Dr. David O'Brochta'ya ve Böcek Genetik Teknolojileri Koordinasyon Araştırma Ağı'nın tüm üyelerine genetik teknolojilerin uygulanması konusunda bize ve diğerlerine sağladıkları yardım ve eğitim için teşekkür ederiz. Özellikle Channa Aluvihare'ye Culex embriyolarının enjekte edilmesine ve yumurtadan çıkmasına izin vermek için mikro yönetim protokolünü optimize ettiği için teşekkür ediyoruz. Ayrıca, Meuti laboratuvarında çalışan lisans öğrencileri Devante Simmons ve Joseph Urso'ya transgenik sivrisineklerin bakım ve taramalarına yardımcı olmaları için ve ITF'den Zora Elmkami'ye sivrisineklerin enjeksiyon için yetiştirilmelerine ve hazırlanmasına yardımcı olmaları için teşekkür ederiz. Bu çalışma, MEM'e sağlanan OSU'daki Bulaşıcı Hastalıklar Enstitüsü'nden disiplinler arası tohumlar hibesi ile desteklendi.

<ol>
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L., 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=Isolation+of+St.+Louis+encephalitis+virus+from+overwintering+Culex+pipiens+mosquitoes.">Isolation of St. Louis encephalitis virus from overwintering Culex pipiens mosquitoes.</a> Science. 199 (4335), 1346-1349 (1978).</li><li>Sabry, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+realistic+index+of+experimental+transmission+efficiency+for+Bancroftian+filariasis.">A new realistic index of experimental transmission efficiency for Bancroftian filariasis.</a> The Journal of Tropical Medicine and Hygiene. 94 (4), 283-290 (1991).</li><li>Cancrini, G., 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=Aedes+albopictus+and+Culex+pipiens+implicated+as+natural+vectors+of+Dirofilaria+repens+in+central+Italy.">Aedes albopictus and Culex pipiens implicated as natural vectors of Dirofilaria repens in central Italy.</a> Journal of Medical Entomology. 44 (6), 1064-1066 (2007).</li><li>Wilton, D. 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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+temperature+and+photoperiod+on+blood-feeding+and+ovarian+development+in+mosquitoes+of+the+Culex+pipiens+complex.">The effect of temperature and photoperiod on blood-feeding and ovarian development in mosquitoes of the Culex pipiens complex.</a> The American Journal of Tropical Medicine and Hygiene. 17 (1), 133-140 (1968).</li><li>Spielman, A., Wong, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environmental+control+of+ovarian+diapause+in+Culex+pipiens.">Environmental control of ovarian diapause in Culex pipiens.</a> Annals of the Entomological Society of America. 66 (4), 905-907 (1973).</li><li>Fritz, M. L., Walker, E. D., Miller, J. R., Severson, D. W., Dworkin, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Divergent+host+preferences+of+above-and+below-ground+Culex+pipiens+mosquitoes+and+their+hybrid+offspring.">Divergent host preferences of above-and below-ground Culex pipiens mosquitoes and their hybrid offspring.</a> Medical and Veterinary Entomology. 29 (2), 115-123 (2015).</li><li>Meola, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+temperature+and+humidity+on+embryonic+longevity+in+Aedes+aegypti.">The influence of temperature and humidity on embryonic longevity in Aedes aegypti.</a> Annals of the Entomological Society of America. 57 (4), 468-472 (1964).</li><li>Dong, S., Lin, J., Held, N. L., Clem, R. J., Passarelli, A. L., Franz, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heritable+CRISPR/Cas9-mediated+genome+editing+in+the+yellow+fever+mosquito,+Aedes+aegypti.">Heritable CRISPR/Cas9-mediated genome editing in the yellow fever mosquito, Aedes aegypti.</a> PloS one. 10 (3), (2015).</li><li>Kyrou, 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=A+CRISPR-Cas9+gene+drive+targeting+doublesex+causes+complete+population+suppression+in+caged+Anopheles+gambiae+mosquitoes.">A CRISPR-Cas9 gene drive targeting doublesex causes complete population suppression in caged Anopheles gambiae mosquitoes.</a> Nature Biotechnology. 36 (11), 1062-1066 (2018).</li><li>Liu, T., 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=Construction+of+an+efficient+genomic+editing+system+with+CRISPR/Cas9+in+the+vector+mosquito+Aedes+albopictus.">Construction of an efficient genomic editing system with CRISPR/Cas9 in the vector mosquito Aedes albopictus.</a> Insect Science. 26 (6), 1045-1054 (2019).</li><li>Itokawa, K., Komagata, O., Kasai, S., Ogawa, K., Tomita, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Testing+the+causality+between+CYP9M10+and+pyrethroid+resistance+using+the+TALEN+and+CRISPR/Cas9+technologies.">Testing the causality between CYP9M10 and pyrethroid resistance using the TALEN and CRISPR/Cas9 technologies.