这项工作得到了UCSD启动基金的一部分支持，这些基金被引导到O.S.A.

1. C. 五角星殖民地饲养
<ol><li>在虫子宿舍笼子里建立多个 C. 五角星 成人的殖民地。 注：殖民地是由劳拉哈林顿博士在康奈尔大学20。饲养 Culex 蚊子的详细方案可以在其他文献21中找到。<ol>
<li>将蚊子保持在 25 ± 1 °C 的湿度为 30%，白天：夜间周期为 12：12 小时。</li>
		<li>通过将带芯的糖溶液容器引入笼子或用糖溶液饱和棉球并将其放入笼子中，提供 20% 的糖溶液。</li>
	</ol>
</li>
	<li>允许蚊子在喂血前交配至少3天（图2A）。</li>
</ol>2. 收集 C. 五角星 前胚芽阶段胚胎
<ol><li>闭合后3-6天后，通过合成膜为女性提供1-2兆加仑的受cite化牛血餐，并在血液喂养系统中加热至约40°C（图2B）。也有替代血液喂养协议，可用于Culex蚊子（例如，见参考21）。 注：C.五角星以偏爱鸟类血液而闻名。一些实验室使用麻醉活禽或禽类血液作为他们的血餐来源21，22，23。然而，如果选择这种功能超过几代人，可以训练/选择以牛血或小啮齿动物为食的菌落。<ol>
<li>允许女性在喂血后至少休息3天，进行生化和卵子成熟，然后诱导卵化进行微注射实验。</li>
	</ol>
</li>
	<li>在胚胎微注射当天，用有机注入的卵泡水创建一个卵泡杯。在24日、25日5天或5天以上，应通过发酵兔粪（50克/升）、分解草（4.5克/升）或鱼食（25克/升）在水中进行排卵。</li>
	<li>将卵泡杯放入笼子中，将整个笼子放入黑暗位置（图2C）。</li>
	<li>每30分钟后，检查杯子是否有蛋筏。<ol>
<li>如果有蛋筏存在，用画笔铲取木筏，放在湿滤纸上（图2D，E）。</li>
	</ol>
</li>
</ol>3. 对齐 C.五角星 前胚芽阶段胚胎
<ol><li>将鸡蛋从木筏上分离出来，压在木筏上，然后用细尖画笔和钳子单独挑逗鸡蛋（图2E）。</li>
	<li>将单个鸡蛋对准放在玻璃滑梯顶部的双面胶带的细条上（图 2F）。</li>
	<li>对齐时，尝试将每个鸡蛋的前侧指向同一方向，以便于访问。 注：在先前公布的 纳索尼亚维特里彭尼斯 胚胎微注射协议26中，可以找到一种没有双面粘胶带的替代卵子对齐方法。</li>
	<li>用卤油混合物盖住鸡蛋。 注：卤碳混合可以提前准备，轻轻混合两个卤碳试剂和水（9：1：20，卤碳700：卤碳27：超纯水），然后在25+C孵育混合物过夜，以促进卤化碳油的水饱和。</li>
</ol>4. 微注射针头制备
<ol><li>使用玻璃微管拉拔器生成氧化铝硅酸盐毛细管玻璃针。<ol>
<li>根据针拔器用户手册的说明，将氧化铝毛细细玻璃放入针拔器中。 注：石英和铜硅酸盐毛细管针也可以使用，但对于这个实验，氧化铝酸盐是首选，因为它的相对承受能力和耐用性。</li>
		<li>将热度设置为 516，速度到 100，延迟到 70，拉到 97，压力到 500 对针拔拔器。</li>
		<li>根据拔针器的说明激活拔针器，并根据需要重复其他针头。 注：在注射过程中，针头经常堵塞或意外断裂，因此，强烈建议为单个实验拔出额外的针头。</li>
	</ol>
</li>
	<li>以 50° 角轻轻触摸旋转的钻石磨料板上拉针的尖端，将针尖倾斜约 10s。 注：将针头打开，让液体流过，同时创建更锋利的尖端，以便更容易地渗透到胚胎中。在先前第26条中可以看到适当的针头的例子。</li>
	<li>将针头嵌入到培养皿中的模具粘土线中，从而将针头粘接并斜接。 注：为确保针尖的最佳质量，针头应尽可能接近注射时间，新鲜拔出并斜面。</li>
</ol>5. 加载注射混合物
<ol><li>准备由基因组修饰试剂（例如，200 ng/μL sgRNA 和 200 ng/μL Cas9 混合物）组成的注射混合物，或首选的注射解决方案，并将其保留在冰上。 注：这种混合物可以在等待产卵时准备。有关Cas9和sgRNA生产和微注射准备的更多细节可以在以前的出版物27，28，29，30找到。</li>
	<li>使用微加载器尖端将 2 μL 的注射混合物装载到注射针中。</li>
</ol>6. 设置微投射
<ol><li>将填充的注射针放入与电子微喷射器相连的微操纵器中。</li>
	<li>将装有对齐鸡蛋的玻璃滑梯放在复合显微镜的舞台上。</li>
	<li>使用显微操纵器和复合显微镜，将针头对齐，以 25-35° 角瞄准胚胎的后端。</li>
</ol>7. 胚胎微注射 （图2G）
<ol><li>小心地将针头插入胚胎，并按胚胎体积的10%（根据卵子大小的大小，为700-800 pL）注入混合物。 注：注射时，卵子应稍微膨胀，但是，如果注入过多的液体，卵子可能会破裂，或者细胞质液体可能会泄漏出来。</li>
	<li>一次注射约20个卵子，然后停止并执行胚胎恢复程序。 注意：注射时，针头堵塞或破裂的可能性很大。如果发生堵塞，这可以通过缺乏注射液流过针头来确定，尝试用电子微喷射器上的清洁功能清理针头，或者尝试重新梳理针头。如果这两个步骤都不起作用，快速换上新针头，同时确保准备好的鸡蛋保持湿润。</li>
</ol>8. 胚胎恢复和孵化
<ol><li>让鸡蛋不受干扰至少5分钟。注射后20分钟内，用干净的画笔轻轻刷抹，小心地去除卤化碳油（图2H）。</li>
	<li>用画笔轻轻提起鸡蛋，放入一杯双蒸馏水中（图2I）。小心把鸡蛋放在水面上。</li>
	<li>每天检查卵子7天进行孵化（图2J）。</li>
	<li>遵循正常的幼虫饲养程序21。</li>
</ol>9. 基因组修饰筛查
<ol><li>使用立体镜（图2K）对注射的蚊子进行变异表型筛查。<ol>
<li>通过 PCR 放大、T7 Endonuclease I 检测和通过对目标区域31进行分包来验证没有易于识别表型的突变。</li>
	</ol>
</li>
	<li>在注射个体之间设置额外的交叉，以检测细菌系内的可染异突变。</li>
</ol>

