We would like to thank the entire Lazzaro lab, and especially Susan Rottschaefer, for their help in both reviewing and testing these protocols. This is a product of their cumulative expertise. Work in the Lazzaro lab is supported by grants R01 AI083932 and R01 AI064950 from the US National Institutes of Health.

<ol>
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None of the authors have competing interests or conflicting interests.

Описанная здесь процедура дает строгий и качественный инфекции дрозофилы. Изображенные примеры, прежде всего, направлены на инфекции Провиденсия rettgeri и Е. палочка, но протокол является весьма гибкой и может быть применен к инфекции с различных бактерий в диапазоне условий выращивания хост и техническое обслуживание. Детали оптимального экспериментальный подход будет зависеть от бактерии, используемой для инфекции, генотипа хозяина, и общих условий эксперимента. Настоятельно рекомендуется пилотного тестирования любые новые экспериментальные условия до начала более амбициозные проекты. Хорошая отправная точка, чтобы проверить три инфекции дозы в диапазоне 100-кратным. Высоко вирулентные патогены часто лучше представлены на очень низких инфекционных доз, порядка 10 - 100 бактериальных клеток на лету. Более умеренные патогены могут быть введены в более высоких дозах около 1000 бактерий на FLу, и не-патогены могут быть введены в дозах до 10000 бактерий на лету. Это часто полезно определить кинетику новых инфекций путем отслеживания патогена нагрузку, хост смертности, и деятельность иммунной системы на продольной временных рядов. Поскольку измерение патогена нагрузки и экспрессии генов хозяина разрушающие анализы, надо заразить различных мух в начале эксперимента для каждой временной точки, которая должна быть измерена. При принятии решения, следует ли использовать укол или микрокапиллярных основе инъекции, важно отметить, что преимущества и ограничения для каждого подхода. Капиллярная впрыска вводит объем жидкости в лету, что обе незначительно увеличивает давление тургора и вводит солей или других молекул, которые, суспендированные или растворенные в носителе. Инъекции капиллярной также требует доступа к инъекции объекта или приобретение необходимого оборудования. не Септик укол булавкой не требует специального оборудования и вводитнезначительные СМИ в лету, и, как правило, более эффективны для заражения большого количества мух. Тем не менее, укол инфекции не позволяют точно контролировать дозу инфекции, что может быть достигнуто с капиллярной инъекции. Настоящий Протокол сосредоточены на инжекционного устройства, которое механически регулирует объем впрыска, но существуют также системы впрыска на основе дискретных импульсов сжатого воздуха. 20,21 Это, как правило, дороже, чем аппарат признакам здесь и требует калибровки воздушного импульса для каждого игла для того, чтобы обеспечить устойчивые объемы инъекций. Существует значительная дискуссия, но очень мало данных о том, как мухи стали системно инфицированных бактериями в дикой природе. Некоторые исследователи постулируют, что большинство природных инфекций происходят, когда дрозофилы глотать патогенные бактерии и бактерии впоследствии удалось избежать кишку, чтобы установить системные инфекции. Тем не менее, есть очень FeW бактерии, известные, чтобы иметь возможность пересечь кишечник D. MELANOGASTER, и те, которые имеют эту способность очень смертельной для мух 22,23. Альтернативная теория, что летит регулярно поддерживать кутикулы повреждения через бегство от неудачных попыток хищничества или нападения эктопаразитарными клещей. Эта гипотеза подтверждается частым сбора дикого D. MELANOGASTER подшипник меланизации пятна, которые свидетельствуют о исцелил раны (неопубликованные наблюдения). Клещи, как было показано для передачи бактериальных инфекций у дрозофилы 24 и раны, оставленные клещей может быть вторичной инфекции бактериями в медоносных пчел 25. Тем не менее, частота в природе клещей-приводом или иначе оппортунистической инфекции в D. MELANOGASTER через кутикулы нарушений не известно. Протокол, описанный здесь, позволяет введение бактерий непосредственно в гемолимфе через количественного инъекции в обход каких-либо эпителиальные барьеры или поведенческие IMMUщества. Методы для кормления патогенных бактерий D. MELANOGASTER были описаны в Vodovar и др. 22 и Nehme и др. 23. Многие бактерии представляют энтомопатогенные мало или вообще не человеческое риск для здоровья, что позволяет исследователям работать с ними удобно. Кроме того, очень немногие бактерии имеют способность инфицировать дрозофилы при контакте без вмешательства при, так что риск &quot;эпидемия&quot; распространения бактериальной инфекции через лаборатории через загрязненных поверхностей или бежали мух, как правило, очень низкий. Тем не менее, желательно, чтобы гарантировать, что адекватные меры по сдерживанию в месте, чтобы предотвратить зараженные мух от побега и отбить любой избежал мух. Лаборатория должна быть оборудована на биологическом уровне безопасности соразмерно, что возбудителей используются, и должны быть использованы стандартные передового опыта в области микробиологии. Экспериментальная infectiпо методу, описанному здесь позволяет инфекции дрозофилы с любой дозе произвольной бактерии. После инфекции было установлено, что это просто для измерения кинетики бактериальной пролиферации или зазор, чтобы отслеживать хоста смертности, и для анализа индукции иммунной системы хозяина. Зараженные мухи могут быть легко применены и другие фенотипические анализы, в том числе испытаний физиологических функций, которые могут форму или иметь форму инфекцией. Процедуры, описанные недороги, требует относительно мало специализированного оборудования, и легко узнаются, что делает их поддаются для использования в различных проектах на широтой научных и учебных лабораторий.