</a> Scientific Reports. 6, 24652 (2016).</li><li>Li, M., Li, T., Liu, N., Raban, R. R., Wang, X., Akbari, O. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+the+generation+of+heritable+germline+mutations+in+the+disease+vector+Culex+quinquefasciatus+using+clustered+regularly+interspaced+short+palindrome+repeats-associated+protein+9.">Methods for the generation of heritable germline mutations in the disease vector Culex quinquefasciatus using clustered regularly interspaced short palindrome repeats-associated protein 9.</a> Insect Molecular Biology. 29, 214-220 (2019).</li><li>Anderson, M. E., 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=CRISPR/Cas9+gene+editing+in+the+West+Nile+Virus+vector,+Culex+quinquefasciatus+Say.">CRISPR/Cas9 gene editing in the West Nile Virus vector, Culex quinquefasciatus Say.</a> PloS one. 14 (11), (2019).</li><li>Van der Oost, J., Jore, M. M., Westra, E. R., Lundgren, M., Brouns, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR-based+adaptive+and+heritable+immunity+in+prokaryotes.">CRISPR-based adaptive and heritable immunity in prokaryotes.</a> Trends in Biochemical Sciences. 34 (8), 401-407 (2009).</li><li>Heyer, W. D., Ehmsen, K. T., Liu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+homologous+recombination+in+eukaryotes.">Regulation of homologous recombination in eukaryotes.</a> Annual Review of Genetics. 44, 113-139 (2010).</li><li>Monnerat, A. T., 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=Anopheles+albitarsis+embryogenesis:+morphological+identification+of+major+events.">Anopheles albitarsis embryogenesis: morphological identification of major events.</a> Memorias do Instituto Oswaldo Cruz. 97 (4), 589-596 (2002).</li><li>Davis, C. W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparative+study+of+larval+embryogenesis+in+the+mosquito+Culex+fatigans+Wiedemann+(Diptera:+Culicidae)+and+the+sheep-fly+Lucilia+sericata+Meigen+(Diptera:+Calliphoridae).">A comparative study of larval embryogenesis in the mosquito Culex fatigans Wiedemann (Diptera: Culicidae) and the sheep-fly Lucilia sericata Meigen (Diptera: Calliphoridae).</a> Australian Journal of Zoology. 15 (3), 547-579 (1967).</li><li>Port, F., Chen, H. M., Lee, T., Bullock, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+CRISPR/Cas+tools+for+efficient+germline+and+somatic+genome+engineering+in+Drosophila.">Optimized CRISPR/Cas tools for efficient germline and somatic genome engineering in Drosophila.</a> Proceedings of the National Academy of Sciences. 111 (29), 2967-2976 (2014).</li><li>Kistler, K. E., Vosshall, L. B., Matthews, B. 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+engineering+with+CRISPR-Cas9+in+the+mosquito+Aedes+aegypti.">Genome engineering with CRISPR-Cas9 in the mosquito Aedes aegypti.</a> Cell Reports. 11 (1), 51-60 (2015).</li><li>Gokcezade, J., Sienski, G., Duchek, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+CRISPR/Cas9+plasmids+for+rapid+and+versatile+genome+editing+in+Drosophila.">Efficient CRISPR/Cas9 plasmids for rapid and versatile genome editing in Drosophila.</a> G3: Genes, Genomes, Genetics. 4 (11), 2279-2282 (2014).</li><li>Hwang, W. 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=Efficient+in+vivo+genome+editing+using+RNA-guided+nucleases.">Efficient in vivo genome editing using RNA-guided nucleases.</a> Nature Biotechnology. 31 (3), 227-229 (2013).</li><li>Basu, 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=Silencing+of+end-joining+repair+for+efficient+site-specific+gene+insertion+after+TALEN/CRISPR+mutagenesis+in+Aedes+aegypti.">Silencing of end-joining repair for efficient site-specific gene insertion after TALEN/CRISPR mutagenesis in Aedes aegypti.</a> Proceedings of the National Academy of Sciences. 112 (13), 4038-4043 (2015).</li><li>Hammond, 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=A+CRISPR-Cas9+gene+drive+system+targeting+female+reproduction+in+the+malaria+mosquito+vector+Anopheles+gambiae.">A CRISPR-Cas9 gene drive system targeting female reproduction in the malaria mosquito vector Anopheles gambiae.</a> Nature Biotechnology. 34 (1), 78-83 (2016).</li><li>Lin, S., Staahl, B. T., Alla, R. K., Doudna, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+homology-directed+human+genome+engineering+by+controlled+timing+of+CRISPR/Cas9+delivery.">Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery.</a> eLife. 3, 04766 (2014).</li></ol>

