Name: mating-based overexpression library screening in yeast
Uploaded: 2018-07-06T14:00:00
Description: Watch this Scientific Journal Video about Mating-based Overexpression Library Screening in Yeast at JoVE.com

हम जू प्रयोगशाला और Zhong प्रयोगशाला के सदस्यों के साथ विचारशील विचार विमर्श के लिए आभारी हैं, और राइट राज्य विश्वविद्यालय से वित्तीय सहायता ।

नोट: प्रोटोकॉल यहां वर्णित स्क्रीनिंग लाइब्रेरी plasmids के लिए डिज़ाइन किया गया है १० ९६-अच्छी तरह से प्लेट में निहित है, लेकिन ऊपर या नीचे तदनुसार बढ़ाया जा सकता है । पूरी लाइब्रेरी स्क्रीनिंग पूरी करने क?...

<ol>
	<li>Dujon, B. A., Louis, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+diversity+and+evolution+in+the+budding+yeasts+(Saccharomycotina).">Genome diversity and evolution in the budding yeasts (Saccharomycotina).</a> Genetics. 206 (2), 717-750 (2017).</li><li>Khurana, V., Lindquist, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modelling+neurodegeneration+in+Saccharomyces+cerevisiae:+why+cook+with+baker's+yeast.">Modelling neurodegeneration in Saccharomyces cerevisiae: why cook with baker's yeast.</a> Nature Reviews Neuroscience. 11 (6), 436-449 (2010).</li><li>Willingham, S., Outeiro, T. F., DeVit, M. J., Lindquist, S. L., Muchowski, P. 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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=Alpha-synuclein+blocks+ER-Golgi+traffic+and+Rab1+rescues+neuron+loss+in+Parkinson's+models.">Alpha-synuclein blocks ER-Golgi traffic and Rab1 rescues neuron loss in Parkinson's models.</a> Science. 313 (5785), 324-328 (2006).</li><li>Treusch, 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=Functional+links+between+Abeta+toxicity,+endocytic+trafficking,+and+Alzheimer's+disease+risk+factors+in+yeast.">Functional links between Abeta toxicity, endocytic trafficking, and Alzheimer's disease risk factors in yeast.</a> Science. 334 (6060), 1241-1245 (2011).</li><li>Ju, 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=A+yeast+model+of+FUS/TLS-dependent+cytotoxicity.">A yeast model of FUS/TLS-dependent cytotoxicity.</a> PLoS Biology. 9 (4), 1001052 (2011).</li><li>Sun, Z., 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=Molecular+determinants+and+genetic+modifiers+of+aggregation+and+toxicity+for+the+ALS+disease+protein+FUS/TLS.">Molecular determinants and genetic modifiers of aggregation and toxicity for the ALS disease protein FUS/TLS.</a> PLoS Biology. 9 (4), 1000614 (2011).</li><li>Johnson, B. S., McCaffery, J. M., Lindquist, S., Gitler, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+yeast+TDP-43+proteinopathy+model:+Exploring+the+molecular+determinants+of+TDP-43+aggregation+and+cellular+toxicity.">A yeast TDP-43 proteinopathy model: Exploring the molecular determinants of TDP-43 aggregation and cellular toxicity.</a> Proceedings of the National Academy of Sciences of the United States of America. 105 (17), 6439-6444 (2008).</li><li>Fleming, M. S., Gitler, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+yeast+plasmid+overexpression+screen.">High-throughput yeast plasmid overexpression screen.</a> Journal of Visualized Experiments. (53), e2836 (2011).</li><li>Herskowitz, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Life+cycle+of+the+budding+yeast+Saccharomyces+cerevisiae.">Life cycle of the budding yeast Saccharomyces cerevisiae.</a> Microbiological Reviews. 52 (4), 536-553 (1988).</li><li>Suter, B., Auerbach, D., Stagljar, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yeast-based+functional+genomics+and+proteomics+technologies:+the+first+15+years+and+beyond.">Yeast-based functional genomics and proteomics technologies: the first 15 years and beyond.</a> Biotechniques. 40 (5), 625-644 (2006).</li><li>Barmada, S. 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यहां, हम एक प्रोटोकॉल का वर्णन करने के लिए खमीर में एक प्लाज्मिड व्यक्त स्क्रीन संभोग का उपयोग करने खमीर मॉडल में प्लाज्मिड पुस्तकालय परिचय प्रदर्शन । इस दृष्टिकोण का उपयोग करना, neurodegenerative रोग प्रोटीन वि?...