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NEB Available from: <a target="_blank" href="https://www.neb.com/protocols/2014/08/11/determining-genome-targeting-efficiency-using-t7-endonuclease-i">https://www.neb.com/protocols/2014/08/11/determining-genome-targeting-efficiency-using-t7-endonuclease-i</a> (2020)</li></ol>

最近，为了生产用于蚊媒控制的基因工程工具，有必要为常见的蚊子病媒开发和优化胚胎微注射方案。虽然已经为 伊蚊 和 阿诺菲勒斯 蚊子开发了方法，但专门为 Culex 设计的协议已经很少被探索。一般蚊子胚胎注射方案可分为3个一般步骤：1）胚胎采集和制备，2）胚胎注射，3）注射后恢复。为了成功生成突变体，必须针对目标物种优化所有三个步骤。这个经过修改的胚胎微注射方案是 库莱克斯 蚊子基因组工程成功的专用方案。
最大化胚胎收集是胚胎微注射协议中常见的瓶颈。为了在短时间内增加收集的卵子数量，蚊子在血餐后2-3天内被限制在卵小板上。需要注意的是，与哺乳动物血源或用哺乳动物血液喂养的膜相比，C.五角星倾向于选择鸟类血源。然而，许多研究人员很难获得可靠的活禽或禽类血液来源供血液喂养，因此实验室库存可以适应以更容易获得的血液来源为食。使用富含营养的水作为卵小板也至关重要，因为它为C. 五角星和大多数其他Culex载体提供卵液提示。有许多方法可以产生富含营养的水，可能需要在实验室之间进行优化，以确保有适当数量的鸡蛋可用于微注射。例如，虽然这种方法最成功，兔粪在去离子水中发酵5天或更长的时间，但其他研究人员更成功地将草或鱼食作为营养来源，有些甚至用蒸馏水21。
由于 Culex 蚊子在木筏上产卵，收集卵子相当简单。鸡蛋只需用画笔铲起整个木筏即可收集。鸡蛋分离更为复杂，但对成功注射至关重要。鸡蛋可以通过用画笔或钳子在鸡蛋之间施加温和的向下压力，小心地从木筏上分离出来。通过一些实践，多个用户能够可靠地将单个鸡蛋从蛋筏中分离出来。分离每个鸡蛋后，鸡蛋在双面胶带上以相同的方向对齐，在微喷射过程中稳定鸡蛋。鸡蛋被注射到锥形的后端，添加光环碳油使鸡蛋在操作过程中保持湿润。
为了限制微注射过程中的胚胎损伤，注射针头需要具有适当的强度，并以适当的角度斜面。氧化铝针以50°角斜面10秒，效果最佳，但氧化硅酸盐和石英针可能也有效，即使它们可能不太耐用，成本更高。此外，适当的针斜面有助于更好的Cas9和sgRNA混合物的流动，并创造一个更尖锐的点，更容易渗透到胚胎。在许多情况下，斜面也是快速修复堵塞针头的有效方法，而不是花费时间和精力来更换堵塞的针头。
注射后，鸡蛋应保持至少5分钟的不受干扰，然后用干净的画笔轻轻刷掉光环碳油。去除卤化碳油后，注射的卵子可以放入水中孵化，通常在注射后3天内发生。通过实践和充足的卵子数量，该协议可以在 C.五角星 中实现一致的体细胞和生殖系突变，并且具有足够的多功能性，因此它应该能够很容易地适应其他 Culex 蚊子。