RH, böcek genetik modifikasyonunda hizmet veren Böcek Dönüşüm Tesisi için çalışmaktadır.

Bu protokol, Culex sivrisineklerinin genomuna belirli mutasyonları sokmak için yöntemler sunar ve diğer sivrisineklerin genomlarını düzenlemek için de kullanılabilir. Protokol, sadece enjeksiyon malzemelerinin nasıl hazırlanacağına dair özel ayrıntılar değil, aynı zamanda sivrisineklerin yumurta bırakmaya nasıl teşviklanacağı ve bu yumurtaların nasıl hazırlanacağı ve enjektelanacağı hakkında ayrıntılı bir video özeti sağlaması açısından önemlidir. Ayrıca, dişi Cx. p...

Culex sivrisinekleri dünyanın ılıman ve tropikal bölgelerine dağılmıştır ve Batı Nil virüsü1, St. Louis ensefaliti2 ve köpek kalp kurdu3 ve elephantiasis4'eneden olan filarial nematodlar da dahil olmak üzere birkaç ölümcül virüs iletir. Cx. quinquefasciatus , Cx. pipiens pipiensve Cx. pipiens molestus içeren Culex pipienskompleksinin üyeleri, biyolojilerinin birçok alanında çarpıcı varyasyonlar göstermektedir. Örneğin, Cx. quinquefasciatus ve Cx. pipiens molestus kışlayan bir dormancy 5,6, Cx. pipiens pipiens girmek için yetersiz iken sağlam mevsimsel tepkiler gösterir ve kısa günlere yanıt olarak diapause girer7,8. Ek olarak, Cx. pipiens molestus daha antropofilik olma eğilimindedir, Cx. pipiens ve Cx. quinquefasciatus ise daha zoofilik6. Bununla birlikte, Amerika Birleşik Devletleri'nde ve dünyadaki diğer birçok yerde, Cx. pipiens pipiens ve Cx. pipiens molestus'un melezleri fırsatçı besleyiciler olduğu ve hem kuşları hem de insanları ısıracağı için hastalık bulaşmasında güçlü etkileri olan bu türler iç içe geçmiştir9, böylece Batı Nil virüsü için köprü vektörleri olarak hizmet eder. Culex sivrisineklerinin biyolojisinin bu ve diğer büyüleyici yönlerini incelemek kısmen engellenmiştir, çünkü Culex sivrisineklerinin laboratuvarda geri yetiştirilmeleri, sessiz ve kuruluğa dayanıklı yumurta10 üreten Aedes sivrisineklerinden biraz daha zordur ve fonksiyonel moleküler araçlar Culex türleri için iyi gelişmemiştir.
CRISPR / Cas9 genom düzenleme, Güney evi sivrisinek, Culex quinquefasciatus14,15,16dahil olmak üzere birkaç önemli sivrisinek türünün biyolojisini değerlendirmek için kullanılan güçlü bir teknolojidir. Jennifer Doudna ve Emmanuelle Charpentier tarafından geliştirilen bu teknoloji, bakteriyel olarak türetilmiş, CRISPR ile ilişkili endonülonikleazlar tarafından virüslere karşı doğal bir bakteriyel savunmadan yararlanır (Cas proteinleri; Van der Oost ve ark.17tarafından incelenmiştir). Hayvan embriyolarına enjekte edildiğinde, Cas9 proteinleri uygun bir kılavuz RNA ile birlikte genom içinde çift iplikli kırılmalar üretebilir. Bu en sık, endononekleaz aktivitesini genomun belirli bir bölgesine yönlendiren kılavuz RNA'larla karmaşık cas9 proteini kullanılarak yapılır. Cas9 proteini bölgeye özgü çift iplikli bir kırılma yarattıktan sonra, hücresel makine iki mekanizmadan birini kullanarak kırılmayı onarmaya çalışır. birincisi, iki ucun homolog olmayan uç birleştirme (NHEJ) yoluyla birbirine bağlanmasını gerektirir, bu da hataya eğilimlidir ve genomda genellikle işlev dışı proteinlerle sonuçlanabilen çerçeve dışı eklemeler ve silmeler üretir, böylece bir nakavt mutasyonu oluşturur. Alternatif olarak, hücresel makineler, molayı doğru bir şekilde onarmak için benzer diziler bularak homolojiye yönelik onarım (HDR) kullanabilir. Benzer dizi organizma içindeki ikinci kromozom tarafından sağlanabilir (bkz. gözden geçirme18). Bununla birlikte, onarılmış dizi orijinal diziyle tam olarak eşleşirse, Cas9 proteini DNA'yı tekrar kesebilecektir. Alternatif olarak, araştırmacılar, hedef dizinin kesim bölgesinin her iki tarafında homolog diziler içeren bir donör plazmidi de içerebilir ve alternatif bir onarım dizisi (genellikle floresan bir işaret proteini, orijinal genin değiştirilmiş versiyonu veya başka bir değişiklik) kopyalanıp genoma eklenebilir veya "girilebilen".
Embriyo enjekte ederken zamanlama kritiktir ve bu özellikle böceklerde mutasyon oluşturmak için CRISPR / Cas9 genom düzenlemesini kullanırken durumdur. Bunun nedeni, Cas9 proteini ve gRNA'ların sadece embriyo senksiyal durumundayken, hücresel zarlar oluşmadan önce ve embriyo içinde birden fazla çekirdeğe erişilmeden önce mutasyon üretme kapasitesine sahip olmasıdır. Sivrisinekler için, çekirdekler, sıcaklık 19'a bağlı olarak yumurtlamadan yaklaşık2-4saat sonra çevreye ulaşır ve bu nedenle bu süreden önce başarılı mikroenjeksiyon gerçekleşmelidir. Ek olarak, Cas9 proteini erişebileceği herhangi bir nükleer DNA'yı kesecektir, böylece enjeksiyondan kaynaklanan birey, bazıları istenen mutasyona sahip, diğerleri ise olmayan bir hücre mozaiği içerecektir. Bu mutasyonların başarıyla kalıtsal olabilmesi için Cas9 proteininin mikrop hattında bulunan ve gelecekteki yumurta ve sperme yol açacak DNA'yı kesmesi gerekir. Mutasyonların mikrop hattında üretilmesini sağlamak için, böcek germline'ın ataları olan embriyo içindeki kutup hücrelerinin bulunduğu yere yakın tüm malzemeleri enjekte etmek en iyisidir. Kutup hücreleri Culex embriyolarının arka ucuna yakın bulunur20. Embriyo enjekte etmenin yanı sıra, istenen mutasyonu tespit etmek için yavruları geçmek ve taramak için dikkatli bir plan geliştirmek zorunludur.
Bu protokol, enjeksiyon karışımlarını hazırlamak için gRNA'ların nasıl üretilip Cas9 proteini ile nasıl karmaşıklaştırılacağını, ayrıca Culex pipiens'in dişi sivrisineklerinin yumurta bırakmaya nasıl teşviklanacağını ve crispr/ Cas9 aracılı genom düzenlemesi için bu yumurtaların nasıl hazırlanacağını ve enjektelanacağını açıklar. Ek olarak, istenen mutasyonun elde edildiğini doğrulamak için enjekte edilen embriyoların ve soylarının nasıl arka, çapraz ve taramadan geçeceğini açıklıyoruz. Bu protokolü kullanarak, bir ilgi geni için boş mutasyonlar ürettik, döngü, Culex pipiensBuckeye suşunda . Bu suş ilk olarak 2013 yılında Columbus, Ohio'da tarlada toplanan sivrisineklerden kuruldu ve Meuti laboratuvarı tarafından korunuyor. Bu protokol, Culex sivrisineklerinin yanı sıra diğer sivrisinek türlerinde CRISPR / Cas9 genom düzenlemesini gerektiren ek çalışmalar için kullanılabilir ve daha genel olarak, crispr / cas9 genom düzenlemesini herhangi bir böcek türüne sağlamakla ilgilidir.

