T3.

<table><tbody><tr><td>pBABE-puro vector</td><td>Addgene</td><td>Plasmid 1764</td><td>cloning vector</td></tr><tr><td>Platinum-E Retroviral Packaging Cell Line, Ecotropic</td><td>Cell Biolabs, Inc.</td><td>RV-101</td><td>cell line for viral production</td></tr><tr><td>NIH 3T3 Cell Line murine</td><td>Sigma-Aldrich</td><td>93061524</td><td>cell line for focus formation assay</td></tr><tr><td>10 ml BD Luer-Lok tip syringe</td><td>BD Biosciences</td><td>309604</td><td>viral production reagent</td></tr><tr><td>0.45 &amp;mu;m Puradisc Syringe Filter</td><td>Whatman</td><td>6750-2504</td><td>viral production reagent</td></tr><tr><td>Polyethylenimine (PEI)</td><td>Polysciences, Inc.</td><td>23966-2</td><td>cell transfection reagent</td></tr><tr><td>Polybrene Infection / Transfection Reagent</td><td>EMD Millipore</td><td>TR-1003-G</td><td>cell transfection reagent</td></tr><tr><td>Crystal Violet</td><td>Fisher Scientific</td><td>C581-25</td><td>cell stain reagent</td></tr><tr><td>Plasmid Plus Midi Kit</td><td>QIAGEN</td><td>12945</td><td>plasmid purification</td></tr><tr><td>BD Falcon Tissue Culture Dishes</td><td>BD Biosciences</td><td>353003</td><td>cell culture supplies</td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium (DMEM)</td><td>Gibco</td><td>11995-065</td><td>cell culture media</td></tr><tr><td>0.05% Trypsin-EDTA</td><td>Gibco</td><td>25300-054</td><td>cell culture supplies</td></tr><tr><td>Opti-MEM I Reduced Serum Medium</td><td>Gibco</td><td>31985-062</td><td>cell culture media</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Gibco</td><td>16000-044</td><td>cell culture media</td></tr></tbody></table>

focus formation: a cell-based assay to determine the oncogenic potential of a gene

Malignant transformation of cells is typically associated with increased proliferation, loss of contact inhibition, acquisition of anchorage-independent growth potential, and the ability to form tumors in experimental animals1. In NIH 3T3 cells, the Ras signal transduction pathway is known to trigger many of these events, what is known as Ras transformation. The introduction of an overexpressed gene in NIH 3T3 cells may promote morphological transformation and loss of contact inhibition, which can help determine the oncogenic potential of that gene of interest. An assay that provides a straightforward method to assess one aspect of the transforming potential of an oncogene is the Focus Formation Assay (FFA)2. When NIH 3T3 cells divide normally in culture, they do so until they reach a confluent monolayer. However, in the presence of an overexpressed oncogene, these cells can begin to grow in dense, multilayered foci1 that can be visualized and quantified by crystal violet or Hema 3 staining. In this article we describe the FFA protocol with retroviral transduction of the gene of interest into NIH 3T3 cells, and how to quantify the number of foci through staining. Retroviral transduction offers a more efficient method of gene delivery than transfection, and the use of an ecotropic murine retrovirus provides a biosafety control when working with potential human oncogenes.

MXD3 هو عامل النسخ الأساسي سحاب الحلزون حلقة حلزون ليسين (bHLHZ) الذي هو عضو من MYC / MAX / شبكة MAD. وهي عضو غير نمطية للأسرة MAD 13-15، وأفيد أن تشارك في التسرطن 16،17. بالمقارنة مع pBABEpuro (المراقبة السلبية) وMYC (مراقبة إيجابية)، كانت أطباق NIH 3T3 حيث تم overexpressed MXD3 أقل بكثير البؤ...

Watch this Scientific Journal Video about Focus Formation: A Cell-based Assay to Determine the Oncogenic Potential of a Gene at JoVE.com

Focus Formation: A Cell-based Assay to Determine the Oncogenic Potential of a Gene

توفر الفحص التركيز تشكيل طريقة واضحة لتقييم إمكانات تحويل من الجين الورمي مرشح.

التركيز تشكيل: A الفحص القائم على خلية لتحديد إمكانية أنكجنيك لجين

Malignant transformation of cells is typically associated with increased proliferation, loss of contact inhibition, acquisition of anchorage-independent ...

focus-formation-cell-based-assay-to-determine-oncogenic-potential

Research

JoVE Journal

Medicine

27.8K Views.  University of California, Davis.  توفر الفحص التركيز تشكيل طريقة واضحة لتقييم إمكانات تحويل من الجين الورمي مرشح. 