Плодовой мушки дрозофилы является одним из ведущих модельных организмов для изучения функции и эволюцию иммунной защиты. Дрозофилы являются недорогой и простой в тылу, очень поддаются экспериментальной манипуляции, и подкреплены обширным научным сообществом, которая сложилась широкая массив исследовательских инструментов. Многие аспекты врожденного иммунитета сохраняются между насекомых и млекопитающих, в том числе передачи сигнала, опосредованного Toll-подобных рецепторов и NF-KB факторов транскрипции, семья ЯК / STAT сигнализации и JNK ответов пути. 1,2 функция этих генов и путей может быть запрошены в D. MELANOGASTER помощью мутаций или РНК-интерференции нокдаун, что увеличение или уменьшение пути деятельности. 3 - 6 Кроме того, дрозофилы могут быть использованы для изучения физиологических последствий инфекции и заболевания, в том числе в контексте эволюционной теории истории жизни 7.- 9 Все такие исследования, однако, зависит от способности надежно инфицировать экспериментальных мух при определенных условиях обработки. Описанная здесь процедура представляет методологическую основу для предоставления надежных и воспроизводимых бактериальных инфекций в дрозофилы, а затем измерения тяжести инфекции и количественной оценки иммунного ответа. Дрозофилы могут быть, естественно, и экспериментально зараженных широкого спектра патогенных микроорганизмов и паразитов, в том числе бактерий, грибков, вирусов, нематод и паразитоид ос. Текущий протокол ориентирован на достижение системной бактериальной инфекции. Много различных бактерий может использоваться, чтобы заразить мух, и выбор экспериментатора должны быть основаны на точных научных вопросов, задаваемых. Например, клинические изоляты человека могут быть использованы для изучения бактериальных механизмов вирулентности 10, или экологически соответствующие изоляты могут быть preferreд эволюционного исследования. 11 Некоторые бактерии являются возбудителями компетентные D. MELANOGASTER, пролиферирующих на инфекции и вызывая хозяина болезнь или смерть. Другие бактерии эффективно управляется иммунной системы хозяина и очищены в течение нескольких дней. В этой демонстрации, Providencia rettgeri будет использоваться в качестве пролиферативного патогена, что может привести к хост смертности и сохраняется в живых хозяев. Кишечной палочки будет использоваться в качестве не патогена, который очищается от иммунной системы хозяина. Заражение будет установлено путем введения бактерий непосредственно в полость тела лету. Этот подход обходит эпителиальные барьеры и защитные поведения, позволяя расследование системной инфекции независимо от естественного режима передачи. Есть два основных метода для создания экспериментально системной инфекции. В-первых, а nanoinjector и вытащил стеклянный капилляр иглы используются, чтобы ввести точное числоБактерии в лету. Этот метод имеет преимущества позволяет большой динамический диапазон инфекции дозах и бытия количественно высокой повторяемостью. Второй подход заключается в предоставлении инфекции с септическим укол. Этот подход имеет преимущества быть быстрым и не требует специального оборудования. После инфекции установлено, становится возможным измерить системную нагрузку патогенных микроорганизмов, хоста смертности и индуцируемый активности иммунной системы. Конечно, любое число дополнительных фенотипов теоретически может быть измерена в инфицированной D. MELANOGASTER, в том числе пост-инфекции плодовитость 12, способность к обучению 13 метаболического статуса 14 или практически любой другой признак, что можно себе представить.