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	<tbody>
		<tr height="38">
			<td height="38" style="height:38px;width:216px;">Artificial Membrane Feeder</td>
			<td style="width:121px;">Hemotek</td>
			<td style="width:119px;">SP5W1-3</td>
			<td style="width:167px;">Company location: Blackburn, UK</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:216px;">ATP</td>
			<td style="width:121px;">Invitrogen</td>
			<td style="width:119px;">18330019</td>
			<td style="width:167px;">Company location: Carlsbad, CA, USA</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:216px;">Borosilicate glass mirocapillary tubes, 1 mm outer diameter</td>
			<td style="width:121px;">World Precision Instruments</td>
			<td style="width:119px;">1B100-6</td>
			<td style="width:167px;">Company Location: Sarasota, FL, USA</td>
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		<tr height="38">
			<td height="38" style="height:38px;width:216px;">BV10 Needle Beveler</td>
			<td style="width:121px;">Sutter Instruments</td>
			<td style="width:119px;">BV-10-B</td>
			<td style="width:167px;">Company Location: Nobato, CA, USA</td>
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		<tr height="38">
			<td height="38" style="height:38px;width:216px;">Whatman Circular filter paper (12.5 cm)</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td style="width:119px;">WHA1001125</td>
			<td style="width:167px;">Company Location: St. Louis, MO, USA</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:216px;">Conical tube (50 mL)</td>
			<td style="width:121px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">339652</td>
			<td style="width:167px;">Company Location: Waltham, MA, USA</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:216px;">Fisherbrand course filter paper with fast flow rate</td>
			<td style="width:121px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">09-800</td>
			<td style="width:167px;">Company Location: Waltham, MA, USA</td>
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		<tr height="38">
			<td height="38" style="height:38px;width:216px;">Cover glass (24 x 40 mm)</td>
			<td style="width:121px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">50-311-20</td>
			<td style="width:167px;">Company Location: Waltham, MA, USA</td>
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			<td height="38" style="height:38px;width:216px;">Dental dam</td>
			<td style="width:121px;">Henry Schein Inc</td>
			<td style="width:119px;">1010171</td>
			<td style="width:167px;">Company Location: Melville, NY USA</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:216px;">Scotch double-sided tape</td>
			<td style="width:121px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">NC0879005</td>
			<td style="width:167px;">Company Location: Waltham, MA, USA</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:216px;">FemtoJet 4i microinjector</td>
			<td style="width:121px;">Eppendorf</td>
			<td style="width:119px;">5252000021</td>
			<td style="width:167px;">Company Location: Hamburg, Germany</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:216px;">Glass vial (2 dram)</td>
			<td style="width:121px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">033401C</td>
			<td style="width:167px;">Company Location: Waltham, MA, USA</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:216px;">Halocarbon oil</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td style="width:119px;">H8898-50ML</td>
			<td style="width:167px;">Company Location: St. Louis, MO, USA</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:216px;">P-2000 Laser Needle Puller</td>
			<td style="width:121px;">Sutter Instruments</td>
			<td style="width:119px;">P-2000/G</td>
			<td style="width:167px;">Company Location: Nobato, CA, USA</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:216px;">Parafilm</td>
			<td style="width:121px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">50-998-944</td>
			<td style="width:167px;">Company Location: Waltham, MA, USA</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:216px;">PC-100 Weighted Needle Puller</td>
			<td style="width:121px;">Narishige</td>
			<td style="width:119px;">PC-100</td>
			<td style="width:167px;">This is compatabile with the earlier PC-10 model, which has been discontinued. Company Location: Amityville, NY, USA</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:216px;">Phire Direct PCR Kit</td>
			<td style="width:121px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">F140WH</td>
			<td style="width:167px;">Company Location: Waltham, MA, USA</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:216px;">Kodak Photo-Flo (1%)</td>
			<td style="width:121px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">50-268-05</td>
			<td style="width:167px;">Company Location: Waltham, MA, USA</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:216px;">Quartz glass mirocapillary tubes, 1 mm outer diameter</td>
			<td style="width:121px;">Capillary Tube Supplies Limited</td>
			<td style="width:119px;">QGCT 1.0</td>
			<td style="width:167px;">Company Location: Cornwall, UK</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:216px;">Guide-it&#8482; sgRNA Screening Kit</td>
			<td style="width:121px;">Takara, Bio USA</td>
			<td style="width:119px;">632639</td>
			<td style="width:167px;">This kit allows you to determine if gRNAs cut DNA sequences in vitro. Company Location: Mountain View, CA, USA</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:216px;">Sigmacote</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td style="width:119px;">SL2-100ML</td>
			<td style="width:167px;">Company Location: St. Louis, MO, USA</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:216px;">Small petri dishes (35X10 mm)</td>
			<td style="width:121px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">50-190-0273</td>
			<td style="width:167px;">Company Location: Waltham, MA, USA</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:216px;">Sodium citrate chicken blood</td>
			<td style="width:121px;">Lampire biologicals</td>
			<td style="width:119px;">7201406</td>
			<td style="width:167px;">Company Location: Everett, PA, USA</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:216px;">Fisherbrand Square petri dish (10 cm x 10 cm)</td>
			<td style="width:121px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">FB0875711A</td>
			<td style="width:167px;">Company Location: Waltham, MA, USA</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:216px;">Tegaderm</td>
			<td style="width:121px;">Henry Schein Inc.</td>
			<td style="width:119px;">7771180</td>
			<td style="width:167px;">Company Location: Melville, NY USA</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:216px;">Tropical fish food</td>
			<td style="width:121px;">Tetramin</td>
			<td style="width:119px;">N/A</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:216px;">Whatman filter paper</td>
			<td style="width:121px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">09-927-826</td>
			<td style="width:167px;">Company Location: Waltham, MA, USA</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:216px;">Whatman filter paper, 4.25 cm</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td style="width:119px;">1001-042</td>
			<td style="width:167px;">Company Location: St. Louis, MO, USA</td>
		</tr>
	</tbody>
</table>