Video: Focus Formation: A Cell-based Assay to Determine the Oncogenic Potential of a Gene

Utilizing Functional Genomics Screening to Identify Potentially Novel Drug Targets in Cancer Cell Spheroid Cultures

The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery of the next generation of novel drug targets. It is becoming apparent that more complex models of cancer are required to fully appreciate the contributing factors that drive tumorigenesis in vivo and increase the efficacy of novel therapies that make the transition from pre-clinical models to clinical trials.
Here we present a methodology for generating uniform and reproducible tumor spheroids that can be subjected to siRNA functional screening. These spheroids display many characteristics that are found in solid tumors that are not present in traditional two-dimension culture. We show that several commonly used breast cancer cell lines are amenable to this protocol. Furthermore, we provide proof-of-principle data utilizing the breast cancer cell line BT474, confirming their dependency on amplification of the epidermal growth factor receptor HER2 and mutation of phosphatidylinositol-4,5-biphosphate 3-kinase (PIK3CA) when grown as tumor spheroids. Finally, we are able to further investigate and confirm the spatial impact of these dependencies using immunohistochemistry.

Identifying novel drug targets that transition from pre-clinical testing to human trials is a scientific priority. To that end, here we describe a functional genomics approach for examining the impact of gene depletion on cancer cell line spheroids, which more appropriately model human cancers in vivo.

الاستفادة من علم الجينوم وظيفية الفرز لتحديد أهداف المخدرات رواية يحتمل في الثقافات كروي الخلية السرطانية

The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery ...

Cancer Research

Colony Forming Cell (CFC) Assay for Human Hematopoietic Cells

Human hematopoietic stem/progenitor cells are usually obtained from bone marrow, cord blood, or peripheral blood and are used to study hematopoiesis and leukemogenesis. They have the capacity to differentiate into lymphoid and myeloid lineages. The colony forming cell (CFC) assay is used to study the proliferation and differentiation pattern of hematopoietic progenitors by their ability to form colonies in a semisolid medium. The number and the morphology of the colonies formed by a fixed number of input cells provide preliminary information about the ability of progenitors to differentiate and proliferate. Cells can be harvested from individual colonies or from the whole plate to further assess their numbers and differentiation states using flow cytometry and morphologic evaluation of Giemsa-stained slides. This assay is useful for assessing myeloid but not lymphoid differentiation. The term myeloid in this context is used in its wider sense to encompass granulocytic, monocytic, erythroid, and megakaryocytic lineages.

We have used this assay to assess the effects of oncogenes on the differentiation of primary human CD34+ cells derived from peripheral blood. For this purpose cells are transduced with either control retroviral construct or a construct expressing the oncogene of interest, in this case NUP98-HOXA9. We employ a commonly used retroviral vector, MSCV-IRES-GFP, that expresses a bicistronic mRNA that produces the gene of interest and a GFP marker. Cells are pre-activated by growing in the presence of cytokines for two days prior to retroviral transduction. After another two days, GFP+ cells are isolated by fluorescence-activated cell sorting (FACS) and mixed with a methylcellulose-containing semisolid medium supplemented with cytokines and incubated till colonies appear on the surface, typically 14 days. The number and morphology of the colonies are documented. Cells are then removed from the plates, washed, counted, and subjected to flow cytometry and morphologic examination. Flow cytometry with antibodies specific to the cell surface markers expressed during hematopoiesis provides information about lineage and maturation stage. Morphological studies of individual cells under a microscope after Wright- Giemsa staining provide further information with regard to lineage and maturation. Comparison of cells transduced with control empty vector to those transduced with an oncogene reveals the effects of the oncogene on hematopoietic differentiation.

Colony Forming Cell CFC Assay for Human Hematopoietic Cells

The colony forming cell (CFC) assay is an in vitro assay in which hematopoietic progenitors form colonies in a semi-solid medium. A combination of colony morphology, cell morphology, and flow cytometry are used to assess the ability of the progenitors to proliferate and differentiate along the different hematopoietic lineages.

تشكيل خلية مستعمرة (CFC) للخلايا المكونة للدم الفحص الإنسان

Human hematopoietic stem/progenitor cells are usually obtained from bone marrow, cord blood, or peripheral blood and are used to study hematopoiesis and ...