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			<td height="21" style="height:21px;width:399px;">Reagent</td>
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			<td style="width:239px;">Powers Scientific, Inc</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Paintbrush</td>
			<td style="width:239px;"></td>
			<td style="width:177px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:399px;">CO2 Flypads</td>
			<td style="width:239px;">FlyStuff</td>
			<td style="width:177px;">59-114</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">CO2</td>
			<td style="width:239px;">Airgas</td>
			<td>CD FG50</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Drosophila rearing mix&#160;</td>
			<td style="width:239px;"></td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">6 oz Square Bottom Bottles, polypropylene</td>
			<td style="width:239px;">Genesee Scientific</td>
			<td style="width:177px;">32-130</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Nosterile Extra Large Cotton Balls</td>
			<td style="width:239px;">Fisher brand</td>
			<td style="width:177px;">22-456-882</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Microscope</td>
			<td style="width:239px;">Olympus Corporation</td>
			<td style="width:177px;">SZ51</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Drosophila Vials polystyrene</td>
			<td style="width:239px;">VWR international</td>
			<td style="width:177px;">89092-720</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Nosterile Large Cotton Balls</td>
			<td style="width:239px;">Fisher brand</td>
			<td style="width:177px;">22-456-883</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">2L flask</td>
			<td style="width:239px;">VWR international</td>
			<td>89000-370</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Petri Dishes with Clear Lids, Raised Ridge; 100 . x 15 mm;</td>
			<td style="width:239px;">VWR international</td>
			<td style="width:177px;">25384-302</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">LB Agar, Miller</td>
			<td style="width:239px;">Difco</td>
			<td style="width:177px;">244520</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Innoculing Loop</td>
			<td style="width:239px;">VWR international</td>
			<td>80094-488</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:399px;">Rainin Clasic Pipettes in various sizes 
			0.1 &#181;l to 2 &#181;l, 
			2 &#181;l to 20 &#181;l, 
			20 &#181;l to 200 &#181;l, 
			100 &#181;l to 1000</td>
			<td style="width:239px;">Rainin</td>
			<td style="width:177px;">PR-2 
			PR-20 
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			PR-1000</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:399px;">Micropipette tips (assorted sizes)</td>
			<td style="width:239px;">VWR international</td>
			<td style="width:177px;">30128-376 
			53503-810 
			16466-008</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Luria Broth Base, Miller</td>
			<td style="width:239px;">Difco</td>
			<td>241420</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Disposable Culture Tubes Borosilicate Glass</td>
			<td style="width:239px;">VWR international</td>
			<td style="width:177px;">47729-576</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">S-500 Orbital Shaker</td>
			<td style="width:239px;">VWR international</td>
			<td style="width:177px;">14005-830</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">centrifuge</td>
			<td style="width:239px;">VWR international</td>
			<td style="width:177px;">37001-300</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">PBS pH 7.4 10x</td>
			<td style="width:239px;">Invitrogen</td>
			<td style="width:177px;">70011044</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">SmartSpec 3000 Spectrophotometer</td>
			<td style="width:239px;">Bio-Rad</td>
			<td style="width:177px;">170-2501</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Semimicrovolume Cuvettes</td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Vertical Capillary Puller</td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">3.5&#39;&#39; Replacement glass Capillaries for Nanojet II</td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Nanoject II</td>
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			<td style="width:177px;">3-000-204</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Forceps</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:399px;">10mL Syringe</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Mineral Oil, White, light</td>
			<td>Macron Fine Chemicals</td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">minutein pins</td>
			<td style="width:239px;">Fine Science Tools</td>
			<td style="width:177px;">26002-10</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">1.5mL&#160; Microcentrifuge tubes; Seal Rite</td>
			<td style="width:239px;">USA Scientific Inc.</td>
			<td style="width:177px;">1615-5500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Motorized Pestle; Talboys Laboratory Stirrer</td>
			<td style="width:239px;">Troemner</td>
			<td style="width:177px;">103</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Talboys High Throughput Homogenizer</td>
			<td style="width:239px;">OPS Diagnostics</td>
			<td style="width:177px;">930145</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">5/32&#39;&#39; Grinding Balls</td>
			<td>OPS Diagnostics</td>
			<td style="width:177px;">GBSS 156-5000-01</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Vortex Genie</td>
			<td style="width:239px;">Scientific indurstries inc.</td>
			<td style="width:177px;">G560</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Multichannel Pipettor (10uL-300uL)</td>
			<td style="width:239px;">Sartorius</td>
			<td>730360</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;"></td>
			<td style="width:239px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">WASP2 Whitley Automated Spiral Plater</td>
			<td style="width:239px;">Microbiology International</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:399px;">ProtoCOL automated colony counter / plate counter/ plate reader&#160;</td>
			<td style="width:239px;">Microbiology International</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">TRIzol</td>
			<td style="width:239px;">Life Technologies</td>
			<td style="width:177px;">15596-026</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">qPCR tubes; Low-Profile 0.2 ml 8-Tube Strips</td>
			<td style="width:239px;">Bio-Rad</td>
			<td style="width:177px;">TLS0801</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">qPCR caps; Optical Flat 8-Cap Strips</td>
			<td style="width:239px;">Bio-Rad</td>
			<td style="width:177px;">TCS0803</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">RQ1 RNase-Free DNase</td>
			<td style="width:239px;">Promega</td>
			<td style="width:177px;">m610a</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">M-MLV Reverse Transcriptase</td>
			<td style="width:239px;">Promega</td>
			<td style="width:177px;">m170b</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">dNTPs</td>
			<td style="width:239px;">Promega</td>
			<td style="width:177px;">U1240</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Oligo-dT</td>
			<td style="width:239px;">IDT</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">&#160;SsoAdvanced SYBR Green Supermix</td>
			<td style="width:239px;">Bio-Rad</td>
			<td style="width:177px;">172-5260</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CFX Connect Real-Time PCR Detection System</td>
			<td style="width:239px;">Bio-Rad</td>
			<td style="width:177px;">185-5200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RNasin Ribonuclease Inhibitor</td>
			<td>Promega</td>
			<td>N2115</td>
		</tr>
	</tbody>
</table>

systemic bacterial infection and immune defense phenotypes in drosophila melanogaster

Плодовой мушки дрозофилы является одним из ведущих модельных организмов для изучения функции и эволюцию иммунной защиты. Многие аспекты врожденного иммунитета сохраняются между насекомых и млекопитающих, а с дрозофилы могут быть легко генетически и экспериментально манипулировать, они являются мощным для изучения функции иммунной системы и физиологические последствия болезни. Процедура продемонстрировали здесь позволяет инфекции мух путем введения бактерий непосредственно в полость тела, минуя эпителиальные барьеры и более пассивные формы защиты и позволяет сосредоточиться на системной инфекции. Процедура включает в себя протоколы для измерения скоростей принимающей смертности, системной нагрузки патогена, и степень индукции иммунной системы хозяина. Эта процедура инфекция недорогой, надежный и количественно повторяемые, и может быть использован в исследованиях функциональных генетики, эволюционной истории жизни и физиологии.