preparing and injecting embryos of culex mosquitoes to generate null mutations using crispr/cas9

Culex sivrisinekleri, Batı Nil virüsü ve köpek kalp kurdu ve elephantasis gibi filarial nematodların neden olduğu hastalıklar da dahil olmak üzere insan ve hayvan sağlığını olumsuz etkileyen çeşitli hastalıkların başlıca vektörleridir. Son zamanlarda, CRISPR / Cas9 genom düzenleme, bir rehber RNA (gRNA) ile karmaşık hale gelen bir Cas9 proteinini, Anopheles ve Aedes cinslerine ait sivrisinekler de dahil olmak üzere çeşitli böcek türlerinin taze döşenmiş embriyolarına enjekte ederek bölgeye yönelik mutasyonları teşvik etmek için kullanılmıştır. Culex sivrisineklerini manipüle etmek ve enjekte etmek biraz daha zordur, çünkü bu sivrisinekler yumurtalarını diğer sivrisinek türleri gibi ayrı ayrı değil, sallara dik bir şekilde bırakırlar. Burada gRNA'ların nasıl tasarlanacağı, Cas9 proteini ile nasıl karmaşıklaşacağı, Culex pipiens'in dişi sivrisineklerinin yumurta bırakmaya nasıl teşvik edeceği ve cas9/gRNA ile mikroenjeksiyon için yeni döşenen embriyoların nasıl hazırlanacağı ve enjekte edeceği anlatılacağı açıklanmaktadır. Ayrıca, istenen mutasyon için enjekte edilen sivrisineklerin nasıl arka ve taramadan geçirildiği de açıklanmaktadır. Temsili sonuçlar, bu tekniğin Culex sivrisineklerinin genomunda bölgeye yönelik mutasyonları teşvik etmek için kullanılabileceğini ve hafif değişikliklerle diğer sivrisinek türlerinde de boş mutantlar oluşturmak için kullanılabileceğini göstermektedir.

Açıklanan protokolü kullanarak, Cx. pipiensembriyolarını başarıyla enjekte edebildik ve enjekte edilen embriyolar arasında yüksek bir hayatta kalma oranı gözlemledik (~% 55, Şekil 1). Daha önceki denemeler daha düşük bir hayatta kalma yüzdesine sahipti, muhtemelen yumurta folikülünün ön kısmı tıbbi pansuman şeridine tutturuldu, sivrisinek larvalarının korondan kaçmasını ve suya başarılı bir şekilde yüzmesini önledi. Ön ucun tıbbi pansuman şerid...

Watch this Scientific Journal Video about Preparing and Injecting Embryos of Culex Mosquitoes to Generate Null Mutations using CRISPR/Cas9 at JoVE.com

Preparing and Injecting Embryos of Culex Mosquitoes to Generate Null Mutations using CRISPR/Cas9

CRISPR/Cas9, model olmayan organizmalarda gen fonksiyonunu karakterize etmek için giderek daha fazla kullanılmaktadır. Bu protokol, enjeksiyon karışımlarının hazırlanmasından sivrisinek embriyolarının elde ve enjektesine kadar Culex pipiens'innakavt hatlarının nasıl oluşturulacağını ve enjekte edilen sivrisineklerin ve bunların istenen mutasyonlar için soylarının nasıl arka, çapraz ve taramadan geçeceğini açıklar.