नवोदित खमीर Saccharomyces cerevisiae व्यापक रूप से बुनियादी वैज्ञानिक अनुसंधान में प्रयोग किया गया है1 सेलुलर प्रक्रियाओं को समझने के लिए सीधे मानव रोगों से संबंधित । इसके अलावा, यह मानव रोग से जुड़े प्रोटीन का अध्ययन करने के लिए एक मॉडल जीव के रूप में इस्तेमाल किया गया है, ऐसे सबसे आम neurodegenerative रोगों से जुड़े लोगों के रूप में, अल्जाइमर रोग, पार्किंसंस रोग, Huntington की बीमारी, और पेशीशोषी सहित पार्श्व स्केलेरोसिस (ALS)2. खमीर मॉडल का एक लाभ के साथ आसानी से जो एक जीनोम के साथ व्यापक स्क्रीन को सेलुलर रास्ते रोग से संबंधित प्रोटीन की विषाक्तता से संबंधित पहचान प्रदर्शन किया जा सकता है, इस प्रकार उनके विषाक्तता के तंत्र में अंतर्दृष्टि दे रही है । ऐसा ही एक स्क्रीन एक एक्सप्रेस पुस्तकालय स्क्रीन, जिसमें एक सरणी पुस्तकालय में ५,५०० खमीर जीन के प्रत्येक एक खमीर मॉडल में तब्दील हो जाता है की पहचान करने के लिए जो जीन विषाक्तता को संशोधित कर सकते है जब व्यक्त की है । इस स्क्रीनिंग पद्धति को सफलतापूर्वक कई neurodegenerative रोग-संबद्ध प्रोटीन के खमीर मॉडल में लागू किया गया है, जिसमें Huntington के रोग के लिए huntingtin3, α-synuclein पार्किंसंस रोग के लिए4,5 , अल्जाइमर रोग के लिए Aβ6, और FUS और टीडीपी-४३ के लिए ALS7,8,9. हालांकि यह आमतौर पर एक उच्च प्रवाह तरीके10में किया जाता है, सबसे श्रम-स्क्रीन के गहन कदम व्यक्तिगत रूप से एक सरणी पुस्तकालय से ५,५०० खमीर जीन बदल रहा है । यह कदम हर बार स्क्रीनिंग दोहराया है प्रदर्शन किया जाना चाहिए, और जब भी एक नए स्थापित खमीर मॉडल का अध्ययन करने की जरूरत है । यह इस कार्य को पूरा करने के लिए एक अधिक कुशल तरीका खोजने के लिए महत्वपूर्ण है ।
खमीर कोशिकाओं अगुणित और द्विगुणित दोनों रूपों में छुरा मौजूद कर सकते हैं । अगुणित कोशिकाओं के दो विपरीत संभोग प्रकार हैं, संभोग प्रकार एक और α. अगुणित कोशिकाओं प्रत्येक संभोग प्रकार के उत्पादन और अपने स्वयं के विशिष्ट संभोग फेरोमोन, जो करने के लिए केवल विपरीत संभोग प्रकार कोशिकाओं का जवाब स्रावित । यह एक और α कोशिकाओं के बीच सहवास करने के लिए स्थिर द्विगुणित कोशिकाओं का उत्पादन की अनुमति देता है, एक/α । इस प्रक्रिया को सहज और अत्यधिक कुशल11है । प्लाज्मिड पुस्तकालय का परिचय देने के लिए हम एस cerevisiae के इस अनूठे जीवन चक्र का लाभ उठा सकते हैं । अधिक विशेष रूप से, सरणी प्लाज्मिड पुस्तकालय में प्रत्येक जीन एक संभोग प्रकार, यानी, α सेल के अगुणित कोशिकाओं में तब्दील हो जाएगा । पुस्तकालय जीन युक्त इन कोशिकाओं को तो ग्लिसरॉल स्टॉक में एक सरणी ९६-अच्छी तरह से प्रारूप में संग्रहीत किया जाएगा । प्रत्येक खमीर मॉडल है कि की जरूरत है के लिए जांच की, पुस्तकालय जीन युक्त खमीर कोशिकाओं ग्लिसरॉल स्टॉक से गल सकता है, और स्क्रीनिंग विपरीत संभोग प्रकार में ब्याज की खमीर मॉडल के साथ संभोग के माध्यम से किया जा सकता है, यानी, संभोग प्रकार एक । एक साथ खमीर में दो जीन लाने के लिए सहवास का उपयोग करने का यह विचार नया नहीं है. यह सफलतापूर्वक उच्च प्रवाह खमीर दो में लागू किया गया है संकर स्क्रीनिंग, जिसमें एक चारा का निर्माण (यानी, Gal4 डीएनए-एक संभोग प्रकार में बंधन डोमेन संलयन) एक शिकार के साथ सहवास के माध्यम से एक साथ लाया जाता है एक सरणी पुस्तकालय से निर्माण 12. हालांकि, इस रणनीति हमेशा पारंपरिक परिवर्तन तरीकों का इस्तेमाल किया है, जो व्यक्त पुस्तकालय की स्क्रीनिंग, में लागू नहीं किया गया है.
हमारी प्रयोगशाला पहले ALS के एक खमीर मॉडल-जुड़े प्रोटीन FUS7की स्थापना की । परिवर्तन विधि का उपयोग कर के माध्यम से व्यक्त पुस्तकालय स्क्रीनिंग हम पांच खमीर जीन की खोज (ECM32, NAM8, SBP1, SKO1, और VHR1) कि FUS के बचाव विषाक्तता जब व्यक्त । इन निष्कर्षों को स्वतंत्र रूप से एक और8समूह द्वारा एक समान अध्ययन के साथ पुष्टि की गई । hUPF1, ECM32के एक मानव homolog, बाद में प्राथमिक न्यूरॉन्स कोशिकाओं में विषाक्तता को दबाने के लिए दिखाया गया था13 और ALS के एक पशु मॉडल में14 के रूप में अच्छी तरह से. सिद्धांत के सबूत के रूप में इन पांच जीनों का प्रयोग, हम प्रदर्शित करता है कि सभी पांच जीन इसी तरह FUS विषाक्तता बचाव जब वे संभोग द्वारा FUS खमीर मॉडल में पेश कर रहे हैं । चूंकि खमीर पुस्तकालय जीन युक्त कोशिकाओं ग्लिसरॉल स्टॉक में स्थाई रूप से संग्रहीत किया जा सकता है और जब भी जरूरत पुनर्जीवित, इस संभोग आधारित विधि समय परिवर्तन की खपत कदम हर बार पुस्तकालय के खिलाफ जांच की जरूरत को हटा देंगे । संभोग के बाद से कोई प्लाज्मिड शामिल परिवर्तन के साथ अत्यधिक कुशल है, इस रणनीति भी काफी शोधन और एक बड़ी प्लाज्मिड पुस्तकालय के परिवर्तन के साथ जुड़े लागत कम हो जाती है । हम सफलतापूर्वक FUS के खमीर मॉडल के खिलाफ एक पुस्तकालय स्क्रीनिंग के लिए इस पद्धति को लागू करेगा ।
संभोग के लिए प्रक्रिया आधारित स्क्रीनिंग संक्षेप में चित्रा 1में वर्णित है । शुरू में, सरणी प्लाज्मिड पुस्तकालय संभोग प्रकार के एक अगुणित खमीर तनाव में तब्दील हो जाता है α एक उच्च प्रवाह खमीर परिवर्तन प्रोटोकॉल जिसमें एक ९६ के एक अच्छी तरह से प्लेट के प्रत्येक कुआं का उपयोग एक विशिष्ट पुस्तकालय प्लाज्मिड के साथ बदल खमीर शामिल हैं । रूपांतरित खमीर का यह संग्रह एक ग्लिसरॉल स्टॉक के रूप में सहेजा गया है जो बाद में उपयोग के लिए गल और पुनर्जीवित किया जा सकता है । ब्याज की खमीर मॉडल, इस मामले FUS विषाक्तता में, विपरीत संभोग प्रकार (संभोग प्रकार एक) के साथ एक अगुणित खमीर तनाव में उत्पंन किया जाना चाहिए । एक उच्च प्रवाह बाँझ ९६-पिन प्रतिकृतियों का उपयोग कर तरीके में, FUS तनाव और प्लाज्मिड पुस्तकालय युक्त खमीर उपभेदों ९६ के लिए स्थानांतरित कर रहे है अच्छी तरह से अमीर मीडिया युक्त प्लेटों और दोस्त को अनुमति दी । संभोग के बाद, संभोग संस्कृति का एक अच्छी तरह से एक छोटी सी मात्रा ९६ को हस्तांतरित-अच्छी तरह से सिंथेटिक dropout मीडिया जिसमें केवल द्विगुणित दोनों FUS और पुस्तकालय जीन युक्त खमीर विकसित कर सकते है युक्त प्लेट है । एक रोबोट खोलना मशीन तो प्रत्येक अच्छी तरह से जहां FUS की अभिव्यक्ति और पुस्तकालय जीन प्रेरित किया जाता है आगर प्लेटों पर से खमीर संस्कृति हस्तांतरण करने के लिए इस्तेमाल होता है ।  इसके अतिरिक्त, खमीर संस्कृति जहां FUS और पुस्तकालय जीन व्यक्त नहीं कर रहे है प्लेटों आगार नियंत्रित करने के लिए देखा जाता है । आगर प्लेटों पर विकास के बाद, जीन है कि बचाव या ख़राब FUS विषाक्तता की पहचान की जाएगी ।