В этом разделе представлены результаты, которые могут быть получены после бактериальной инфекции дрозофилы. 1 показано, что инфицирование доза изменяется в зависимости от оптической плотности бактериальной суспензии, используемой для инъекций, и что доза может быть доставлен надежно оценена путем гомогенизации и обшивки летит сразу после инъекции , Как показано на рисунке 2, различные патогены могут вызывать различные уровни принимающей смертности (фиг.2А) и принимающей смертности может быть дозозависимое (фиг.2В). Важно отметить, что этот протокол позволяет различные виды инфекций должны быть достигнуты: Провиденсия rettgeri может вызвать хроническое, суб-смертельная инфекция, которая сохраняется в течение 20 дней и более (рис 3а). Тем не менее, другие бактерии, такие как Escerichia палочки в основном будут удалены принимающей лету в течение шести часов после заражения (рис 3b). Индукция иммунной SYSTEM может быть оценена с помощью выделения РНК и последующей Qrt-PCR антибактериальных пептидных транскриптов (фиг.4А и Б). Аналогично, но менее количественно, мухи выражают GFP под контролем антимикробного пептида промоторов генов может быть использован для визуализации индукции иммунной системы (фиг.4С). <img alt="Фигура 1" src="/files/ftp_upload/52613/52613fig1.jpg" /> . Рисунок 1: Определение инфекционная доза Мухи вводили 50 NL бактериальных суспензий, охватывающих широкий круг оптических плотностей (0,0001 - 0,05). Мухи были немедленно гомогенизировали и высевали для определения количества бактериальной введены путем инъекции. Первоначальная бактериальная нагрузка сильно коррелирует с начальным ОД вводят (R 2 = 0,96) <img alt="Рисунок 2" src="/files/ftp_upload/52613/52613fig2.jpg" /> <sЧонг&gt; Рисунок 2:. Выживание после инъекций с бактериальными патогенами пяти до семи дней старые самцы вводили 50 NL либо бактериальной суспензии или стерильные массовой информации и мониторинг за выживание. (А) Дикие мухи типа вводили приблизительно 5000 бактерий из одного из трех разных видов. Скорость принимающей смертности в значительной степени зависит от вида бактерий, используемых для инфекции. (B) Тип Дикие мухи вводили в трех различных дозах Enterococcous аесаИз - 50, 500 и 5000 бактерий на лету. Мухи умирают быстрее при инфицировании выше инфекционных доз (C) иммунной системы и ее мутант дикого типа изогенных линии управления вводили примерно 500 Burkholderia cepacia. Попарно сравнение Войти ранга показывает, что дикого типа мухи выживает инфекции значительно лучше, чем мутанта (χ 2 = 59,02, DF = 1, р &lt;0,0001). Рисунок 3: бактериальной нагрузки. После инъекции мух вводили примерно (A) 5000 P. rettgeri (Б) 3400 Е. палочка. Бактериальной нагрузки была определена сразу же после инъекции и в различных последующих моментов времени. Каждая точка данных представляет собой бактериальную нагрузку одной лету. Е. палочка быстро очищается от зараженных хостов в то время как P. rettgeri сохраняется в течение оставшейся жизни хозяина ,. <img alt="Рисунок 4" src="/files/ftp_upload/52613/52613fig4.jpg" /> Рисунок 4: Индукция экспрессии генов иммунной после инъекций. (A) Мухи вводили 50 нл Р. rettgeri или стерильной PBS, либо в брюшной полости или грудной клетки, или остались в стороне от unmanipulated CO <sUB&gt; 2 анестезии. Через шесть часов после инфекции, мухи были собраны для выделения РНК и экспрессии гена Diptericin была определена с использованием QRT-PCR. (A) Уровни экспрессии графически как отношение Diptericin транскрипт к rp49 транскрипта и масштабируется таким образом, что управление СО 2 определен как имеющий уровень экспрессии 1. бары представляют собой среднее и стандартную ошибку коэффициентов из каждого состояния (п = 4). Уровни (Б) Экспрессия графически в виде 2 ΔΔCT со средним значением С Т от СО 2 мух управления эстезия, используемых в качестве стандартного неиндуцированном состоянии. Столбики представляют среднее и стандартную ошибку ΔΔC T от каждого состояния (п = 4). Хотя нет никакой разницы в индукции транскрипта из-месте инъекции, сравнение панели А и В показывает, как метод ΔΔC Т может приводить к завышению уровня индукции. (С) Мухи выражая GFP под контролем промотора Diptericin вводили 50 нл Р. rettgeri (OD = 0,1), а затем отображены 7 дней. GFP панель показывает экспрессию GFP в брюшной жир тела зараженных мух, указывая активации Diptericin промоутер в бактериально-инфицированных мух, но не СМИ впрыском управления. <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> Ген </td><td> Вперед </td><td> Обратный </td></tr><tr><td> rp49 (также называется rpL32) </td><td> 5 &#39;AGGCCCAAGATCGTGAAGAA 3&#39; </td><td> 5 &#39;GACGCACTCTGTTGTCGATACC 3&#39; </td></tr><tr><td> Diptericin </td><td> 5 &#39;GCGGCGATGGTTTTGG 3&#39; </td><td> 5 &#39;CGCTGGTCCACACCTTCTG 3&#39; </td></tr><tr><td> Drosomycin </td><td> 5 &#39;CTGCCTGTCCGGAAGATACAA 3 &#39; </td><td> 5 &#39;TCCCTCCTCCTTGCACACA 3&#39; </td></tr><tr><td> Дефенсина </td><td> 5 &#39;GAGGATCATGTCCTGGTGCAT 3&#39; </td><td> 5 &#39;TCGCTTCTGGCGGCTATG 3&#39; </td></tr><tr><td> Attacin </td><td> 5 &#39;CGTTTGGATCTGACCAAGG 3&#39; </td><td> 5 &#39;AAAGTTCCGCCAGGTGTGAC 3&#39; </td></tr><tr><td> Metchnikowan </td><td> 5 &#39;AACTTAATCTTGGAGCGATTTTTCTG 3&#39; </td><td> 5 &#39;ACGGCCTCGTATCGAAAATG 3&#39; </td></tr></tbody></table> Таблица 1: Праймеры для Qrt-PCR.