CRISPR/Cas9 Kullanarak Boş Mutasyonlar Oluşturmak için Culex Sivrisineklerinin Embriyolarının Hazırlanması ve EnjekteSi

Culex mosquitoes are the major vectors of several diseases that negatively impact human and animal health including West Nile virus and diseases caused by ...

preparing-injecting-embryos-culex-mosquitoes-to-generate-null

Research

JoVE Journal

Biology

Preparing and Injecting Embryos of Culex Mosquitoes to Generate Null Mutations using CRISPR/Cas9

6.7K Görüntüleme.  The Ohio State University. CRISPR/Cas9, model olmayan organizmalarda gen fonksiyonunu karakterize etmek için giderek daha fazla kullanılmaktadır. Bu protokol, enjeksiyon karışımlarının hazırlanmasından sivrisinek embriyolarının elde ve enjektesine kadar Culex pipiens'innakavt hatlarının nasıl oluşturulacağını ve enjekte edilen sivrisineklerin ve bunların istenen mutasyonlar için soylarının nasıl arka, çapraz ve taramadan geçeceğini açıklar.

Video: Preparing and Injecting Embryos of Culex Mosquitoes to Generate Null Mutations using CRISPR/Cas9

Injection of An. stephensi Embryos to Generate Malaria-resistant Mosquitoes

The introduction of exogenous genes into the genomes of mosquitoes requires microinjection techniques tailored to the specific species of interest. This video protocol demonstrates a method used by the James laboratory to microinject DNA constructs into Anopheles stephensi embryos for the generation of transformed mosquitoes. Techniques for preparing microinjection needles, collecting and preparing embryos and performing the microinjection are illustrated.

Anopheles stephensi mosquitoes are vectors for malaria inhabiting India and throughout Asia. This video demonstrates the technique for performing microinjections of this species with transgenes that will confer resistance to the malaria to the mosquito. Much of the methodology demonstrated in this video is applicable to microinjection techniques of other mosquito species.

Bir, Enjeksiyon. stephensi Embriyolar Sıtma dayanıklı Sivrisinekler oluşturun

The introduction of exogenous genes into the genomes of mosquitoes requires microinjection techniques tailored to the specific species of interest. This ...

Biyoloji

Microinjection of A. aegypti Embryos to Obtain Transgenic Mosquitoes

In this video, Nijole Jasinskiene demonstrates the methodology employed to generate transgenic Aedes aegypti mosquitoes, which are vectors for dengue fever.  The techniques for correctly preparing microinjection needles, dessicating embryos, and performing microinjection are demonstrated.

In this video, Nijole Jasinskiene demonstrates the methodology employed to generate transgenic Aedes aegypti mosquitoes, which are vectors for dengue fever. The techniques for correctly preparing microinjection needles, dessicating embryos, and performing microinjection are demonstrated.

Transgenik Sivrisinekler Edinme A. aegypti Embriyolar Mikroenjeksiyon

In this video, Nijole Jasinskiene demonstrates the methodology employed to generate transgenic Aedes aegypti mosquitoes, which are vectors for dengue ...

Harvesting and Preparing Drosophila Embryos for Electrophysiological Recording and Other Procedures

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite isolated from each other, with largely independent histories and scientific communities. However, the interface between these usually disparate fields is the developmental programs underlying acquisition of functional electrical signaling properties and differentiation of functional chemical synapses during the final phases of neural circuit formation. This interface is a critically important area for investigation. In Drosophila, these phases of functional development occur during a period of &lt;8 hours (at 25&deg;C) during the last third of embryogenesis. This late developmental period was long considered intractable to investigation owing to the deposition of a tough, impermeable epidermal cuticle. A breakthrough advance was the application of water-polymerizing surgical glue that can be locally applied to the cuticle to enable controlled dissection of late-stage embryos. With a dorsal longitudinal incision, the embryo can be laid flat, exposing the ventral nerve cord and body wall musculature to experimental investigation. This system has been heavily used to isolate and characterize genetic mutants that impair embryonic synapse formation, and thus reveal the molecular mechanisms governing the specification and differentiation of synapse connections and functional synaptic signaling properties.

Harvesting and Preparing Drosophila Embryos for Electrophysiological Recording and Other Procedures

This technique exposes the Drosophila embryonic neuromusculature for immunohistochemistry or electrophysiological recording. It is useful for studying early events in neuromuscular development or performing electrophysiology in mutants that cannot hatch.

Hasat ve hazırlanması Drosophila</emElektrofizyolojik Kayıt ve Diğer İşlemler> Embriyolar

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite ...

Generation of Genomic Deletions in Mammalian Cell Lines via CRISPR/Cas9

The prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system may be re-purposed for site-specific eukaryotic genome engineering. CRISPR/Cas9 is an inexpensive, facile, and efficient genome editing tool that allows genetic perturbation of genes and genetic elements. Here we present a simple methodology for CRISPR design, cloning, and delivery for the production of genomic deletions. In addition, we describe techniques for deletion, identification, and characterization. This strategy relies on cellular delivery of a pair of chimeric single guide RNAs (sgRNAs) to create two double strand breaks (DSBs) at a locus in order to delete the intervening DNA segment by non-homologous end joining (NHEJ) repair. Deletions have potential advantages as compared to single-site small indels given the efficiency of biallelic modification, ease of rapid identification by PCR, predictability of loss-of-function, and utility for the study of non-coding elements. This approach can be used for efficient loss-of-function studies of genes and genetic elements in mammalian cell lines.

CRISPR/Cas9 is a robust system to produce disruption of genes and genetic elements. Here we describe a protocol for the efficient creation of genomic deletions in mammalian cell lines using CRISPR/Cas9.