Immunology and Infection

Methods for Evaluating the Role of c-Fos and Dusp1 in Oncogene Dependence

The demonstration of tyrosine kinase inhibitors (TKIs) in treating chronic myeloid leukemia (CML) has heralded a new era in cancer therapeutics. However, a small population of cells does not respond to TKI treatment, resulting in minimal residual disease (MRD); even the most potent TKIs fail to eradicate these cells. These MRD cells serve as a reservoir to develop resistance to therapy. Why TKI treatment is ineffective against MRD cells is not known. Growth factor signaling is implicated in supporting the survival of MRD cells during TKI treatment, but a mechanistic understanding is lacking. Recent studies demonstrated that an elevated c-Fos and Dusp1 expression as a result of convergent oncogenic and growth factor signaling in MRD cells mediate TKI resistance. The genetic and chemical inhibition of c-Fos and Dusp1 renders CML exquisitely sensitive to TKIs and cures CML in both genetic and humanized mouse models. We identified these target genes using multiple microarrays from TKI-sensitive and -resistant cells. Here, we provide methods for target validation using in vitro and in vivo mouse models. These methods can easily be applied to any target for genetic validation and therapeutic development.

Here, we describe protocols for the genetic and chemical validation of c-Fos and Dusp1 as a drug target in leukemia using in vitro and in vivo genetic and humanized mouse models. This method can be applied to any target for genetic validation and therapeutic development.

أساليب لتقييم دور ج-المنحدر و Dusp1 في الاعتماد على السرطاني

The demonstration of tyrosine kinase inhibitors (TKIs) in treating chronic myeloid leukemia (CML) has heralded a new era in cancer therapeutics. However, ...

Yeast As a Chassis for Developing Functional Assays to Study Human P53

The finding that the well-known mammalian P53 protein can act as a transcription factor (TF) in the yeast S. cerevisiae has allowed for the development of different functional assays to study the impacts of 1) binding site [i.e., response element (RE)] sequence variants on P53 transactivation specificity or 2) TP53 mutations, co-expressed cofactors, or small molecules on P53 transactivation activity. Different basic and translational research applications have been developed. Experimentally, these approaches exploit two major advantages of the yeast model. On one hand, the ease of genome editing enables quick construction of qualitative or quantitative reporter systems by exploiting isogenic strains that differ only at the level of a specific P53-RE to investigate sequence-specificity of P53-dependent transactivation. On the other hand, the availability of regulated systems for ectopic P53 expression allows the evaluation of transactivation in a wide range of protein expression. Reviewed in this report are extensively used systems that are based on color reporter genes, luciferase, and the growth of yeast to illustrate their main methodological steps and to critically assess their predictive power. Moreover, the extreme versatility of these approaches can be easily exploited to study different TFs including P63 and P73, which are other members of TP53 gene family.

Presented here are four protocols to construct and exploit yeast Saccharomyces cerevisiae reporter strains to study human P53 transactivation potential, impacts of its various cancer-associated mutations, co-expressed interacting proteins, and the effects of specific small molecules.

الخميرة كهيكل لتطوير الاختبارات الوظيفية لدراسة P53 الإنسان

The finding that the well-known mammalian P53 protein can act as a transcription factor (TF) in the yeast S. cerevisiae has allowed for the development of ...

Oncogenic Gene Fusion Detection Using Anchored Multiplex Polymerase Chain Reaction Followed by Next Generation Sequencing

Gene fusions frequently contribute to the oncogenic phenotype of many different types of cancer. Additionally, the presence of certain fusions in samples from cancer patients often directly influences diagnosis, prognosis, and/or therapy selection. As a result, the accurate detection of gene fusions has become a critical component of clinical management for many disease types. Until recently, clinical gene fusion detection was predominantly accomplished through the use of single-gene assays. However, the ever-growing list of gene fusions with clinical significance has created a need for assessing fusion status of multiple genes simultaneously. Next generation sequencing (NGS)-based testing has met this demand through the ability to sequence nucleic acid in massively parallel fashion. Multiple NGS-based approaches that employ different strategies for gene target enrichment are now available for use in clinical molecular diagnostics, each with its own strengths and weaknesses. This article describes the use of anchored multiplex PCR (AMP)-based target enrichment and library preparation followed by NGS to assess for gene fusions in clinical solid tumor specimens. AMP is unique among amplicon-based enrichment approaches in that it identifies gene fusions regardless of the identity of the fusion partner. Detailed here are both the wet-bench and data analysis steps that ensure accurate gene fusion detection from clinical samples.

This article details the use of an anchored multiplex polymerase chain reaction-based library preparation kit followed by next-generation sequencing to assess for oncogenic gene fusions in clinical solid tumor samples. Both wet-bench and data analysis steps are described.