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			<td height="42" style="height:42px;width:183px;">salmon Sperm DNA (SS-DNA)</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td style="width:99px;">&#160; D1626</td>
			<td style="width:147px;"></td>
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			<td height="84" style="height:84px;width:183px;">YPD broth</td>
			<td style="width:127px;">Research Products International (RPI)</td>
			<td style="width:99px;">Y20090</td>
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mating-based overexpression library screening in yeast

नवोदित खमीर व्यापक रूप से मानव रोगों के साथ जुड़े प्रोटीन का अध्ययन में एक मॉडल के रूप में इस्तेमाल किया गया है । जीनोम-वाइड आनुवंशिक स्क्रीनिंग एक शक्तिशाली आमतौर पर खमीर अध्ययन में प्रयोग किया जाता उपकरण है । neurodegenerative रोग के एक नंबर की अभिव्यक्ति खमीर में प्रोटीन जुड़े cytotoxicity और समग्र गठन, recapitulating इन विकारों के साथ रोगियों में देखा निष्कर्षों का कारण बनता है । यहां, हम अपनी विषाक्तता के संशोधक के लिए पेशीशोषी पार्श्व स्केलेरोसिस-जुड़े प्रोटीन FUS के एक खमीर मॉडल स्क्रीनिंग के लिए एक विधि का वर्णन । इसके बजाय परिवर्तन का उपयोग कर के, इस नए स्क्रीनिंग मंच खमीर के सहवास पर निर्भर करता है खमीर मॉडल में plasmids की एक सरणी पुस्तकालय परिचय । सहवास विधि में दो स्पष्ट लाभ हैं: पहला, यह अत्यधिक कुशल है; दूसरा, पूर्व plasmids की सरणी पुस्तकालय बदल एक ग्लिसरॉल शेयर के रूप में लंबी अवधि के लिए संग्रहीत किया जा सकता है, और जल्दी से श्रम के बिना अंय स्क्रीन पर लागू-खमीर मॉडल में परिवर्तन के गहन कदम हर बार । हम प्रदर्शन कैसे इस विधि सफलतापूर्वक जीन है कि FUS की विषाक्तता को संशोधित करने के लिए स्क्रीन करने के लिए इस्तेमाल किया जा सकता है ।

ALS-जुड़े प्रोटीन FUS, एक आरएनए/डीएनए बाइंडिंग प्रोटीन, पहले अगुणित खमीर7,8में अध्ययन किया गया था । आनुवंशिक परिवर्तन-विधि आधारित स्क्रीनिंग का उपयोग कई खमीर जीन है कि FUS वि...