CRISPR / Cas9 üzerinden Memeli Hücre Hatlarında Genomik Delesyonlar Üretimi

The prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system may be re-purposed for site-specific ...

Silencing the Spark: CRISPR/Cas9 Genome Editing in Weakly Electric Fish

Electroreception and electrogenesis have changed in the evolutionary history of vertebrates. There is a striking degree of convergence in these independently derived phenotypes, which share a common genetic architecture. This is perhaps best exemplified by the numerous convergent features of gymnotiforms and mormyrids, two species-rich teleost clades that produce and detect weak electric fields and are called weakly electric fish. In the 50 years since the discovery that weakly electric fish use electricity to sense their surroundings and communicate, a growing community of scientists has gained tremendous insights into evolution of development, systems and circuits neuroscience, cellular physiology, ecology, evolutionary biology, and behavior. More recently, there has been a proliferation of genomic resources for electric fish. Use of these resources has already facilitated important insights with regards to the connection between genotype and phenotype in these species. A major obstacle to integrating genomics data with phenotypic data of weakly electric fish is a present lack of functional genomics tools. We report here a full protocol for performing CRISPR/Cas9 mutagenesis that utilizes endogenous DNA repair mechanisms in weakly electric fish. We demonstrate that this protocol is equally effective in both the mormyrid species Brienomyrus brachyistius and the gymnotiform Brachyhypopomus gauderio by using CRISPR/Cas9 to target indels and point mutations in the first exon of the sodium channel gene scn4aa. Using this protocol, embryos from both species were obtained and genotyped to confirm that the predicted mutations in the first exon of the sodium channel scn4aa were present. The knock-out success phenotype was confirmed with recordings showing reduced electric organ discharge amplitudes when compared to uninjected size-matched controls.

Here, a protocol is presented to produce and rear CRISPR/Cas9 genome knockout electric fish. Outlined in detail are the required molecular biology, breeding, and husbandry requirements for both a gymnotiform and a mormyrid, and injection techniques to produce Cas9-induced indel F0 larvae.

Kıvılcım Susturma: CRISPR/Cas9 Zayıf Elektrikli Balıklarda Genom Düzenleme

Electroreception and electrogenesis have changed in the evolutionary history of vertebrates. There is a striking degree of convergence in these ...

Genome Engineering of Primary Human B Cells Using CRISPR/Cas9

B cells are lymphocytes derived from hematopoietic stem cells and are a key component of the humoral arm of the adaptive immune system. They make attractive candidates for cell-based therapies because of their ease of isolation from peripheral blood, their ability to expand in vitro, and their longevity in vivo. Additionally, their normal biological function&#8212;to produce large amounts of antibodies&#8212;can be utilized to express very large amounts of a therapeutic protein, such as a recombinant antibody to fight infection, or an enzyme for the treatment of enzymopathies. Here, we provide detailed methods for isolating primary human B cells from peripheral blood mononuclear cells (PBMCs) and activating/expanding isolated B cells in vitro. We then demonstrate the steps involved in using the CRISPR/Cas9 system for site-specific KO of endogenous genes in B cells. This method allows for efficient KO of various genes, which can be used to study the biological functions of genes of interest. We then demonstrate the steps for using the CRISPR/Cas9 system together with a recombinant, adeno-associated, viral (rAAV) vector for efficient site-specific integration of a transgene expression cassette in B cells. Together, this protocol provides a step-by-step engineering platform that can be used in primary human B cells to study biological functions of genes as well as for the development of B-cell therapeutics.

Here we provide a detailed, step-by-step protocol for CRISPR/Cas9-based genome engineering of primary human B cells for gene knockout (KO) and knock-in (KI) to study biological functions of genes in B cells and the development of B-cell therapeutics.

CRISPR/Cas9 Kullanarak Birincil İnsan B Hücrelerinin Genom Mühendisliği

B cells are lymphocytes derived from hematopoietic stem cells and are a key component of the humoral arm of the adaptive immune system. They make ...

TGF-&#946;-mediated Endothelial to Mesenchymal Transition (EndMT) and the Functional Assessment of EndMT Effectors using CRISPR/Cas9 Gene Editing

In response to specific external cues and the activation of certain transcription factors, endothelial cells can differentiate into a mesenchymal-like phenotype, a process that is termed endothelial to mesenchymal transition (EndMT). Emerging results have suggested that EndMT is causally linked to multiple human diseases, such as fibrosis and cancer. In addition, endothelial-derived mesenchymal cells may be applied in tissue regeneration procedures, as they can be further differentiated into various cell types (e.g., osteoblasts and chondrocytes). Thus, the selective manipulation of EndMT may have clinical potential. Like epithelial-mesenchymal transition (EMT), EndMT can be strongly induced by the secreted cytokine transforming growth factor-beta (TGF-&#946;), which stimulates the expression of so-called EndMT transcription factors (EndMT-TFs), including Snail and Slug. These EndMT-TFs then up- and downregulate the levels of mesenchymal and endothelial proteins, respectively. Here, we describe methods to investigate TGF-&#946;-induced EndMT in vitro, including a protocol to study the role of particular TFs in TGF-&#946;-induced EndMT. Using these techniques, we provide evidence that TGF-&#946;2 stimulates EndMT in murine pancreatic microvascular endothelial cells (MS-1 cells), and that the genetic depletion of Snail using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated gene editing, abrogates this phenomenon. This approach may serve as a model to interrogate potential modulators of endothelial biology, and can be used to perform genetic or pharmacological screens in order to identify novel regulators of EndMT, with potential application in human disease.