الكشف عن الانصهار الجيني الأنسيوجيني باستخدام تفاعل سلسلة بوليميراز متعددة المضاعفات متبوعاً بتسلسل الجيل التالي

Gene fusions frequently contribute to the oncogenic phenotype of many different types of cancer. Additionally, the presence of certain fusions in samples ...

Mapping the Structure-Function Relationships of Disordered Oncogenic Transcription Factors Using Transcriptomic Analysis

Many cancers are characterized by chromosomal translocations which result in the expression of oncogenic fusion transcription factors. Typically, these proteins contain an intrinsically disordered domain (IDD) fused with the DNA-binding domain (DBD) of another protein and orchestrate widespread transcriptional changes to promote malignancy. These fusions are often the sole recurring genomic aberration in the cancers they cause, making them attractive therapeutic targets. However, targeting oncogenic transcription factors requires a better understanding of the mechanistic role that low-complexity, IDDs play in their function. The N-terminal domain of EWSR1 is an IDD involved in a variety of oncogenic fusion transcription factors, including EWS/FLI, EWS/ATF, and EWS/WT1. Here, we use RNA-sequencing to investigate the structural features of the EWS domain important for transcriptional function of EWS/FLI in Ewing sarcoma. First shRNA-mediated depletion of the endogenous fusion from Ewing sarcoma cells paired with ectopic expression of a variety of EWS-mutant constructs is performed. Then RNA-sequencing is used to analyze the transcriptomes of cells expressing these constructs to characterize the functional deficits associated with mutations in the EWS domain. By integrating the transcriptomic analyses with previously published information about EWS/FLI DNA binding motifs, and genomic localization, as well as functional assays for transforming ability, we were able to identify structural features of EWS/FLI important for oncogenesis and define a novel set of EWS/FLI target genes critical for Ewing sarcoma. This paper demonstrates the use of RNA-sequencing as a method to map the structure-function relationship of the intrinsically disordered domain of oncogenic transcription factors.

Intrinsically disordered domains are important for oncogenic fusion transcription factor function. To therapeutically target these proteins, a more detailed understanding of the regulatory mechanisms employed by these domains is required. Here, we use transcriptomics to map important structural features of the intrinsically disordered EWS domain in Ewing sarcoma.

رسم خرائط للعلاقات بين الهيكل والوظيفة لعوامل النسخ غير المنضبطة باستخدام التحليل النسخي

Many cancers are characterized by chromosomal translocations which result in the expression of oncogenic fusion transcription factors. Typically, these ...

A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

Genome-scale interrogation of gene function using RNA interference (RNAi) holds tremendous promise for the rapid identification of chemically tractable cancer cell vulnerabilities. Limiting the potential of this technology is the inability to rapidly delineate the mechanistic basis of phenotypic outcomes and thus inform the development of molecularly targeted therapeutic strategies. We outline here methods to deconstruct cellular phenotypes induced by RNAi-mediated gene targeting using multiplexed reporter systems that allow monitoring of key cancer cell-associated processes. This high-content screening methodology is versatile and can be readily adapted for the screening of other types of large molecular libraries.

Achieving a systems level understanding of cellular processes is a goal of modern-day cell biology. We describe here strategies for multiplexing luciferase reporters of various cellular function end-points to interrogate gene function using genome-scale RNAi libraries.

A تعدد في منصة الفرز القائم على luciferase المراسل لاستجواب المرتبطة بالسرطان نقل الإشارة في الخلايا المستزرعة

Genome-scale interrogation of gene function using RNA interference (RNAi)  holds tremendous promise for the rapid identification of chemically tractable  ...

Proximity Ligation Assay to Study Oncogene-Derived Transcription-Replication Conflicts

Both DNA replication and RNA transcription utilize genomic DNA as their template, necessitating spatial and temporal separation of these processes. Conflicts between the replication and transcription machinery, termed transcription-replication conflicts (TRCs), pose a considerable risk to genome stability, a critical factor in cancer development. While several factors regulating these collisions have been identified, pinpointing primary causes remains difficult due to limited tools for direct visualization and clear interpretation. In this study, we directly visualize TRCs using a proximity ligation assay (PLA), leveraging antibodies specific to PCNA and phosphorylated CTD of RNA polymerase II. This approach allows precise measurement of TRCs between replication and transcription processes mediated by RNA polymerase II. The method is further enhanced through DNA primers conjugated covalently to these antibodies, coupled with PCR amplification using fluorescent probes, providing a highly sensitive and specific means of detecting endogenous TRCs. Fluorescence microscopy enables the visualization of these conflicts, offering a powerful tool to study genome instability mechanisms associated with cancer. This technique addresses the gap in direct TRC visualization, allowing for a more comprehensive analysis and understanding of the underlying processes driving genome instability in cells.