Watch this Scientific Journal Video about Systemic Bacterial Infection and Immune Defense Phenotypes in Drosophila Melanogaster at JoVE.com

Systemic Bacterial Infection and Immune Defense Phenotypes in Drosophila Melanogaster

Drosophila melanogaster is an outstanding model organism for studying innate immune systems and the physiological consequences of infection and disease. This protocol describes how to deliver robust and quantitatively repeatable bacterial infections to D. melanogaster, and how to subsequently measure infection severity and quantify the host immune response.

Системная бактериальная инфекция и иммунная Фенотипы обороны в<em&gt; Дрозофилы</em

The fruit fly Drosophila melanogaster is one of the premier model organisms for studying the function and evolution of immune defense. Many aspects of ...

systemic-bacterial-infection-immune-defense-phenotypes-drosophila

Research

JoVE Journal

Immunology and Infection

Systemic Bacterial Infection and Immune Defense Phenotypes in Drosophila Melanogaster

24.3K Views.  Cornell University. Drosophila melanogaster is an outstanding model organism for studying innate immune systems and the physiological consequences of infection and disease. This protocol describes how to deliver robust and quantitatively repeatable bacterial infections to D. melanogaster, and how to subsequently measure infection severity and quantify the host immune response.

Video: Systemic Bacterial Infection and Immune Defense Phenotypes in Drosophila Melanogaster

Seven Steps to Stellate Cells

Hepatic stellate cells are liver-resident cells of star-like morphology and are located in the space of Disse between liver sinusoidal endothelial cells and hepatocytes1,2. Stellate cells are derived from bone marrow precursors and store up to 80% of the total body vitamin A1, 2. Upon activation, stellate cells differentiate into myofibroblasts to produce extracellular matrix, thus contributing to liver fibrosis3. Based on their ability to contract, myofibroblastic stellate cells can regulate the vascular tone associated with portal hypertension4. Recently, we demonstrated that hepatic stellate cells are potent antigen presenting cells and can activate NKT cells as well as conventional T lymphocytes5. 

 Here we present a method for the efficient preparation of hepatic stellate cells from mouse liver. Due to their perisinusoidal localization, the isolation of hepatic stellate cells is a multi-step process. In order to render stellate cells accessible to isolation from the space of Disse, mouse livers are perfused in situ with the digestive enzymes Pronase E and Collagenase P. Following perfusion, the liver tissue is subjected to additional enzymatic treatment with Pronase E and Collagenase P in vitro. Subsequently, the method takes advantage of the massive amount of vitamin A-storing lipid droplets in hepatic stellate cells. This feature allows the separation of stellate cells from other hepatic cell types by centrifugation on an 8% Nycodenz gradient. The protocol described here yields a highly pure and homogenous population of stellate cells. Purity of preparations can be assessed by staining for the marker molecule glial fibrillary acidic protein (GFAP), prior to analysis by fluorescence microscopy or flow cytometry. Further, light microscopy reveals the unique appearance of star-shaped hepatic stellate cells that harbor high amounts of lipid droplets. 


 Taken together, we present a detailed protocol for the efficient isolation of hepatic stellate cells, including representative images of their morphological appearance and GFAP expression that help to define the stellate cell entity.

Here we describe a method for the isolation of hepatic stellate cells from mouse liver. For stellate cell purification, mouse livers are digested in situ and in vitro by pronase-collagenase treatment prior to density gradient centrifugation. This technique yields highly pure hepatic stellate cells.

Семь шагов к звездчатых клеток

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of Listeria, which results in systemic infection2. After injection, Listeria quickly disseminates to the spleen and liver due to uptake by CD8&alpha;+ dendritic cells and Kupffer cells3,4. Once phagocytosed, various bacterial proteins enable Listeria to escape the phagosome, survive within the cytosol, and infect neighboring cells5. During the first three days of infection, different innate immune cells (e.g. monocytes, neutrophils, NK cells, dendritic cells) mediate bactericidal mechanisms that minimize Listeria proliferation. CD8+ T cells are subsequently recruited and responsible for the eventual clearance of Listeria from the host, typically within 10 days of infection6.
Successful clearance of Listeria from infected mice depends on the appropriate onset of host immune responses6	. There is a broad range of sensitivities amongst inbred mouse strains7,8. Generally, mice with increased susceptibility to Listeria infection are less able to control bacterial proliferation, demonstrating increased bacterial load and/or delayed clearance compared to resistant mice. Genetic studies, including linkage analyses and knockout mouse strains, have identified various genes for which sequence variation affects host responses to Listeria infection6,8-14. Determination and comparison of infection kinetics between different mouse strains is therefore an important method for identifying host genetic factors that contribute to immune responses against Listeria. Comparison of host responses to different Listeria strains is also an effective way to identify bacterial virulence factors that may serve as potential targets for antibiotic therapy or vaccine design.
We describe here a straightforward method for measuring bacterial load (colony forming units [CFU] per tissue) and preparing single-cell suspensions of the liver and spleen for FACS analysis of immune responses in Listeria-infected mice. This method is particularly useful for initial characterization of Listeria infection in novel mouse strains, as well as comparison of immune responses between different mouse strains infected with Listeria. We use the Listeria monocytogenes EGD strain15 that, when cultured on blood agar, exhibits a characteristic halo zone around each colony due to &beta;-hemolysis1 (Figure 1). Bacterial load and immune responses can be determined at any time-point after infection by culturing tissue homogenate on blood agar plates and preparing tissue cell suspensions for FACS analysis using the protocols described below. We would note that individuals who are immunocompromised or pregnant should not handle Listeria, and the relevant institutional biosafety committee and animal facility management should be consulted before work commences.