TGF-&#946;-mediated Endothelial to Mesenchymal Transition EndMT and the Functional Assessment of EndMT Effectors using CRISPR/Cas9 Gene Editing

We describe methods to investigate TGF-&#946;2-induced EndMT in endothelial cells by observing cell morphology changes and examining the expression EndMT-related marker changes using immunofluorescence staining. CRISPR/Cas9 gene editing was described and used to deplete the gene encoding Snail to investigate its role in TGF-&#946;2-induced EndMT.

TGF-β aracılı Endotelden Mezenkimal Geçişe (EndMT) ve CRISPR/Cas9 Gen Düzenleme Kullanarak EndMT Efektörlerinin Fonksiyonel Değerlendirmesi

In response to specific external cues and the activation of certain transcription factors, endothelial cells can differentiate into a mesenchymal-like ...

Preparing Irradiated and Marked Male Aedes aegypti Mosquitoes for Release in an Operational Sterile Insect Technique Program

The control of such human diseases as dengue, Zika, and chikungunya relies on the control of their vector, the Aedes aegypti mosquito, because there is no prevention. Control of mosquito vectors can rely on chemicals applied to the immature and adult stages, which can contribute to the mortality of non-targets and more importantly, lead to insecticide resistance in the vector. The sterile insect technique (SIT) is a method of controlling populations of pests through the release of sterilized adult males that mate with wild females to produce non-viable offspring. This paper describes the process of producing sterile males for use in an operational SIT program for the control of Aedes aegypti mosquitoes. Outlined here are the steps used in the program including rearing and maintaining a colony, separating male and female pupae, irradiating and marking adult males, and shipping Aedes aegypti males to the release site. Also discussed are procedural caveats, program limitations, and future objectives.

Preparing Irradiated and Marked Male Aedes aegypti Mosquitoes for Release in an Operational Sterile Insect Technique Program

The sterile insect technique (SIT) is used to control specific, medically important mosquito populations that may be resistant to chemical controls. Here, we describe a method of mass rearing and preparation of sterile male mosquitoes for release in an operational SIT program targeting the Aedes aegypti mosquito.

Işınlanmış ve İşaretlenmiş Erkek Aedes aegypti Sivrisineklerinin Operasyonel Steril Böcek Tekniği Programında Salınıma Hazırlanması

The control of such human diseases as dengue, Zika, and chikungunya relies on the control of their vector, the Aedes aegypti mosquito, because there is no ...

Construction of Homozygous Mutants of Migratory Locust Using CRISPR/Cas9 Technology

The migratory locust, Locusta migratoria, is not only one of the worldwide plague locusts that caused huge economic losses to human beings but also an important research model for insect metamorphosis. The CRISPR/Cas9 system can accurately locate at a specific DNA locus and cleave within the target site, efficiently introducing double-strand breaks to induce target gene knockout or integrate new gene fragments into the specific locus. CRISPR/Cas9-mediated genome editing is a powerful tool for addressing questions encountered in locust research as well as a promising technology for locust control. This study provides a systematic protocol for CRISPR/Cas9-mediated gene knockout with the complex of Cas9 protein and single guide RNAs (sgRNAs) in migratory locusts. The selection of target sites and design of sgRNA are described in detail, followed by in vitro synthesis and verification of the sgRNAs. Subsequent procedures include egg raft collection and tanned-egg separation to achieve successful microinjection with low mortality rate, egg culture,&#160;preliminary estimation of the mutation rate, locust breeding as well as detection, preservation, and passage of the mutants to ensure population stability of the edited locusts. This method can be used as a reference for CRISPR/Cas9 based gene editing applications in migratory locusts as well as in other insects.

This study provides a systematically optimized procedure of CRISPR/Cas9 ribonuclease-based construction of homozygous locust mutants as well as a detailed method for cryopreservation and resuscitation of the locust eggs.

CRISPR/Cas9 Teknolojisi Kullanılarak Göçmen Locust'un Homozigot Mutantlarının İnşası

The migratory locust, Locusta migratoria, is not only one of the worldwide plague locusts that caused huge economic losses to human beings but also an ...

CRISPR/Cas9 Gene Editing of Hematopoietic Stem and Progenitor Cells for Gene Therapy Applications

CRISPR/Cas9 is a highly versatile and efficient gene-editing tool adopted widely to correct various genetic mutations. The feasibility of gene manipulation of hematopoietic stem and progenitor cells (HSPCs) in vitro makes HSPCs an ideal target cell for gene therapy. However, HSPCs moderately lose their engraftment and multilineage repopulation potential in ex vivo culture. In the present study, ideal culture conditions are described that improves HSPC engraftment and generate an increased number of gene-modified cells in vivo. The current report displays optimized in vitro culture conditions, including the type of culture media, unique small molecule cocktail supplementation, cytokine concentration, cell culture plates, and culture density. In addition to that, an optimized HSPC gene-editing procedure, along with the validation of the gene-editing events, are provided. For in vivo validation, the gene-edited HSPCs infusion and post-engraftment analysis in mouse recipients are displayed. The results demonstrated that the culture system increased the frequency of functional HSCs in vitro, resulting in robust engraftment of gene-edited cells in vivo.

The present protocol describes an optimized hematopoietic stem and progenitor cell (HSPC) culture procedure for the robust engraftment of gene-edited cells in vivo.