Watch this Scientific Journal Video about Mating-based Overexpression Library Screening in Yeast at JoVE.com

Mating-based Overexpression Library Screening in Yeast

यह लेख एक संभोग आधारित विधि प्रस्तुत करने के लिए नवोदित खमीर में व्यक्त स्क्रीनिंग की सुविधा एक सरणी प्लाज्मिड पुस्तकालय का उपयोग कर ।

सहवास आधारित खमीर में एक्सप्रेस पुस्तकालय स्क्रीनिंग

Budding yeast has been widely used as a model in studying proteins associated with human diseases. Genome-wide genetic screening is a powerful tool ...

This method can help answer key questions in the neurodegenerative disease field, such as what genetic factors and pathways regulate the toxicity of disease-associated proteins. The main advantage of this technique is that the highly efficient process of yeast mating is used to screen yeast models against a large collection of library genes. Though this method can provide insight into the toxicity of neurodegenerative disease proteins, it can also be applied to the study of other cellular processes in yeast, such as tolerance of other stress conditions.Begin this procedure by aliquoting five microliters per well of plasmid DNA from an arrayed plasmid library to round bottom 96-well plates. Keep the plates at four degrees Celsius. Next, inoculate 150 milliliters of yeast peptone dextrose or YPD medium in a 500 milliliter flask with a colony of the haploid yeast strain W303 Alpha.Incubate the culture at 30 degrees Celsius with shaking at 200 rpm overnight. On the following morning, after measuring the OD600 of the overnight culture, dilute the culture to an OD600 of 0.1 in two liters of YPD. Incubate at 30 degrees Celsius with shaking at 200 rpm for about five hours, until the culture reaches an OD600 of 0.4 to 0.6.To harvest the yeast culture, fill eight sterile 250 milliliter centrifuge bottles and centrifuge at 3000 xG for 10 minutes at room temperature. Pour off the supernatant without disrupting the pellet. Wash the yeast as described in the text protocol, and consolidate the cells into two centrifuge tubes.Resuspend each cell pellet in 25 milliliters of a 0.1 molar lithium acetate solution. Combine the resuspended cells, add five milliliters of salmon sperm DNA, and transfer them to a 150 milliliter flask. Incubate at 30 degrees Celsius with shaking at 225 rpm for 30 minutes.After 30 minutes, pour the cell mixture into a sterile disposable reagent reservoir. Using a multichannel pipette, transfer 35 microliters of the cell mixture to each well of the round bottom 96-well plates, containing the library plasmid DNA prepared earlier. Vortex the 96-well plates at 1000 rpm for one minute.Incubate the plates at 30 degrees Celsius without shaking for 30 minutes. Do not stack the plates, so the heat can transfer more efficiently. Remove the 96-well plates from the 30 degree Celsius incubator and mix them at 1000 rpm for 30 seconds.Add 125 microliters of transformation buffer to each well, and then vortex the plates at 1000 rpm for one minute. Incubate the plates at 30 degrees Celsius for 30 minutes. Then heat shock the yeast by placing the plates in a 42 degree Celsius incubator for 15 minutes.Centrifuge the plates at 3000 xG for five minutes. Remove the transformation buffer from the wells by inverting the plates over a waste bin and forcefully dumping the buffer from the plates. Quickly blot the inverted plates on a clean paper towel to remove any liquid on top of the plates.Rinse the cells by adding to each well, 200 microliters of minimal dropout medium corresponding to the selectable marker on the library plasmid. Synthetic ura minus medium is used here. Centrifuge the plates at 3000 xG for five minutes.Remove the supernatant over a waste bin as demonstrated earlier. Add 160 microliters of minimal ura minus medium, containing 2%glucose to each well and vortex the plates at 1000 rpm for one minute. Incubate the plates without shaking at 30 degrees Celsius for 48 hours.After 48 hours, a pellet of transformed yeast should be visible at the bottom of each well. Using a liquid dispenser, add 100 microliters of ura minus medium containing glucose into each well of a new set of 96-well plates. Vortex the plates containing the transformed yeast at 1000 rpm for 30 seconds.Using a sterile plastic 96-pin replicator, place the pins into the wells containing the transformed yeast and then inoculate them into the corresponding wells of the new plates filled with media. Incubate the new plates at 30 degrees Celsius for 24 hours. After 24 hours, a pellet of yeast should be visible at the bottom of each well that contains successfully transformed yeast.To save these yeast strains as glycerol stocks, add 50 microliters of 50%glycerol to each well, vortex the plates at 1000 rpm for 30 seconds, seal the plates with aluminum foil, and freeze at minus 80 degrees Celsius. To revive the library strains from the glycerol stock, take the 96-well plates out of the minus 80 degrees Celsius freezer, remove the aluminum foil, and let the yeast thaw at room temperature for approximately 30 minutes. Once the yeast has thawed, use a sterile plastic 96 pin replicator to inoculate the glycerol stocks to 160 microliters of fresh ura minus media containing 2%glucose in 96-well plates and incubate at 30 degrees Celsius for 24 hours.Immediately after the library strains are used, seal the plates, and return them to the minus 80 degrees Celsius freezer. On the same day that the library strains are thawed, inoculate the query yeast strain in 50 milliliters of YPD and grow at 30 degrees Celsius with shaking at 250 rpm overnight. On the following morning, pour the query yeast strain into a sterile disposable reagent reservoir and aliquot 160 microliters of the query strain to each well of a 96-well plate.Using the liquid dispenser, dispense 160 microliters per well of YPD media into 96-well plates. Briefly vortex the 96-well plates containing the query strain and the library strain plates and then use a sterile 96-pin replicator to transfer the library strains to the YPD plates that have been inoculated with the query strain. Incubate the YPD plates at 30 degrees Celsius for 24 hours.After 24 hours a pellet of yeast will be visible at the bottom of each well. Fill 96-well plates with minimal droupout medium containing 2%Raffinose corresponding to the selectable markers on the plasmid in the query strain, as well as on the library plasmid. Use a sterile 96-pin replicator to transfer yeast from the YPD mating cultures to the selective medium.Incubate the 96-well plates at 30 degrees Celsius for 48 hours. Only yeast cells that have mated and formed diploid cells and therefore contain both the query plasmid and the library plasmid will be able to grow in this medium. After 48 hours of growth in the Raffinose containing selective medium, observe that a pellet is visible at the bottom of the wells that contain diploid cells.Vortex the 96-well plates at 1000 rpm for one minute. Use a robotic spotting machine to spot the yeast on agar plates in quadruplicate to ura-his-droupout plates containing 2%galactose and 2%agar, and to ura-his-droupout plates containing 2%glucose and 2%agar. After spotting, let the agar plates dry and then place them upside down in a 30 degrees Celsius incubator for four days.Every 24 hours, photograph the agar plates to record the growth of the yeast. Toxicity of the ALS associated protein FUS was first examined in diploid yeast. As indicated by the agar plate containing galactose, which induces FUS, FUS toxicity is consistent in both diplod and haploid yeast.Five genes previously identified as suppressors of FUS toxicity from a transformation based method was tested by the mating method. Lane one is a control yeast strain transformed with two empty vectors. Lane two is the diploid FUS yeast strain with an empty vector where the expression of FUS is very toxic.Lanes three to seven show diploid yeast expressing FUS as well as a suppression gene. All five genes rescue the toxicity of FUS in diploid yeast, indicating that the mating method was effective. This mating based method was then applied to an over-expression library screening of 940 genes.And a photo of one representative plate is shown. As indicated on the galactose plate, where FUS and library genes are expressed, FUS was toxic to the diplod yeast cell. The library gene, indicated by the green square, rescued FUS toxicity.While the library gene, indicated by the red square, enhanced toxicity. Once mastered, this technique can be done in 120 hours, if it is performed properly. While attempting this procedure, it is important to remember to check beforehand that the phenotype used for screening is not haploid mating type dependent.After watching this video, you should have a good understanding of how to identify genetic factors upon overexpression that rescue the toxicity of disease associated proteins in yeast. After its development, this technique can pave the way for researchers in the field of neurodegenerative disease, to explore mechanisms of cytotoxicity of disease associated proteins in the yeast model. Following this procedure, other methods such as the beta galactosidase assay can be performed in order to answer additional questions, like whether the rescue is specific.