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes is a model organism for studying immune responses and genetic susceptibility to intracellular bacteria in mice. This method enables one to measure bacterial load and generate single-cell suspensions of the liver and spleen from mice for FACS analysis to determine changes in immune cells due to Listeria infection.

Измерение бактериальной нагрузки и иммунного ответа в мышей, инфицированных Listeria моноцитогенес</em

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of ...

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

Представляем напряжения сдвига в изучении бактериальной адгезии

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. 1A-C), females of the genus Leptopilina (Fig. 1D, 1F, 1G) and Ganaspis (Fig. 1E) attack the larval stages. We use these parasites to study the molecular basis of a biological arms race. Parasitic wasps have tremendous value as biocontrol agents. Most of them carry virulence and other factors that modify host physiology and immunity. Analysis of Drosophila wasps is providing insights into how species-specific interactions shape the genetic structures of natural communities. These studies also serve as a model for understanding the hosts' immune physiology and how coordinated immune reactions are thwarted by this class of parasites.
The larval/pupal cuticle serves as the first line of defense. The wasp ovipositor is a sharp needle-like structure that efficiently delivers eggs into the host hemocoel. Oviposition is followed by a wound healing reaction at the cuticle (Fig. 1C, arrowheads). Some wasps can insert two or more eggs into the same host, although the development of only one egg succeeds. Supernumerary eggs or developing larvae are eliminated by a process that is not yet understood. These wasps are therefore referred to as solitary parasitoids.
Depending on the fly strain and the wasp species, the wasp egg has one of two fates. It is either encapsulated, so that its development is blocked (host emerges; Fig. 2 left); or the wasp egg hatches, develops, molts, and grows into an adult (wasp emerges; Fig. 2 right). L. heterotoma is one of the best-studied species of Drosophila parasitic wasps. It is a "generalist," which means that it can utilize most Drosophila species as hosts1. L. heterotoma and L. victoriae are sister species and they produce virus-like particles that actively interfere with the encapsulation response2. Unlike L. heterotoma, L. boulardi is a specialist parasite and the range of Drosophila species it utilizes is relatively limited1. Strains of L. boulardi also produce virus-like particles3 although they differ significantly in their ability to succeed on D. melanogaster1. Some of these L. boulardi strains are difficult to grow on D. melanogaster1 as the fly host frequently succeeds in encapsulating their eggs. Thus, it is important to have the knowledge of both partners in specific experimental protocols.
In addition to barrier tissues (cuticle, gut and trachea), Drosophila larvae have systemic cellular and humoral immune responses that arise from functions of blood cells and the fat body, respectively. Oviposition by L. boulardi activates both immune arms1,4. Blood cells are found in circulation, in sessile populations under the segmented cuticle, and in the lymph gland. The lymph gland is a small hematopoietic organ on the dorsal side of the larva. Clusters of hematopoietic cells, called lobes, are arranged segmentally in pairs along the dorsal vessel that runs along the anterior-posterior axis of the animal (Fig. 3A). The fat body is a large multifunctional organ (Fig. 3B). It secretes antimicrobial peptides in response to microbial and metazoan infections.
Wasp infection activates immune signaling (Fig. 4)4. At the cellular level, it triggers division and differentiation of blood cells. In self defense, aggregates and capsules develop in the hemocoel of infected animals (Fig. 5)5,6. Activated blood cells migrate toward the wasp egg (or wasp larva) and begin to form a capsule around it (Fig. 5A-F). Some blood cells aggregate to form nodules (Fig. 5G-H). Careful analysis reveals that wasp infection induces the anterior-most lymph gland lobes to disperse at their peripheries (Fig. 6C, D).
We present representative data with Toll signal transduction pathway components Dorsal and Sp&auml;tzle (Figs. 4,5,7), and its target Drosomycin (Fig. 6), to illustrate how specific changes in the lymph gland and hemocoel can be studied after wasp infection. The dissection protocols described here also yield the wasp eggs (or developing stages of wasps) from the host hemolymph (Fig. 8).

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Parasitoid (parasitic) wasps constitute a major class of natural enemies of many insects including Drosophila melanogaster. We will introduce the techniques to propagate these parasites in Drosophila spp. and demonstrate how to analyze their effects on immune tissues of Drosophila larvae.

Введение в паразитических наездников<em&gt; Drosophila</em&gt; И противопаразитарной иммунного ответа

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. ...

