Wir danken Dr. David O'Brochta und allen Mitgliedern des Insect Genetic Technologies Coordination Research Network für die Hilfe und Schulung, die sie uns und anderen bei der Implementierung genetischer Technologien bieten. Wir danken Channa Aluvihare besonders für die Optimierung des Mikromanipulationsprotokolls, damit Culex-Embryonen injiziert und geschlüpft werden können. Wir danken auch Devante Simmons und Joseph Urso, Studenten, die im Meuti-Labor arbeiten, für ihre Unterstützung bei der Pflege und dem Screening transgener Moskitos und Zora Elmkami von der ITF für die Unterstützung bei der Aufzucht und Vorbereitung von Moskitos für die Injektion. Diese Arbeit wurde durch ein Interdisciplinary Seeds Grant des Infectious Diseases Institute der OSU unterstützt, das MEM zur Verfügung gestellt wurde.

<ol>
	<li>Hamer, G. 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=Culex+pipiens+(Diptera:+Culicidae):+a+bridge+vector+of+West+Nile+virus+to+humans.">Culex pipiens (Diptera: Culicidae): a bridge vector of West Nile virus to humans.</a> Journal of Medical Entomology. 45 (1), 125-128 (2008).</li><li>Bailey, C. 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. P., Smith, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovarian+diapause+in+three+geographic+strains+of+Culex+pipiens+(Diptera:+Culicidae).">Ovarian diapause in three geographic strains of Culex pipiens (Diptera: Culicidae).</a> Journal of Medical Entomology. 22 (5), 524-528 (1985).</li><li>Mattingly, P. 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+Culex+pipiens+complex.">The Culex pipiens complex.</a> Transactions of the Royal Entomological Society of London. 102 (7), 331-342 (1951).</li><li>Eldridge, B. 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 arbeitet für die Insect Transformation Facility, die Dienstleistungen in der genetischen Veränderung von Insekten anbietet.

Dieses Protokoll stellt Methoden vor, um spezifische Mutationen in das Genom von Culex-Moskitos einzuführen und kann verwendet werden, um auch das Genom anderer Moskitos zu bearbeiten. Das Protokoll ist insoweit von Bedeutung, als es nicht nur spezifische Details zur Vorbereitung der Injektionsmaterialien enthält, sondern auch einen detaillierten Videoüberblick darüber, wie Moskitos dazu veranlasst werden, Eier zu legen, sowie wie diese Eier zubereitet und injiziert werden. Wir fassen auch zusammen, wie man...