C. 五角星，俗称南方家蚊，是多种病原体的合格载体，包括西尼罗河病毒（WNV）、日本脑炎、圣路易斯脑炎和东方马脑炎。特别是，自1999年在纽约首次发现WNV以来，WNV已成为美国大陆（美国）的主要病媒传播疾病，1999年至2018年间，超过50，000例人类病例报告，导致约2，300人死亡，2008-2019年间报告了4，500多例马匹病例。此外，在北美发现的至少23种鸟类受到WNV感染的影响，至少有12种被归类为无法恢复的物种，由于WNV3。WNV对人类、马匹和鸟类种群的影响是由于其载体的机会性喂养行为造成的。通常，鸟类是WNV的主要宿主，人类和马匹是偶然的或死胡同的宿主。一些病原体由C.五角星病媒，只感染鸟类，如禽流感寄生虫，疟原虫。在夏威夷，C.五角星是禽流感的主要传播媒介，并导致许多本地鸟类物种4，5的灭绝。
为了控制由C.五角星传播的疾病，研究人员和病媒控制机构已经使用了常见的蚊虫种群控制工具，如杀虫剂应用6，但是，这些方法成本高昂，不针对物种，而且效果有限，因为许多C.五角星种群的抗杀虫能力很高，6、7、8、9。其他控制技术，如基于沃尔巴契亚的人口控制策略近年来已经开发了10，11，但与沃尔巴契亚感染相关的健身成本限制了这种方法的可行性，为这个载体12。其他蚊子物种，如伊蚊13号、14号、阿诺菲斯甘比亚15号和阿诺菲斯·斯蒂芬西16号，也都开发了基于遗传的控制方法，包括开发抗病原体蚊子17、18、19，如果必要的基因组工程工具是的话，这些方法也可以为C.quinquefasciatus开发 为这个物种开发。然而，C.五角星生物学与其他伊蚊和阿诺菲勒斯蚊子病媒有很大的不同，这使得这种病媒中类似遗传技术的发展变得困难。随着基于CRISPR的基因组工程技术的出现，精确的基因组工程变得越来越琐碎、经济、适应能力强，从而导致各种物种中新颖的遗传工具的发展。
为了利用CRISPR技术产生突变，Cas9蛋白质和合成导引RNA（sgRNA）的混合物被微注入到爆炸前阶段胚胎中。由于 C.五角星 雌性在漂浮的木筏结构（图1）中分组产卵，而不是卵巢个体卵子， 即伊蚊 和 阿诺菲斯 蚊子的特征，胚胎微注射在这个物种中变得越来越复杂。 Culex 幼虫也从每个卵子的前侧出现，这是与水面接触（图1），所以鸡蛋定向后操作在这个物种中很重要。在这里，我们描述了一个详细的协议，旨在将Cas9蛋白质和sgRNA微注射到 C.五角星 胚胎中。该协议旨在适应 Culex 生物学特有的特征，以便通过某些步骤提高胚胎存活率和基因组突变率，这些步骤是及时采集卵子和卵子存活的关键。