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Throughout the last years, the contribution of alveolar type II epithelial cells (AECII) to various aspects of immune regulation in the lung has been increasingly recognized. AECII have been shown to participate in cytokine production in inflamed airways and to even act as antigen-presenting cells in both infection and T-cell mediated autoimmunity 1-8. Therefore, they are especially interesting also in clinical contexts such as airway hyper-reactivity to foreign and self-antigens as well as infections that directly or indirectly target AECII. However, our understanding of the detailed immunologic functions served by alveolar type II epithelial cells in the healthy lung as well as in inflammation remains fragmentary. Many studies regarding AECII function are performed using mouse or human alveolar epithelial cell lines 9-12. Working with cell lines certainly offers a range of benefits, such as the availability of large numbers of cells for extensive analyses. However, we believe the use of primary murine AECII allows a better understanding of the role of this cell type in complex processes like infection or autoimmune inflammation. Primary murine AECII can be isolated directly from animals suffering from such respiratory conditions, meaning they have been subject to all additional extrinsic factors playing a role in the analyzed setting. As an example, viable AECII can be isolated from mice intranasally infected with influenza A virus, which primarily targets these cells for replication 13. Importantly, through ex vivo infection of AECII isolated from healthy mice, studies of the cellular responses mounted upon infection can be further extended.
Our protocol for the isolation of primary murine AECII is based on enzymatic digestion of the mouse lung followed by labeling of the resulting cell suspension with antibodies specific for CD11c, CD11b, F4/80, CD19, CD45 and CD16/CD32. Granular AECII are then identified as the unlabeled and sideward scatter high (SSChigh) cell population and are separated by fluorescence activated cell sorting 3.
In comparison to alternative methods of isolating primary epithelial cells from mouse lungs, our protocol for flow cytometric isolation of AECII by negative selection yields untouched, highly viable and pure AECII in relatively short time. Additionally, and in contrast to conventional methods of isolation by panning and depletion of lymphocytes via binding of antibody-coupled magnetic beads 14, 15, flow cytometric cell-sorting allows discrimination by means of cell size and granularity. Given that instrumentation for flow cytometric cell sorting is available, the described procedure can be applied at relatively low costs. Next to standard antibodies and enzymes for lung disintegration, no additional reagents such as magnetic beads are required. The isolated cells are suitable for a wide range of functional and molecular studies, which include in vitro culture and T-cell stimulation assays as well as transcriptome, proteome or secretome analyses 3, 4.

We describe the rapid isolation of primary murine type II alveolar epithelial cells (AECII) by flow cytometric negative selection. These AECII show high viability and purity and are suitable for a wide range of functional and molecular studies regarding their role in respiratory conditions such as autoimmune or infectious diseases.

Проточной цитометрии Выделение первичных мышиных Type II альвеолярных эпителиальных клеток для функционального и молекулярных исследований

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

Quantitative Measurement of the Immune Response and Sleep in Drosophila

A complex interaction between the immune response and host behavior has been described in a wide range of species. Excess sleep, in particular, is known to occur as a response to infection in mammals 1 and has also recently been described in Drosophila melanogaster2. It is generally accepted that sleep is beneficial to the host during an infection and that it is important for the maintenance of a robust immune system3,4. However, experimental evidence that supports this hypothesis is limited4, and the function of excess sleep during an immune response remains unclear. We have used a multidisciplinary approach to address this complex problem, and have conducted studies in the simple genetic model system, the fruitfly Drosophila melanogaster. We use a standard assay for measuring locomotor behavior and sleep in flies, and demonstrate how this assay is used to measure behavior in flies infected with a pathogenic strain of bacteria. This assay is also useful for monitoring the duration of survival in individual flies during an infection. Additional measures of immune function include the ability of flies to clear an infection and the activation of NF&kappa;B, a key transcription factor that is central to the innate immune response in Drosophila. Both survival outcome and bacterial clearance during infection together are indicators of resistance and tolerance to infection. Resistance refers to the ability of flies to clear an infection, while tolerance is defined as the ability of the host to limit damage from an infection and thereby survive despite high levels of pathogen within the system5. Real-time monitoring of NF&kappa;B activity during infection provides insight into a molecular mechanism of survival during infection. The use of Drosophila in these straightforward assays facilitates the genetic and molecular analyses of sleep and the immune response and how these two complex systems are reciprocally influenced.

Quantitative Measurement of the Immune Response and Sleep in Drosophila

To understand a link between the immune response and behavior, we describe a method to measure locomotor behavior in Drosophila during bacterial infection as well as the ability of flies to mount an immune response by monitoring survival, bacterial load, and real-time activity of a key regulator of innate immunity, NF&kappa;B.

Количественные измерения иммунного ответа и сна в<em&gt; Drosophila</em

A complex interaction between the immune response and  host behavior has been described in a wide range of species. Excess sleep, in  particular, is known ...

Bacterial Leaf Infiltration Assay for Fine Characterization of Plant Defense Responses using the Arabidopsis thaliana-Pseudomonas syringae Pathosystem

In the absence of specialized mobile immune cells, plants utilize their localized programmed cell death and Systemic Acquired Resistance to defend themselves against pathogen attack. The contribution of a specific Arabidopsis gene to the overall plant immune response can be specifically and quantitatively assessed by assaying the pathogen growth within the infected tissue. For over three decades, the hemibiotrophic bacterium Pseudomonas syringae pv. maculicola ES4326 (Psm ES4326) has been widely applied as the model pathogen to investigate the molecular mechanisms underlying the Arabidopsis immune response. To deliver pathogens into the leaf tissue, multiple inoculation methods have been established, e.g., syringe infiltration, dip inoculation, spray, vacuum infiltration, and flood inoculation. The following protocol describes an optimized syringe infiltration method to deliver virulent Psm ES4326 into leaves of adult soil-grown Arabidopsis plants and accurately screen for enhanced disease susceptibility (EDS) towards this pathogen. In addition, this protocol can be supplemented with multiple pre-treatments to further dissect specific immune defects within different layers of plant defense, including Salicylic Acid (SA)-Triggered Immunity (STI) and MAMP-Triggered Immunity (MTI).