Culex-Moskitos sind in den gemäßigten und tropischen Regionen der Welt verbreitet und übertragen mehrere tödliche Viren, darunter West-Nil-Virus1,St. Louis-Enzephalitis2 sowie filariale Nematoden, die Hundeherzwurm3 und Elephantiasis4verursachen. Mitglieder des Culex pipiens-Komplexes, zu dem Cx. quinquefasciatus, Cx. pipiens pipiens und Cx. pipiens molestusgehören , zeigen auffällige Variationen in vielen Aspekten ihrer Biologie. Zum Beispiel, während Cx. quinquefasciatus und Cx. pipiens molestus nicht in der Lage sind, in eine überwinternde Ruhephase5,6, Cx. pipiens pipiens zeigen robuste saisonale Reaktionen und treten in die Diapause als Reaktion auf kurze Tage7,8ein. Darüber hinaus neigen Cx. pipiens molestus dazu, anthropophiler zu sein, während Cx. pipiens und Cx. quinquefasciatus zoophiler sind6. In den Vereinigten Staaten und an vielen anderen Orten der Welt kreuzen sich diese Arten jedoch, was starke Auswirkungen auf die Krankheitsübertragung hat, da Hybriden der Cx. pipiens pipiens und Cx. pipiens molestus opportunistische Feeder sind und sowohl Vögel als auch Menschenbeißen 9und somit als Brückenvektoren für das West-Nil-Virus dienen. Das Studium dieser und anderer faszinierender Aspekte der Biologie von Culex-Moskitos wurde zum Teil behindert, weil Culex-Moskitos im Labor etwas schwieriger zu züchten sind als Aedes-Moskitos, die ruhe- und austrocknungsresistente Eier produzieren10 und weil funktionelle molekulare Werkzeuge für Culex-Arten nicht so gut entwickelt sind.
CRISPR / Cas9 Genom-Editing ist eine leistungsstarke Technologie, die verwendet wurde, um die Biologie mehrerer wichtiger Mückenarten11,12,13, einschließlich der Südlichen Hausmücke, Culex quinquefasciatus14,15,16. Diese von Jennifer Doudna und Emmanuelle Charpentier entwickelte Technologie nutzt eine natürliche bakterielle Abwehr gegen Viren durch bakteriell abgeleitete, CRISPR-assoziierte Endonukleasen (Cas-Proteine; siehe Review von Van der Oost et al.17). Bei der Injektion in tierische Embryonen können die Cas9-Proteine in Kombination mit einer geeigneten Leit-RNA doppelsträngige Brüche im Genom erzeugen. Dies geschieht am häufigsten durch die Verwendung des Cas9-Proteins, das mit Leit-RNAs komplexiert ist, die die Endonukleaseaktivität auf eine bestimmte Region des Genoms lenken. Nachdem das Cas9-Protein einen ortsspezifischen Doppelstrangbruch erzeugt hat, versucht die zelluläre Maschinerie, den Bruch mit einem von zwei Mechanismen zu reparieren. Die erste beinhaltet die Ligatur der beiden Enden durch nicht-homologe End-Joining (NHEJ), die fehleranfällig ist und oft Out-Frame-Insertionen und Deletionen im Genom erzeugt, die zu nicht-funktionellen Proteinen führen können, wodurch eine Knock-out-Mutation erzeugt wird. Alternativ kann die zelluläre Maschinerie die homologiegerichtete Reparatur (HDR) verwenden, indem sie ähnliche Sequenzen findet, um den Bruch korrekt zu reparieren. Die ähnliche Sequenz kann durch das zweite Chromosom im Organismus bereitgestellt werden (siehe Review18). Wenn die reparierte Sequenz jedoch genau mit der ursprünglichen Sequenz übereinstimmt, kann das Cas9-Protein die DNA erneut schneiden. Alternativ können Forscher auch ein Spenderplasmid einschließen, das homologe Sequenzen auf beiden Seiten der Schnittstelle der Zielsequenz mit einer alternativen Reparatursequenz enthält - oft ein fluoreszierendes Markerprotein, eine modifizierte Version des ursprünglichen Gens oder eine andere Modifikation -, die kopiert und in das Genom eingefügt oder "eingeklopft" werden kann.
Das Timing ist bei der Injektion von Embryonen entscheidend, und dies ist insbesondere der Fall, wenn CRISPR / Cas9-Genom-Editing verwendet wird, um Mutationen bei Insekten zu erzeugen. Dies liegt daran, dass das Cas9-Protein und gRNAs die größte Fähigkeit haben, Mutationen nur dann zu erzeugen, wenn sich der Embryo in seinem synzytialen Zustand befindet, bevor sich Zellmembranen gebildet haben und wenn mehrere Kerne im Embryo zugänglich sind. Bei Moskitos erreichen die Kerne die Peripherie ~ 2-4 Stunden nach der Eiablage, abhängig von der Temperatur19, und daher muss vor dieser Zeit eine erfolgreiche Mikroinjektion erfolgen. Darüber hinaus schneidet das Cas9-Protein jede Kern-DNA, auf die es zugreifen kann, so dass das Individuum, das aus der Injektion resultiert, ein Mosaik von Zellen enthält, von denen einige die gewünschte Mutation aufweisen und andere nicht. Damit diese Mutationen erfolgreich vererbt werden können, muss das Cas9-Protein DNA schneiden, die sich in der Keimbahn befindet und die zukünftigen Eier und Spermien hervorbringt. Um sicherzustellen, dass Mutationen in der Keimbahn erzeugt werden, ist es am besten, alle Materialien in der Nähe der Position der Polzellen im Embryo zu injizieren, die die Vorläufer der Insektenkeimbahn sind. Die Polzellen befinden sich in der Nähe des hinteren Endes der Culex-Embryonen 20. Neben der Injektion von Embryonen ist es unerlässlich, einen sorgfältigen Plan für die Kreuzung und das Screening von Nachkommen zu entwickeln, um die gewünschte Mutation zu erkennen.
Dieses Protokoll beschreibt, wie gRNAs erzeugt und mit Cas9-Protein komplexiert werden, um Injektionsmischungen herzustellen, sowie wie weibliche Moskitos von Culex pipiens dazu veranlasst werden, Eier zu legen und wie diese Eier für die CRISPR / Cas9-vermittelte Genombearbeitung vorbereitet und injiziert werden. Darüber hinaus beschreiben wir, wie injizierte Embryonen und ihre Nachkommen aufgezogen, gekreuzt und gescreent werden, um zu bestätigen, dass die gewünschte Mutation erhalten wurde. Mit diesem Protokoll generierten wir Nullmutationen für ein Gen von Interesse, Zyklus,im Buckeye-Stamm von Culex pipiens. Dieser Stamm wurde ursprünglich 2013 aus feldgesammelten Moskitos in Columbus, Ohio, entwickelt und wird vom Meuti-Labor gepflegt. Dieses Protokoll kann für zusätzliche Studien verwendet werden, die eine CRISPR/Cas9-Genombearbeitung bei Culex-Moskitos sowie anderen Mückenarten erfordern, und ist im Allgemeinen für die Verwendung von CRISPR/Cas9-Genom-Editing bei jeder Insektenart relevant.