<table>
	<tbody>
		<tr>
			<td>9 oz clear plastic pet cup</td>
			<td>Karat</td>
			<td>C-KC9</td>
			<td>Insect Rearing and Egg Collection</td>
		</tr>
		<tr>
			<td>Aluminosilicate glass capillary tubing 1mm(outside diameter) X 0.58mm (inner diameter)</td>
			<td>Sutter Instruments</td>
			<td>BF100-58-10</td>
			<td>Microinjection, borosilicate and quartz needles could also be used but we prefered aluminosilicate</td>
		</tr>
		<tr>
			<td>Blood</td>
			<td>Colorado Serum Company</td>
			<td>31025</td>
			<td>The colony took multiple generations to adapt to this blood source. Other blood sources are likely just as appropriate. See protocol notes on blood source selection.</td>
		</tr>
		<tr>
			<td>Bugdorm</td>
			<td>Bugdorm</td>
			<td>4F2222</td>
			<td>Insect Rearing Cage</td>
		</tr>
		<tr>
			<td>Cas9 Protein with NLS</td>
			<td>PNABio</td>
			<td>CP01</td>
			<td>Microinjection</td>
		</tr>
		<tr>
			<td>Compound Microscope</td>
			<td>Olympus BX41</td>
			<td></td>
			<td>Microinjection, for embryo injection</td>
		</tr>
		<tr>
			<td>Diamond abrasive plate (0.7u to 2.0u tip sizes)</td>
			<td>Sutter Instruments</td>
			<td>104E</td>
			<td>Microinjection, to be used with beveler</td>
		</tr>
		<tr>
			<td>DNase/RNase-Free distilled Water</td>
			<td>Invitrogen</td>
			<td>10977-015</td>
			<td>Microinjection</td>
		</tr>
		<tr>
			<td>Double-sided Tape</td>
			<td>Scotch</td>
			<td>B084NVQGXD</td>
			<td>Microinjection, embryo alignment</td>
		</tr>
		<tr>
			<td>Femtojet 4x or 4i programmable microinjector</td>
			<td>Eppendorf</td>
			<td></td>
			<td>Microinjection</td>
		</tr>
		<tr>
			<td>Femtotips Microloader tips</td>
			<td>Fisher Scientific</td>
			<td>E5242956003</td>
			<td>Microinjection</td>
		</tr>
		<tr>
			<td>Filter Paper</td>
			<td>Whatman</td>
			<td>1001-090</td>
			<td>Microinjection, embryo collection</td>
		</tr>
		<tr>
			<td>Fine-tip paintbrush</td>
			<td>ZEM</td>
			<td>2595</td>
			<td>Microinjection, embryo alignment</td>
		</tr>
		<tr>
			<td>Halocarbon oil 27</td>
			<td>Sigma-Aldrich</td>
			<td>H8773</td>
			<td>Microinjection, embryo alignment</td>
		</tr>
		<tr>
			<td>Halocarbon oil 700</td>
			<td>Sigma-Aldrich</td>
			<td>H8898</td>
			<td>Microinjection, embryo alignment</td>
		</tr>
		<tr>
			<td>Hemotek</td>
			<td>Hemotek</td>
			<td>PS5</td>
			<td>Line Maintenance</td>
		</tr>
		<tr>
			<td>Microelectrode Beveler</td>
			<td>Sutter Instruments</td>
			<td>BV10</td>
			<td>Microinjection, needle beveling</td>
		</tr>
		<tr>
			<td>Micropipette Puller</td>
			<td>Sutter Instruments</td>
			<td>P-1000 or P-2000</td>
			<td>Microinjection, needle pulling</td>
		</tr>
		<tr>
			<td>Microscope Slides</td>
			<td>Fisherbrand</td>
			<td>12-550-A3</td>
			<td>Microinjection, embryo alignment</td>
		</tr>
		<tr>
			<td>Non-Drying Modeling Clay</td>
			<td>Jovi</td>
			<td>B0025Z71IM</td>
			<td>Microinjection, needle storage</td>
		</tr>
		<tr>
			<td>Stereo Microscope</td>
			<td>Olympus</td>
			<td>SZ51</td>
			<td>Microinjection, for embryo alignment</td>
		</tr>
		<tr>
			<td>Sugar</td>
			<td>Domino</td>
			<td></td>
			<td>20% sugar solution for adult sugar source.</td>
		</tr>
		<tr>
			<td>T7 Endonuclease I</td>
			<td>NEB</td>
			<td>M0302</td>
			<td>Preparation of microinjection materials</td>
		</tr>
		<tr>
			<td>TOPO TA Cloning Kit</td>
			<td>ThermoFischer Scientific</td>
			<td>K451020</td>
			<td>Preparation of microinjection materials</td>
		</tr>
		<tr>
			<td>Ultra-fine tip forceps</td>
			<td>Fisher Scientific</td>
			<td>16-100-121</td>
			<td>Microinjection, embryo alignment</td>
		</tr>
	</tbody>
</table>

embryo microinjection techniques for efficient site-specific mutagenesis in culex quinquefasciatus

Culex 五角星 是多种病媒传播疾病的载体，如禽流感、西尼罗河病毒 （WNV）、日本脑炎、东马脑炎、淋巴丝虫病和圣路易斯脑炎。值得注意的是，禽流感在众多特有岛屿鸟类物种的灭绝中发挥了重要作用，而WNV已成为美国重要的病媒传播疾病。为了进一步深入了解 C.五角星 生物学，并扩大其基因控制工具箱，我们需要开发更有效和负担得起的方法，在这个物种的基因组工程。然而 ，Culex 蚊子特有的一些生物特征，特别是它们的卵筏，使得基因组工程所需的微注射程序变得困难。为了应对这些挑战，我们开发了一个优化的胚胎微注射方案，重点是缓解与 Culex 蚊子的独特特性相关的技术障碍。这些程序演示了鸡蛋收集、蛋筏分离和其他处理程序的优化方法，这些方法对于 C.五角星 的显微注射是必不可少的。当与CRISPR/Cas9基因组编辑技术相结合时，这些程序使我们能够实现特定地点、高效和遗传性生殖系突变，这些突变是执行高级基因组工程和开发这一重要但目前研究不足的疾病载体的遗传控制技术所必需的。