Bacterial Leaf Infiltration Assay for Fine Characterization of Plant Defense Responses using the Arabidopsis thaliana-Pseudomonas syringae Pathosystem

Quantification of pathogen growth is a powerful tool to characterize various Arabidopsis thaliana (hereafter: Arabidopsis) immune responses. The method described here presents an optimized syringe infiltration assay to quantify the Pseudomonas syringae pv. maculicola ES4326 growth in adult Arabidopsis leaves.

Бактериальный лист инфильтрация Анализ на тонкой Характеристика защитных реакций растений с помощью<em&gt; Резуховидка Таля-Pseudomonas syringae</em&gt; Pathosystem

In the absence of specialized mobile immune cells, plants utilize their localized programmed cell death and Systemic Acquired Resistance to defend ...

The Ex Vivo Colon Organ Culture and Its Use in Antimicrobial Host Defense Studies

The intestine displays an architecture of repetitive crypt structures consisting of different types of epithelial cells, lamina propia containing immune cells, and stroma. All of these heterogeneous cells contribute to intestinal homeostasis and participate in antimicrobial host defense. Therefore, identifying a surrogate model for studying immune response and antimicrobial activity of the intestine in an in vitro setting is extremely challenging. In vitro studies using immortalized intestinal epithelial cell lines or even primary crypt organoid culture do not represent the exact physiology of normal intestine and its microenvironment. Here, we discuss a method of culturing mouse colon tissue in a culture dish and how this ex vivo organ culture system can be implemented in studies related to antimicrobial host defense responses. In representative experiments, we showed that colons in organ culture express antimicrobial peptides in response to exogenous IL-1&#946; and IL-18. Further, the antimicrobial effector molecules produced by the colon tissues in the organ culture efficiently kill Escherichia coli in vitro. This approach, therefore, can be utilized to dissect the role of pathogen- and danger-associated molecular patterns and their cellular receptors in regulating intestinal innate immune responses and antimicrobial host defense responses.

The Ex Vivo Colon Organ Culture and Its Use in Antimicrobial Host Defense Studies

The ex vivo organ culture allows investigation of biological processes in the context of the intact tissue architecture. Here, we introduce a method of ex vivo culture of the mouse colon, which can be used to study innate immunity and antimicrobial host defense in the intestine.

<em&gt; Ex Vivo</em&gt; Colon органной культуры и ее использование в Антимикробная хост оборонных исследований

The intestine displays an architecture of repetitive crypt structures consisting of different types of epithelial cells, lamina propia containing immune ...

Extraction of Hemocytes from Drosophila melanogaster Larvae for Microbial Infection and Analysis

During the pathogenic infection of Drosophila melanogaster, hemocytes play an important role in the immune response throughout the infection. Thus, the goal of this protocol is to develop a method to visualize the pathogen invasion in a specific immune compartment of flies, namely hemocytes. Using the method presented here, up to 3 &#215; 106 live hemocytes can be obtained from 200 Drosophila 3rd instar larvae in 30 min for ex vivo infection. Alternatively, hemocytes can be infected in vivo through injection of 3rd instar larvae followed by hemocyte extraction up to 24 h post-infection. These infected primary cells were fixed, stained, and imaged using confocal microscopy. Then, 3D representations were generated from the images to definitively show pathogen invasion. Additionally, high-quality RNA for qRT-PCR can be obtained for the detection of pathogen mRNA following infection, and sufficient protein can be extracted from these cells for Western blot analysis. Taken together, we present a method for definite reconciliation of pathogen invasion and confirmation of infection using bacterial and viral pathogen types and an efficient method for hemocyte extraction to obtain enough live hemocytes from Drosophila larvae for ex vivo and in vivo infection experiments.

Extraction of Hemocytes from Drosophila melanogaster Larvae for Microbial Infection and Analysis

This method demonstrates how to visualize pathogen invasion into insect cells with three-dimensional (3D) models. Hemocytes from Drosophila larvae were infected with viral or bacterial pathogens, either ex vivo or in vivo. Infected hemocytes were then fixed and stained for imaging with a confocal microscope and subsequent 3D cellular reconstruction.

Извлечение Hemocytes из личинки Drosophila melanogaster микробной инфекции и анализа

During the pathogenic infection of Drosophila melanogaster, hemocytes play an important role in the immune response throughout the infection. Thus, the ...

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our knowledge of prevention and treatment of viral infection. The fruit fly Drosophila melanogaster has proven to be one of the most efficient and productive model organisms to screen for antiviral factors and investigate virus-host interaction, due to powerful genetic tools and highly conserved innate immune signaling pathways. The procedure described here demonstrates a nano-injection method to establish viral infection and induce systemic antiviral responses in adult flies. The precise control of the viral injection dose in this method enables high experimental reproducibility. Protocols described in this study include the preparation of flies and the virus, the injection method, survival rate analysis, the virus load measurement, and an antiviral pathway assessment. The influence effects of viral infection by the flies' background were mentioned here. This infection method is easy to perform and quantitatively repeatable; it can be applied to screen for host/viral factors involved in virus-host interaction and to dissect the crosstalk between innate immune signaling and other biological pathways in response to viral infection.

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

This protocol describes how to establish viral infection in vivo in Drosophila melanogaster using the nano-injection method and basic techniques to analyze virus-host interaction.

Создание вирусной инфекции и анализа взаимодействия хост вирус в Drosophila Melanogaster

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our ...