<table border="0" cellpadding="0" cellspacing="0" style="width:622px;" width="623">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<tr height="38">
			<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-Moskitos sind die Hauptvektoren mehrerer Krankheiten, die sich negativ auf die Gesundheit von Mensch und Tier auswirken, einschließlich west-Nil-Virus und Krankheiten, die durch filariale Nematoden wie Hundeherzwurm und Elefantenasis verursacht werden. Vor kurzem wurde CRISPR / Cas9-Genom-Editing verwendet, um ortsgerichtete Mutationen zu induzieren, indem ein Cas9-Protein, das mit einer Leit-RNA (gRNA) komplexiert wurde, in frisch gelegte Embryonen mehrerer Insektenarten injiziert wurde, einschließlich Moskitos, die zu den Gattungen Anopheles und Aedesgehören. Die Manipulation und Injektion von Culex-Moskitos ist etwas schwieriger, da diese Moskitos ihre Eier aufrecht in Flößen ablegen und nicht einzeln wie andere Mückenarten. Hier beschreiben wir, wie man gRNAs entwirft, sie mit Cas9-Protein komplexiert, weibliche Moskitos von Culex pipiens dazu bringt, Eier zu legen, und wie man neu gelegte Embryonen für die Mikroinjektion mit Cas9 / gRNA vorbereitet und injiziert. Wir beschreiben auch, wie man injizierte Moskitos auf die gewünschte Mutation rückt und abschirmt. Die repräsentativen Ergebnisse zeigen, dass diese Technik verwendet werden kann, um ortsgerichtete Mutationen im Genom von Culex-Moskitos zu induzieren und mit leichten Modifikationen auch bei anderen Mückenarten Nullmutanten zu erzeugen.

Mit dem beschriebenen Protokoll konnten wir erfolgreich Embryonen von Cx. pipiensinjizieren und beobachteten eine hohe Überlebensrate unter den injizierten Embryonen (~ 55%, Abbildung 1). Frühere Studien hatten einen geringeren Prozentsatz des Überlebens, wahrscheinlich, weil der vordere Teil des Eifollikels am medizinischen Verbandsstreifen befestigt war, was verhinderte, dass Mückenlarven aus dem Chorion entweichen und erfolgreich ins Wasser schwammen. Die Sicherstellung, dass...

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 wird zunehmend zur Charakterisierung der Genfunktion in Nicht-Modellorganismen eingesetzt. Dieses Protokoll beschreibt, wie Man Knock-out-Linien von Culex pipienserzeugt, von der Vorbereitung von Injektionsmischungen über die Gewinnung und Injektion von Moskitoembryonen bis hin zur Aufzucht, Kreuzung und dem Screening injizierter Moskitos und ihrer Nachkommen auf gewünschte Mutationen.

Vorbereitung und Injektion von Embryonen von Culex-Moskitos zur Erzeugung von Nullmutationen mit CRISPR/Cas9

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 Aufrufe.  The Ohio State University. CRISPR/Cas9 wird zunehmend zur Charakterisierung der Genfunktion in Nicht-Modellorganismen eingesetzt. Dieses Protokoll beschreibt, wie Man Knock-out-Linien von Culex pipienserzeugt, von der Vorbereitung von Injektionsmischungen über die Gewinnung und Injektion von Moskitoembryonen bis hin zur Aufzucht, Kreuzung und dem Screening injizierter Moskitos und ihrer Nachkommen auf gewünschte Mutationen.

Serie: 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.

Injektion eines. stephensi Embryonen zu generieren Malaria-resistenter Moskitos

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

Forschung

Biologie

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.

Mikroinjektion von A. aegypti Embryonen, um transgene Moskitos erhalten

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.

Ernte und Vorbereitung Drosophila Embryonen für die elektrophysiologische Ableitung und andere Verfahren

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.

Generierung von Genomic Löschungen in Säugerzelllinien über CRISPR / Cas9

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.

Den Funken zum Schweigen bringen: 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 ...

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.

Genom-Engineering von primären menschlichen B-Zellen mit 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 ...

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-vermittelter endothelialer zu mesenchymaler Übergang (EndMT) und die funktionelle Bewertung von EndMT-Effektoren mittels CRISPR/Cas9-Gen-Editing

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.

Vorbereitung bestrahlter und markierter männlicher Aedes aegypti-Moskitos für die Freisetzung in einem operationellen sterilen Insektentechnikprogramm

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.

Konstruktion homozygoter Mutanten der Wanderheuschrecke mittels CRISPR/Cas9-Technologie

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.

Methods Article

Vorbereitung und Injektion von Embryonen von Culex-Moskitos zur Erzeugung von Nullmutationen mit CRISPR/Cas9