在先前发表的实验中，该方法用于成功生成对黑眼色素相容（CPIJ005542）30（表1）发育至关重要的基因的体细胞和生殖系突变。CRISPR/Cas9产生的体突变是通过筛查注射个体（G0）的阴唇阶段眼睛色素沉着损失而得分的。体细胞突变通常以马赛克表型存在，其中有些（但不是所有细胞）都有突变表型。例如，白色基因的体质突变导致奥马提亚与缺乏色素沉着的表型和具有野生型色素表型30的混合物。然而，当白基因发生生殖系突变时，下一代（G1）继承了全白眼表型。细菌线突变率是由交叉马赛克G0个体和得分为完全白眼的后代（G1）决定的。这些实验的结果是胚胎存活率为64%至82%，体细胞突变率为37%至57%，生殖系突变率为>61%（表1）。通过多重sgRNA瞄准同一基因中的多重位点，体质和生殖系突变率上升到高达86%（表1）。此外，几代人以来，在实验室中成功地保存了白突变体的可行同源种群。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61375/61375fig01.jpg"> 图 1：C.五角 星蛋筏和单卵形态。 多萨尔（A），横向（B） 和通风口（C） 视图 C 五角 星蛋筏。 C. 五角星 雌性产卵时，球状前侧接触水面越多，而后部离水面越尖（D）。A- 前：P- 后请 <a href="https://www.jove.com/files/ftp_upload/61375/61375fig01large.jpg" target="_blank">点击这里查看此图的较大版本。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61375/61375fig2v2.jpg"> 图2：通过微注射创建 C.五角星 突变体的时间轴。 C.五角星群落制备和胚胎收集的微注射（A-D）时间表。收集的鸡蛋然后分离（E） 对齐在同一方向，并覆盖光环碳油（F）。然后注射鸡蛋（G），并允许休息至少5分钟，然后去除卤化碳油（H）。然后将卵子转移到清洁的水源（I）孵化（J），并筛选突变表型（K）。<a href="https://www.jove.com/files/ftp_upload/61375/61375fig2v2large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td></td>
			<td></td>
			<td colspan="3">生存</td>
			<td colspan="3">躯体马赛克 （G0）</td>
			<td>格姆林突变体 （G1）</td>
		</tr>
<tr>
<td>斯格纳</td>
			<td>#injected</td>
			<td>♂</td>
			<td>♀</td>
			<td>总计（%）</td>
			<td>♂</td>
			<td>♀</td>
			<td>总计（%）</td>
			<td>G1 突变体 （%）</td>
		</tr>
<tr>
<td>wsgrna-1</td>
			<td>50</td>
			<td>15</td>
			<td>20</td>
			<td>35(70)</td>
			<td>7(47)</td>
			<td>13(65)</td>
			<td>20(57)</td>
			<td>128(69)</td>
		</tr>
<tr>
<td>wsgrna-2</td>
			<td>50</td>
			<td>9</td>
			<td>32</td>
			<td>41(82)</td>
			<td>3(33)</td>
			<td>12(38)</td>
			<td>15(37)</td>
			<td>51(61)</td>
		</tr>
<tr>
<td>wsgrna-3</td>
			<td>50</td>
			<td>17</td>
			<td>15</td>
			<td>32(64)</td>
			<td>7(41)</td>
			<td>8(53)</td>
			<td>15(47)</td>
			<td>157(72)</td>
		</tr>
<tr>
<td>wsgRNA-1/wsgRNA-2</td>
			<td>50</td>
			<td>7</td>
			<td>16</td>
			<td>23(46)</td>
			<td>5(71)</td>
			<td>12(75)</td>
			<td>17(74)</td>
			<td>123(79)</td>
		</tr>
<tr>
<td>wsgRNA-1/wsgRNA-3</td>
			<td>50</td>
			<td>13</td>
			<td>9</td>
			<td>22(44)</td>
			<td>10(77)</td>
			<td>6(67)</td>
			<td>16(73)</td>
			<td>72(81)</td>
		</tr>
<tr>
<td>wsgRNA-2/wsgRNA-3</td>
			<td>50</td>
			<td>17</td>
			<td>10</td>
			<td>27(54)</td>
			<td>13(76)</td>
			<td>8(80)</td>
			<td>21(78)</td>
			<td>101(85)</td>
		</tr>
<tr>
<td>wsgRNA-1/wsgRNA-2/wsgRNA-3</td>
			<td>50</td>
			<td>11</td>
			<td>10</td>
			<td>21(42)</td>
			<td>9(82)</td>
			<td>9(90)</td>
			<td>18(86)</td>
			<td>149(86)</td>
		</tr>
</tbody></table>表1：注射单片和多重GRNA的胚胎的存活率和突变率。表从30日起转载

Watch this Scientific Journal Video about Embryo Microinjection Techniques for Efficient Site-Specific Mutagenesis in Culex quinquefasciatus at JoVE.com

Embryo Microinjection Techniques for Efficient Site-Specific Mutagenesis in Culex quinquefasciatus

该协议描述了 Culex五角星胚胎的 微注射程序，这些胚胎通过CRISPR/Cas9基因编辑工具进行优化。该技术可以有效地生成特定地点、遗传的生殖系突变，可用于在这种研究不足的疾病载体中构建遗传技术。

胚胎微注射技术，用于在库莱克斯五角星高效位点特异性突变体

Culex quinquefasciatus is a vector of a diverse range of vector-borne diseases such as avian malaria, West Nile virus (WNV), Japanese encephalitis, ...