Here, we present a sensitive and specific method to detect oncogene-induced transcription-replication conflicts (TRCs) using the proximity ligation assay (PLA). This approach leverages specific antibodies targeting PCNA and phospho-CTD RNAPII, enabling the assessment of TRC prevalence between RNA polymerase II transcription and DNA replication machinery.

مقايسة ربط القرب لدراسة تعارضات النسخ والنسخ المتماثل المشتق من الأورام

Both DNA replication and RNA transcription utilize genomic DNA as their template, necessitating spatial and temporal separation of these processes. ...

Oncogene Expression Analysis with Alterations in pH in a Pancreatic Ductal Cell Line

The fourth leading cause of cancer-related death, pancreatic ductal adenocarcinoma (PDAC), has a 12% five-year survival rate. This disease has a poor prognosis and is characterized by a rigid stromal microenvironment, which represents a tangible challenge in its treatment. Chronic pancreatitis patients have a 10-fold greater risk of developing PDAC; in these patients, the ductal pH decreases from pH 8.0 to pH 6.0 due to bicarbonate insufficiency and the inflammatory milieu. Our goal was to understand the role of the acidic environment observed in chronic pancreatitis on oncogene expression in a pancreatic ductal cell line.
Therefore, 80% confluent human pancreatic ductal epithelial cells were incubated at pH 6.0 to pH 7.2 for 6 h. Total RNA from the cells was processed to enrich the total mRNA in the samples. Gene expression was evaluated via next-generation sequencing (NGS) of biological replicates. RNA-seq analysis was carried out with the aid of an online tool, and the differentially expressed genes (FCs &#60; &#177; 2.0) were identified; there were 90, 148, and 109 upregulated genes and 20, 14, and 23 downregulated genes at pH 6.0, 6.5, and 7.0, respectively. Four oncogenes were upregulated at pH 6.0, seven were upregulated at pH 6.5, and seven were upregulated at pH 7.0. The common genes that were upregulated at pH 6.0, pH 6.5, and pH 7.0 were lymphocyte cell-specific protein-tyrosine kinase (LCK) [pH 6.0, FC: 2.93; pH 6.5, FC: 2.93; pH 7.0, FC: 3.32], FGR proto-oncogene, Src family tyrosine kinase (FGR) [pH 6.0, FC: 4.17; pH 6.5, FC: 5.25; pH 7.0, FC: 5.09], and ArfGAP With SH3 domain, ankyrin repeat, and PH domain 3 (ASAP3) [pH 6.0, FC: 2.37; pH 6.5, FC: 3.84; pH 7.0, FC: 2.51]. The acidic environment triggers the activation of proto-oncogenes, which may trigger tumor initiation in chronic pancreatitis.

This study describes a protocol for exploring the effects of altered pH on oncogene expression via RNA-seq analysis of a pancreatic ductal cell line.

تحليل تعبير الجينات المسرطنة مع التغيرات في الأس الهيدروجيني في خط خلايا قنية البنكرياس

The fourth leading cause of cancer-related death, pancreatic ductal adenocarcinoma (PDAC), has a 12% five-year survival rate. This disease has a poor ...

The Soft Agar Colony Formation Assay

Anchorage-independent growth is the ability of transformed cells to grow independently of a solid surface, and is a hallmark of carcinogenesis. The soft agar colony formation assay is a well-established method for characterizing this capability in vitro and is considered to be one of the most stringent tests for malignant transformation in cells. This assay also allows for semi-quantitative evaluation of this capability in response to various treatment conditions. Here, we will demonstrate the soft agar colony formation assay using a murine lung carcinoma cell line, CMT167, to demonstrate the tumor suppressive effects of two members of the Wnt signaling pathway, Wnt7A and Frizzled-9 (Fzd-9). Concurrent overexpression of Wnt7a and Fzd-9 caused an inhibition of colony formation in CMT167 cells. This shows that expression of Wnt7a ligand and its Frizzled-9 receptor is sufficient to suppress tumor growth in a murine lung carcinoma model.

The soft agar colony formation assay is a method used to confirm cellular anchorage-independent growth in vitro. The goal of this protocol is to illustrate a stringent method for the detection of the tumorigenic potential of transformed cells and the tumor suppressive effects of proteins on transformed cells.

تشكيل مستعمرة الفحص لينة آجار

Anchorage-independent growth is the ability of transformed cells to grow independently of a solid surface, and is a hallmark of carcinogenesis. The soft ...