mating-based-overexpression-library-screening-in-yeast

Research

JoVE Journal

Genetics

Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast

We describe a PCR- and homologous recombination-based system for generating targeted mutations in histone genes in budding yeast cells. The resulting mutant alleles reside at their endogenous genomic sites and no exogenous DNA sequences are left in the genome following the procedure. Since in haploid yeast cells each of the four core histone proteins is encoded by two non-allelic genes with highly homologous open reading frames (ORFs), targeting mutagenesis specifically to one of two genes encoding a particular histone protein can be problematic. The strategy we describe here bypasses this problem by utilizing sequences outside, rather than within, the ORF of the target genes for the homologous recombination step. Another feature of this system is that the regions of DNA driving the homologous recombination steps can be made to be very extensive, thus increasing the likelihood of successful integration events. These features make this strategy particularly well-suited for histone gene mutagenesis, but can also be adapted for mutagenesis of other genes in the yeast genome.

Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast

A strategy for generating mutations in histone genes at their endogenous location in Saccharomyces cerevisiae is presented.

लक्षित<em&gt; बगल में</emनवोदित खमीर में&gt; mutagenesis Histone के जीन

We describe a PCR- and homologous recombination-based system for generating targeted mutations in histone genes in budding yeast cells. The resulting ...

Gene-targeted Random Mutagenesis to Select Heterochromatin-destabilizing Proteasome Mutants in Fission Yeast

Random mutagenesis of a target gene is commonly used to identify mutations that yield the desired phenotype. Of the methods that may be used to achieve random mutagenesis, error-prone PCR is a convenient and efficient strategy for generating a diverse pool of mutants (i.e., a mutant library). Error-prone PCR is the method of choice when a researcher seeks to mutate a pre-defined region, such as the coding region of a gene while leaving other genomic regions unaffected. After the mutant library is amplified by error-prone PCR, it must be cloned into a suitable plasmid. The size of the library generated by error-prone PCR is constrained by the efficiency of the cloning step. However, in the fission yeast, Schizosaccharomyces pombe, the cloning step can be replaced by the use of a highly efficient one-step fusion PCR to generate constructs for transformation. Mutants of desired phenotypes may then be selected using appropriate reporters. Here, we describe this strategy in detail, taking as an example, a reporter inserted at centromeric heterochromatin.

This article describes a detailed methodology for random mutagenesis of a target gene in fission yeast. As an example, we target rpt4+, which encodes a subunit of the 19S proteasome, and screen for mutations that destabilize heterochromatin.

जीन लक्षित यादृच्छिक Mutagenesis का चयन करने के लिए Heterochromatin-स्थिर Proteasome म्यूटेंट विखंडन खमीर में

Random mutagenesis of a target gene is commonly used to identify mutations that yield the desired phenotype. Of the methods that may be used to achieve ...