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Embryo Microinjection Techniques for Efficient Site-Specific Mutagenesis in Culex quinquefasciatus

5.3K 次观看.  University of California, San Diego. 该协议描述了 Culex五角星胚胎的 微注射程序，这些胚胎通过CRISPR/Cas9基因编辑工具进行优化。该技术可以有效地生成特定地点、遗传的生殖系突变，可用于在这种研究不足的疾病载体中构建遗传技术。

视频: Embryo Microinjection Techniques for Efficient Site-Specific Mutagenesis in Culex quinquefasciatus

Laser-assisted Cytoplasmic Microinjection in Livestock Zygotes

Cytoplasmic microinjection into one-cell embryos is a very powerful technique. As an example, it enables the delivery of genome editing tools that can create genetic modifications that will be present in every cell of an adult organism. It can also be used to deliver siRNA, mRNAs or blocking antibodies to study gene function in preimplantation embryos. The conventional technique for microinjecting embryos used in rodents consists of a very thin micropipette that directly penetrates the plasma membrane when advanced into the embryo. When this technique is applied to livestock animals it usually results in low efficiency. This is mainly because in contrast to mice and rats, bovine, ovine, and porcine zygotes have a very dark cytoplasm and a highly elastic plasma membrane that makes visualization during injection and penetration of the plasma membrane hard to achieve. In this protocol, we describe a suitable microinjection method for the delivery of solutions into the cytoplasm of cattle zygotes that has proved to be successful for sheep and pig embryos as well. First, a laser is used to create a hole in the zona pellucida. Then a blunt-end glass micropipette is introduced through the hole and advanced until the tip of the needle reaches about 3/4 into the embryo. Then, the plasma membrane is broken by aspiration of cytoplasmic content inside the needle. Finally, the aspirated cytoplasmic content followed by the solution of interest is injected back into the embryonic cytoplasm. This protocol has been successfully used for the delivery of different solutions into bovine and ovine zygotes with 100% efficiency, minimal lysis, and normal blastocysts development rates.

This protocol shows how to perform cytoplasmic microinjection in farm animal zygotes. This technique can be used to deliver any solution into the one-cell embryo such as genome editing tools to generate knockout animals.

激光辅助畜禽受精卵细胞质显微注射

Cytoplasmic microinjection into one-cell embryos is a very powerful technique. As an example, it enables the delivery of genome editing tools that can ...

科研

遗传学

Delivery of Nucleic Acids through Embryo Microinjection in the Worldwide Agricultural Pest Insect, Ceratitis capitata

The Mediterranean fruit fly (medfly) Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) is a pest species with extremely high agricultural relevance. This is due to its reproductive behavior: females damage the external surface of fruits and vegetables when they lay eggs and the hatched larvae feed on their pulp. Wild C. capitata populations are traditionally controlled through insecticide spraying and/or eco-friendly approaches, the most successful being the Sterile Insect Technique (SIT). The SIT relies on mass-rearing, radiation-based sterilization and field release of males that retain their capacity to mate but are not able to generate fertile progeny. The advent and the subsequent rapid development of biotechnological tools, together with the availability of the medfly genome sequence, has greatly boosted our understanding of the biology of this species. This favored the proliferation of new strategies for genome manipulation, which can be applied to population control.
In this context, embryo microinjection plays a dual role in expanding the toolbox for medfly control. The ability to interfere with the function of genes that regulate key biological processes, indeed, expands our understanding of the molecular machinery underlying medfly invasiveness. Furthermore, the ability to achieve germ-line transformation facilitates the production of multiple transgenic strains that can be tested for future field applications in novel SIT settings. Indeed, genetic manipulation can be used to confer desirable traits that can, for example, be used to monitor sterile male performance in the field, or that can result in early life-stage lethality. Here we describe a method to microinject nucleic acids into medfly embryos to achieve these two main goals.

Delivery of Nucleic Acids through Embryo Microinjection in the Worldwide Agricultural Pest Insect, Ceratitis capitata

The Mediterranean fruit fly (medfly) Ceratitis capitata (Diptera: Tephritidae) is a worldwide pest of agriculture. A deeper understanding of its biology is key to control medfly populations and thus reduce economic impact. Embryo microinjection is a fundamental tool allowing both germ-line transformation and reverse genetics studies in this species.

核酸的传递通过显微注射胚胎在世界范围内的农业害虫，<em&gt;地中海实蝇</em

The Mediterranean fruit fly (medfly) Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) is a pest species with extremely high agricultural relevance. ...

Tools to Study the Role of Architectural Protein HMGB1 in the Processing of Helix Distorting, Site-specific DNA Interstrand Crosslinks

High mobility group box 1 (HMGB1) protein is a non-histone architectural protein that is involved in regulating many important functions in the genome, such as transcription, DNA replication, and DNA repair. HMGB1 binds to structurally distorted DNA with higher affinity than to canonical B-DNA. For example, we found that HMGB1 binds to DNA interstrand crosslinks (ICLs), which covalently link the two strands of the DNA, cause distortion of the helix, and if left unrepaired can cause cell death. Due to their cytotoxic potential, several ICL-inducing agents are currently used as chemotherapeutic agents in the clinic. While ICL-forming agents show preferences for certain base sequences (e.g., 5&#39;-TA-3&#39; is the preferred crosslinking site for psoralen), they largely induce DNA damage in an indiscriminate fashion. However, by covalently coupling the ICL-inducing agent to a triplex-forming oligonucleotide (TFO), which binds to DNA in a sequence-specific manner, targeted DNA damage can be achieved. Here, we use a TFO covalently conjugated on the 5&#39; end to a 4&#39;-hydroxymethyl-4,5&#39;,8-trimethylpsoralen (HMT) psoralen to generate a site-specific ICL on a mutation-reporter plasmid to use as a tool to study the architectural modification, processing, and repair of complex DNA lesions by HMGB1 in human cells. We describe experimental techniques to prepare TFO-directed ICLs on reporter plasmids, and to interrogate the association of HMGB1 with the TFO-directed ICLs in a cellular context using chromatin immunoprecipitation assays. In addition, we describe DNA supercoiling assays to assess specific architectural modification of the damaged DNA by measuring the amount of superhelical turns introduced on the psoralen-crosslinked plasmid by HMGB1. These techniques can be used to study the roles of other proteins involved in the processing and repair of TFO-directed ICLs or other targeted DNA damage in any cell line of interest.

Targeted DNA damage can be achieved by tethering a DNA damaging agent to a triplex-forming oligonucleotide (TFO). Using modified TFOs, DNA damage-specific protein association, and DNA topology modification can be studied in human cells by the utilization of modified chromatin immunoprecipitation assays and DNA supercoiling assays described herein.

工具来研究蛋白质建筑HMGB1的作用，螺旋窜改，站点特定的DNA间交联处理

High mobility group box 1 (HMGB1) protein is a non-histone architectural protein that is involved in regulating many important functions in the genome, ...

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most mutagenesis-based screens, researchers have relied on the use of toxic chemicals, carcinogens, or irradiation, which requires designated equipment, safety setup, and/or disposal of hazardous materials. We have developed a simple approach to induce heritable mutations in C. elegans using germline-expressed histone-miniSOG, a light-inducible potent generator of reactive oxygen species. This mutagenesis method is free of toxic chemicals and requires minimal laboratory safety and waste management. The induced DNA modifications include single-nucleotide changes and small deletions, and complement those caused by classical chemical mutagenesis. This methodology can also be used to induce integration of extrachromosomal transgenes. Here, we provide the details of the LED setup and protocols for standard mutagenesis and transgene integration.

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Genetically-encoded histone-miniSOG induces genome-wide heritable mutations in a blue&#160;light-dependent manner. This mutagenesis method is simple, fast, free of toxic chemicals, and well-suited for forward genetic screening and transgene integration.

光遗传学随机突变用组蛋白miniSOG在<em&gt;℃。线虫</em

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most ...

Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast

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

Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast

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

目标<em&gt;原位</em&gt;组蛋白突变的基因在芽殖酵母

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

Embryo Microinjection and Transplantation Technique for Nasonia vitripennis Genome Manipulation

The jewel wasp Nasonia vitripennis has emerged as an effective model system for the study of processes including sex determination, haplo-diploid sex determination, venom synthesis, and host-symbiont interactions, among others. A major limitation of working with this organism is the lack of effective protocols to perform directed genome modifications. An important part of genome modification is delivery of editing reagents, including CRISPR/Cas9 molecules, into embryos through microinjection. While microinjection is well established in many model organisms, this technique is particularly challenging to perform in N. vitripennis primarily due to its small embryo size, and the fact that embryonic development occurs entirely within a parasitized blowfly pupa. The following procedure overcomes these significant challenges while demonstrating a streamlined, visual procedure for effectively removing wasp embryos from parasitized host pupae, microinjecting them, and carefully transplanting them back into the host for continuation and completion of development. This protocol will strongly enhance the capability of research groups to perform advanced genome modifications in this organism.

Embryo Microinjection and Transplantation Technique for Nasonia vitripennis Genome Manipulation

Microinjection of Nasonia vitripennis embryos is an essential method for generating heritable genome modifications. Described here is a detailed procedure for microinjection and transplantation of Nasonia vitripennis embryos, which will greatly facilitate future genome manipulation in this organism.

金小蜂金小蜂基因组操作的胚胎显微注射和移植技术

The jewel wasp Nasonia vitripennis has emerged as an effective model system for the study of processes including sex determination, haplo-diploid sex ...