Ultralow Input Genome Sequencing Library Preparation from a Single Tardigrade Specimen

Tardigrades are microscopic animals that enter an ametabolic state called anhydrobiosis when facing desiccation and can return to their original state when water is supplied. The genomic sequencing of microscopic animals such as tardigrades risks bacterial contamination that sometimes leads to erroneous interpretations, for example, regarding the extent of horizontal gene transfer in these animals. Here, we provide an ultralow input method to sequence the genome of the tardigrade, Hypsibius dujardini, from a single specimen. By employing rigorous washing and contaminant exclusion along with an efficient extraction of the 50 ~ 200 pg genomic DNA from a single individual, we constructed a library sequenced with a DNA sequencing instrument. These libraries were highly reproducible and unbiased, and an informatics analysis of the sequenced reads with other H. dujardini genomes showed a minimal amount of contamination. This method can be applied to unculturable tardigrades that could not be sequenced using previous methods.

Contamination during the genomic sequencing of microscopic organisms remains a large problem. Here, we show a method to sequence the genome of a tardigrade from a single specimen with as little as 50 pg of genomic DNA without whole genome amplification to minimize the risk of contamination.

एक एकल Tardigrade नमूना से Ultralow इनपुट जीनोम अनुक्रमण पुस्तकालय तैयारी

Tardigrades are microscopic animals that enter an ametabolic state called anhydrobiosis when facing desiccation and can return to their original state ...

Single Molecule Fluorescence In Situ Hybridization (smFISH) Analysis in Budding Yeast Vegetative Growth and Meiosis

Single molecule fluorescence in situ hybridization (smFISH) is a powerful technique to study gene expression in single cells due to its ability to detect and count individual RNA molecules. Complementary to deep sequencing-based methods, smFISH provides information about the cell-to-cell variation in transcript abundance and the subcellular localization of a given RNA. Recently, we have used smFISH to study the expression of the gene&#160;NDC80&#160;during meiosis in budding yeast, in which two transcript isoforms exist and the short transcript isoform has its entire sequence shared with the long isoform. To confidently identify each transcript isoform, we optimized known smFISH protocols and obtained high consistency and quality of smFISH data for the samples acquired during budding yeast meiosis. Here, we describe this optimized protocol, the criteria that we use to determine whether high quality of smFISH data is obtained, and some tips for implementing this protocol in&#160;other yeast strains and growth conditions.

Single Molecule Fluorescence In Situ Hybridization smFISH Analysis in Budding Yeast Vegetative Growth and Meiosis

This single molecule fluorescence in situ hybridization protocol is optimized to quantify the number of RNA molecules in budding yeast during vegetative growth and meiosis.

एकल अणु प्रतिदीप्ति में सीटू संकरण (smFISH) विश्लेषण में नवोदित खमीर वनस्पति विकास और अर्धसूत्रीविभाजन

Single molecule fluorescence in situ hybridization (smFISH) is a powerful technique to study gene expression in single cells due to its ability to detect ...

Characterizing Histone Post-translational Modification Alterations in Yeast Neurodegenerative Proteinopathy Models

Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson&#39;s disease (PD), cause the loss of hundreds of thousands of lives each year. Effective treatment options able to halt disease progression are lacking. Despite the extensive sequencing efforts in large patient populations, the majority of ALS and PD cases remain unexplained by genetic mutations alone. Epigenetics mechanisms, such as the post-translational modification of histone proteins, may be involved in neurodegenerative disease etiology and progression and lead to new targets for pharmaceutical intervention. Mammalian in vivo and in vitro models of ALS and PD are costly and often require prolonged and laborious experimental protocols. Here, we outline a practical, fast, and cost-effective approach to determining genome-wide alterations in histone modification levels using Saccharomyces cerevisiae as a model system. This protocol allows for comprehensive investigations into epigenetic changes connected to neurodegenerative proteinopathies that corroborate previous findings in different model systems while significantly expanding our knowledge of the neurodegenerative disease epigenome.

This protocol outlines experimental procedures to characterize genome-wide changes in the levels of histone post-translational modifications (PTM) occurring in connection with the overexpression of proteins associated with ALS and Parkinson&#39;s disease in Saccharomyces cerevisiae models. After SDS-PAGE separation, individual histone PTM levels are detected with modification-specific antibodies via Western blotting.

विशेषता हिस्one पोस्ट-translational में संशोधन परिवर्तन खमीर न्यूरोडेजेनेरेटिव प्रोटिनोपैथी मॉडल

Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson&#39;s disease (PD), cause the loss of hundreds of thousands of lives ...

A Deep-sequencing-assisted, Spontaneous Suppressor Screen in the Fission Yeast Schizosaccharomyces pombe

A genetic screen for mutant alleles that suppress phenotypic defects caused by a mutation is a powerful approach to identify genes that belong to closely related biochemical pathways. Previous methods such as the Synthetic Genetic Array (SGA) analysis, and random mutagenesis techniques using ultraviolet (UV) or chemicals like ethyl methanesulfonate (EMS) or N-ethyl-N- nitrosourea (ENU), have been widely used but are often costly and laborious. Also, these mutagen-based screening methods are frequently associated with severe side effects on the organism, inducing multiple mutations that add to the complexity of isolating the suppressors. Here, we present a simple and effective protocol to identify suppressor mutations in mutants which confer a growth defect in Schizosaccharomyces pombe. The fitness of cells with a growth deficiency in standard rich liquid media or synthetic liquid media can be monitored for recovery using an automated 96-well plate reader over an extended period. Once a cell acquires a suppressor mutation in the culture, its descendants outcompete those of the parental cells. The recovered cells that have a competitive growth advantage over the parental cells can then be isolated and backcrossed with the parental cells. The suppressor mutations are then identified using whole-genome sequencing. Using this approach, we have successfully isolated multiple suppressors that alleviate the severe growth defects caused by loss of Elf1, an AAA+ family ATPase that is important in nuclear mRNA transport and maintenance of genomic stability. There are currently over 400 genes in S. pombe with mutants conferring a growth defect. As many of these genes are uncharacterized, we propose that our method will hasten the identification of novel functional interactions with this user-friendly, high-throughput approach.