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

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

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

基因靶向随机诱变在裂变酵母中选择染色质稳定蛋白酶突变体

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

Sand Fly (Phlebotomus papatasi) Embryo Microinjection for CRISPR/Cas9 Mutagenesis

Sand flies are the natural vectors for Leishmania species, protozoan parasites producing a broad spectrum of symptoms ranging from cutaneous lesions to visceral pathology. Deciphering the nature of the vector/parasite interactions is of primary importance for better understanding of Leishmania transmission to their hosts. Among the parameters controlling the sand fly vector competence (i.e. their ability to carry and transmit pathogens), parameters intrinsic to these insects were shown to play a key role. Insect immune response, for example, impacts sand fly vector competence to Leishmania. The study of such parameters has been limited by the lack of methods of gene expression modification adapted for use in these non-model organisms. Gene downregulation by small interfering RNA (siRNA) is possible, but in addition to being technically challenging, the silencing leads to only a partial loss of function, which cannot be transmitted from generation to generation. Targeted mutagenesis by CRISPR/Cas9 technology was recently adapted to the Phlebotomus papatasi sand fly. This technique leads to the generation of transmissible mutations in a specifically chosen locus, allowing to study the genes of interest. The CRISPR/Cas9 system relies on the induction of targeted double-strand DNA breaks, later repaired by either Non-Homologous End Joining (NHEJ) or by Homology Driven Repair (HDR). NHEJ consists of a simple closure of the break and frequently leads to small insertion/deletion events. In contrast, HDR uses the presence of a donor DNA molecule sharing homology with the target DNA as a template for repair. Here, we present a sand fly embryo microinjection method for targeted mutagenesis by CRISPR/Cas9 using NHEJ, which is the only genome modification technique adapted to sand fly vectors to date.

Sand Fly Phlebotomus papatasi Embryo Microinjection for CRISPR/Cas9 Mutagenesis

This protocol details the steps of CRISPR/Cas9 targeted mutagenesis in sand flies: embryo collection, injection, insect rearing, and identification as well as selection of mutations of interest.

沙飞（木薯）胚胎微注射为CRISPR/Cas9穆塔赫内西

Sand flies are the natural vectors for Leishmania species, protozoan parasites producing a broad spectrum of symptoms ranging from cutaneous lesions to ...

Embryo Injections for CRISPR-Mediated Mutagenesis in the Ant Harpegnathos saltator

The unique traits of eusocial insects, such as social behavior and reproductive division of labor, are controlled by their genetic system. To address how genes regulate social traits, we have developed mutant ants via delivery of CRISPR complex into young embryos during their syncytial stage. Here, we provide a protocol of CRISPR-mediated mutagenesis in Harpegnathos saltator, a ponerine ant species that displays striking phenotypic plasticity. H. saltator ants are readily reared in a laboratory setting. Embryos are collected for microinjection with Cas9 proteins and in vitro synthesized small guide RNAs (sgRNAs) using home-made quartz needles. Post-injection embryos are reared outside the colony. Following emergence of the first larva, all embryos and larvae are transported to a nest box with a few nursing workers for further development. This protocol is suitable for inducing mutagenesis for analysis of caste-specific physiology and social behavior in ants, but may also be applied to a broader spectrum of hymenopterans and other insects.

Embryo Injections for CRISPR-Mediated Mutagenesis in the Ant Harpegnathos saltator

Many characteristics of insect eusociality rely on within-colony communication and division of labor. Genetic manipulation of key regulatory genes in ant embryos via microinjection and CRISPR-mediated mutagenesis provides insights into the nature of altruistic behavior in eusocial insects.

胚胎注射用于CRISPR介导的诱变在蚂蚁Harpegnathos盐化剂中

The unique traits of eusocial insects, such as social behavior and reproductive division of labor, are controlled by their genetic system. To address how ...

Microinjection Method for Anopheles gambiae Embryos

Embryo microinjection techniques are essential for many molecular and genetic studies of insect species. They provide a means to introduce exogenous DNA fragments encoding genes of interest as well as favorable traits into the insect germline in a stable and heritable manner. The resulting transgenic strains can be studied for phenotypic changes resulting from the expression of the integrated DNA to answer basic questions or used in practical applications. Although the technology is straightforward, it requires of the investigator patience and practice to achieve a level of skill that maximizes efficiency. Shown here is a method for microinjection of embryos of the African malaria mosquito, Anopheles gambiae. The objective is to deliver by microinjection exogenous DNA to the embryo so that it can be taken up in the developing germline (pole) cells. Expression from the injected DNA of transposases, integrases, recombinases, or other nucleases (for example CRISPR-associated proteins, Cas) can trigger events that lead to its covalent insertion into chromosomes. Transgenic An. gambiae generated from these technologies have been used for basic studies of immune system components, genes involved in blood-feeding, and elements of the olfactory system. In addition, these techniques have been used to produce An. gambiae strains with traits that may help control the transmission of malaria parasites.

Microinjection Method for Anopheles gambiae Embryos

Microinjection techniques are essential to introduce exogenous genes into the genomes of mosquitoes. This protocol explains a method used by the James laboratory to microinject DNA constructs into Anopheles gambiae embryos to generate transformed mosquitoes.

冈 比亚按蚊 胚胎的显微注射方法

Embryo microinjection techniques are essential for many molecular and genetic studies of insect species. They provide a means to introduce exogenous DNA ...