A Deep-sequencing-assisted, Spontaneous Suppressor Screen in the Fission Yeast Schizosaccharomyces pombe

We present a simple suppressor screen protocol in fission yeast. This method is efficient, mutagen-free, and&#160; selective&#160; for&#160; mutations that&#160; often&#160; occur at&#160; &#160;a&#160; single&#160; genomic&#160; locus.&#160; The protocol is suitable for isolating suppressors that alleviate growth defects in liquid culture that are caused by a mutation or a drug.

विखंडन खमीर schizosaccharomyces pombe में एक गहरी sequencing-असिस्टेड, सहज दबानेवाला स्क्रीन

A genetic screen for mutant alleles that suppress phenotypic defects caused by a mutation is a powerful approach to identify genes that belong to closely ...

HOX Loci Focused CRISPR/sgRNA Library Screening Identifying Critical CTCF Boundaries

CCCTC-binding factor (CTCF)-mediated stable topologically associating domains (TADs) play a critical role in constraining interactions of DNA elements that are located in neighboring TADs. CTCF plays an important role in regulating the spatial and temporal expression of HOX genes that control embryonic development, body patterning, hematopoiesis, and leukemogenesis. However, it remains largely unknown whether and how HOX loci associated CTCF boundaries regulate chromatin organization and HOX gene expression. In the current protocol, a specific sgRNA pooled library targeting all CTCF binding sites in the HOXA/B/C/D loci has been generated to examine the effects of disrupting CTCF-associated chromatin boundaries on TAD formation and HOX gene expression. Through CRISPR-Cas9 genetic screening, the CTCF binding site located between HOXA7/HOXA9 genes (CBS7/9) has been identified as a critical regulator of oncogenic chromatin domain, as well as being important for maintaining ectopic HOX gene expression patterns in MLL-rearranged acute myeloid leukemia (AML). Thus, this sgRNA library screening approach provides novel insights into CTCF mediated genome organization in specific gene loci and also provides a basis for the functional characterization of the annotated genetic regulatory elements, both coding and noncoding, during normal biological processes in the post-human genome project era.

HOX Loci Focused CRISPR/sgRNA Library Screening Identifying Critical CTCF Boundaries

A CRISPR/sgRNA library has been applied to interrogating protein-coding genes.However, the feasibility of a sgRNA library to uncover the function of a CTCF boundary in gene regulation remains unexplored. Here, we describe a HOX loci specific sgRNA library to elucidate the function of CTCF boundaries in HOX loci.

Hox Loci केंद्रित CRISPR/sgRNA पुस्तकालय स्क्रीनिंग महत्वपूर्ण CTCF सीमाओं की पहचान

CCCTC-binding factor (CTCF)-mediated stable topologically associating domains (TADs) play a critical role in constraining interactions of DNA elements ...

ACT1-CUP1 Assays Determine the Substrate-Specific Sensitivities of Spliceosomal Mutants in Budding Yeast

Mutations introduced in the spliceosome or its substrate have significantly contributed to our understanding of the intricacies of spliceosomal function. Whether disease-related or functionally selected, many of these mutations have been studied using growth assays in the model organism Saccharomyces cerevisiae (yeast). The splicing-specific copper growth assay, or ACT1-CUP1 assay, provides a comprehensive analysis of mutation at the phenotypic level. The ACT1-CUP1 assay utilizes reporters that confer copper tolerance when correctly spliced. Thus, in the presence of copper, changes in yeast viability correlate to changes in mRNA production through splicing. In a typical experiment, the yeast spliceosome is challenged with different non-consensus splicing reporters and the splicing factor mutation of interest to detect any synergetic or antithetical impact on splicing. Here a full description of copper plate preparation, plating of yeast cells, and data evaluation are given. A selection of complimentary experiments is described, highlighting the versatility of the ACT1-CUP1 reporters. The ACT1-CUP1 assay is a handy tool in the splicing toolbox thanks to the direct read-out of mutational effect(s) and the comparative possibilities from the continuing use in the field.

The ACT1-CUP1 assay, a copper growth assay, provides a quick readout of precursor messenger RNA (pre-mRNA) splicing and the impact mutant splicing factors have on spliceosomal function. This study provides a protocol and highlights the customization possible to address the splicing question of interest.

ACT1-CUP1 परख नवोदित खमीर में स्प्लिसोसोमल उत्परिवर्ती की सब्सट्रेट-विशिष्ट संवेदनशीलता निर्धारित करता है

Mutations introduced in the spliceosome or its substrate have significantly contributed to our understanding of the intricacies of spliceosomal function. ...

Screening Sperm for the Rapid Isolation of Germline Edits in Zebrafish

The advent of targeted CRISPR-Cas nuclease technologies has revolutionized the ability to perform precise genome editing in both established and emerging model systems. CRISPR-Cas genome editing systems use a synthetic guide RNA (sgRNA) to target a CRISPR-associated (Cas) endonuclease to specific genomic DNA loci, where the Cas endonuclease generates a double-strand break. The repair of double-strand breaks by intrinsic error-prone mechanisms leads to insertions and/or deletions, disrupting the locus. Alternatively, the inclusion of double-stranded DNA donors or single-stranded DNA oligonucleotides in this process can elicit the inclusion of precise genome edits ranging from single nucleotide polymorphisms to small immunological tags or even large fluorescent protein constructs. However, a major bottleneck in this procedure can be finding and isolating the desired edit in the germline. This protocol outlines a robust method for screening and isolating germline mutations at specific loci in Danio rerio (zebrafish); however, these principles may be adaptable in any model where in vivo sperm collection is possible.

CRISPR-Cas technologies have revolutionized the field of genome editing. However, finding and isolating the desired germline edit remains a major bottleneck. Therefore, this protocol describes a robust method for quickly screening F0 CRISPR-injected zebrafish sperm for germline edits using standard PCR, restriction digest, and gel electrophoresis techniques.

ज़ेबराफ़िश में जर्मलाइन एडिट के तेजी से अलगाव के लिए स्क्रीनिंग शुक्राणु

The advent of targeted CRISPR-Cas nuclease technologies has revolutionized the ability to perform precise genome editing in both established and emerging ...

Generation and Purification of Chromosome Conformation Capture (3C) Library

On day one after chromatin cross-linking and nuclei collection from 5, 000 young adult Caenorhabditis Elegans worms, transfer the nuclei pellet re-suspended in 450 microliters of clean water to a clean 1.5 milliliter micro centrifuge tube. Then add 60 microliters of 10X DPN2 restriction enzyme buffer to it and mix well. Incubate the samples with 15 microliters of 10%sodium dodecyl sulfate at 37 degrees Celsius for one hour with agitation.Quench the reaction by adding 75 microliters of 20%triton X 100 and incubating as demonstrated previously. Aliquot 10 microliters from the sample, as the undigested control, and store it at four degrees Celsius. After adding 400 units of DPN 2 to the rest of the sample, incubate it overnight at 37 degrees Celsius with agitation for chromatin digestion.On day two, add an additional 200 units of DPN 2 to the sample. To complete the digestion of the cross-linked sample, incubate it at 37 degrees Celsius with agitation for four hours. Heat inactivate the restriction enzyme by incubating the sample for 20 minutes at 65 degrees Celsius.If the enzyme cannot be inactivated by heat, add 80 microliters of 10%sodium dodecyl sulfate and incubate. Then, add 375 microliters of 20%Triton X 100 to the sample and mix by swirling. Keep aside a 10 microliter aliquot from the sample as the digestion control.For chromatin ligation, transfer the sample to a clean 50 milliliter conical tube. Adjust the sample volume to 5.7 milliliters with molecular grade water and mix by swirling. Then, add 700 microliters of 10 X T4 ligase buffer, followed by 60 units of T4 DNA ligase, and incubate the sample overnight at 16 degrees Celsius.On day three, proceed with protein digestion and reverse cross-linking. Add 30 microliters of 10 milligrams per milliliter Proteinase K to the 3C sample and incubate at 65 degrees Celsius overnight with agitation. To begin purification of the 3C library on day four, add 30 microliters of 10 milligrams per milliliter RNase A to the sample and mix by swirling.After incubation, add seven milliliters of phenyl chloroform to the samples and mix by shaking. Centrifuge the sample for 15 minutes at 3, 270 G and room temperature. Collect and transfer the aqueous phase to a clean 50 milliliter conical tube.Add equal volumes of chloroform and mix the sample by shaking. After centrifuging the sample again as demonstrated, transfer the aqueous phase to another clean 50 milliliter conical tube. Add 7.5 milliliters of molecular grade water, 35 milliliters of 100%ethanol and seven microliters of one milligram per milliliter Glycogen.Freeze the properly mixed sample at minus 80 degrees Celsius. While the sample freezes, begin purifying the control samples by adjusting the volume of the controls to 500 microliters using molecular grade water. Add two microliters of 10 milligrams per milliliter RNase A and mix by flicking the tube.Next, add one milliliter of phenyl chloroform to the tubes containing the controls and mix by shaking. Centrifuge the tubes. After transferring the aqueous phase to a clean 1.5 milliliter micro centrifuge tube, add equal volumes of chloroform.Centrifuge as previously demonstrated before collecting the aqueous phase. To the collected aqueous phase, add one milliliter of ethanol and two microliters of one milligram per milliliter glycogen. After mixing the sample by shaking, incubate it at minus 80 degrees Celsius for 30 minutes.Next, centrifuge the sample for 15 minutes at 3, 270 G at four degrees Celsius. After removing the supernatant, add 750 microliters of chilled 70%ethanol and centrifuge. Re-suspend the air dried pellet in 50 microliters of molecular grade water.The suspension can be frozen here if not proceeding further immediately. To continue with the 3C sample purification, remove the sample from the freezer and centrifuge at 3, 270 G for 30 minutes. Add 10 milliliters of chilled 70%ethanol and break up the DNA pellet.After centrifuging and removing the supernatant, partially air dry the sample at room temperature. Re suspend the pellet in 150 microliters of 10 millimolar tris HCL at pH 7.5 by pipetting up and down to obtain the purified 3C library. QPCR performed on the one experimental and two control 3C samples helped generate the digestion efficiency data.The 3C sample had an approximately 88%digestion efficiency across the seven genomic loci tested.