这份出版物的研究报告得到了国家卫生研究院的过敏和传染性疾病研究所 (NIAID) 的支持, 其奖项编号是 1R41OD021979 (对此) 和下一代 BioGreen 21 的研究补助金。计划, 大韩民国, 格兰特号PJ01107704。PJ01107703 (对 IK)。内容完全是作者的责任, 并不一定代表国家卫生研究院或 BioGreen 21 的官方观点。我们感谢 Dr. Nikolas Robl 编辑手稿。

本议定书所述程序由犹他州立大学动物保育和使用委员会 (IACUC) 批准 (IACUC 议定书: 2484)。本议定书使用的仓鼠是成人 (6-10 周龄) LVG 应变金黄叙利亚仓鼠。所有仓鼠都安置在犹他州立大学 Bioinnovation 中心的 vivarium。室温设置在23° c, 湿度设置为 40-50%, 光周期设置为 14 l:10 d (浅色: 深色)。只要有可能, 胚胎操作和 surugical 程序都应该用无菌技术进行。
1. sgRNA 和 Cas9/sgRNA 均一 (RNP) 的制备
<ol><li>合成 sgRNAs 后的体外转录程序在手册中描述的综合试剂盒 (材料表, 补充 1)。</li>
	<li>要组装均一, 混合2µg Cas9 与1µg sgRNA 和孵化混合在室温下为 10-15 分钟。对于 PN 注射液, 在浓度为 100 ng/µL Cas9 和 50 ng/µL sgRNA 与 10 mM TE 缓冲器 (核糖核酸自由,材料表) 的情况下, 制作一个工作解决方案。</li>
</ol>2. 输精管结扎术的准备
注: 输精管结扎术是在 6-8 周龄的雄性仓鼠身上进行的。手术应在第一次交配前10-14 天进行。不孕是证实的失败怀孕的交配与肥沃的女性。输精管男性通常可以使用一年前, 他们变得较少性活跃。
<ol><li>麻醉雄性经腹腔注射氯胺酮/嗪, 剂量为40毫克/千克 (氯胺酮) 和10毫克/千克 (嗪)。通过缺乏踏板反射 (脚趾捏) 来确认麻醉。</li>
	<li>用干纸巾把卧床的仓鼠放在背上。用70% 酒精和聚维酮碘之间交替的外科擦洗解决方案冲洗腹部三轮防腐处理。</li>
	<li>做一个垂直切口与外科剪刀开始 1.5 cm 头骨对从中间线延伸 1 cm 到每边的包皮 (图 1a)。切开皮肤、皮下组织和式, 进入腹膜间隙 (图 1b)。</li>
	<li>对阴囊的尾部施加轻柔的压力, 以迫使睾丸和脂肪组织出来。用镊子轻轻抓住输精管, 用第二组钳 (图 1c) 仔细地将血管从 vas 中分离出来。</li>
	<li>用钳夹住输精管形成一个循环。在火焰中加热另一个钳子的尖端, 直到它是红色的, 然后用它来除去输精管的回路。插入脂肪组织和睾丸回腹部。用相同的程序将对方的输精管切除。</li>
	<li>先缝合肌肉, 然后缝合皮肤。将动物放在笼中的暖垫上30分钟, 直到动物恢复。观察动物直到它恢复正常的活动。</li>
</ol>3. 供方/受援仓鼠准备时间表
<ol><li>监测和记录动情周期。 注: 雌性仓鼠有一个稳定的4天的动情周期, 在分娩后立即维持。雌性在发情的第一天可以通过不透明、黄色和粘稠的阴道分泌物 (图 2a) 来识别。4天发情的雌性可以通过透明的粘液 (图 2b) 进行识别。</li>
	<li>对于供体仓鼠的超数排卵, superovulate 在动情周期的第一天 (> 6 周), 在 9-上午12点之间, 通过 IP 注射妊娠母马血清促性腺激素 (PMSG; 在 PBS 中溶解为50毫升) (表 1)。 注: PMSG 用于诱导排卵, 但不适用于发情周期同步。</li>
	<li>80-84 h 在注射 PMSG (天 4, 6-下午8点) 以后, 安置女性在笼子与男性为交配。由于仓鼠交配不会导致交配插头, 确保成功的交配通过观看交配。</li>
	<li>在 4 天的发情与输精管男配性成熟女性准备 pseudopregnant 雌性。在表 2中显示了准备捐赠者和受赠女性的时间安排。 注意: 真正的怀孕女性也可以作为接受者,即, 女性与颜色不同的颜色与金色的颜色可育的雄性。从胚胎移植派生的幼崽可以根据涂层颜色来识别。使用真正的孕妇的一个潜在的优势是, 内生产的胚胎将确保成功的怀孕和帮助 PN 注射胚胎植入;使用真正的孕妇作为受体的潜在缺陷是, PN 注射胚胎需要与内生产的胚胎进行竞争。</li>
</ol>4. 受精卵分离
<ol><li>准备培养基 (HECM-9; 配方在补充2中描述), 如前面的麦基尔南和 Bavister12中所述。</li>
	<li>在 pn 注射前一天, 准备受精卵处理皿 (25 µL/滴) 和 pn 注射液滴 (100 µL) 在一个35毫米菜的盖子与 HECM-9。然后用矿物油覆盖中滴。在恒温箱中平衡培养基在以下条件下过夜: 37.5 ° c, 10% CO2, 5% O2, 85% N2和100% 湿度。</li>
	<li>在计划进行 PN 注射的当天, 用 HECM-9 准备胚胎冲洗皿 (1 碟/供体仓鼠), 并将所有的菜肴存放在孵化器中直到使用。</li>
	<li>安乐排仓鼠与 CO2。</li>
	<li>输卵管隔离<ol>
<li>将一只被安乐死的雌性仓鼠放在手术悬垂或纸巾上, 用70% 乙醇喷雾卧床腹部。</li>
		<li>在中线上牢牢握住皮肤和腹部肌肉层, 并进行切口。切开腹膜以暴露腹部器官。</li>
		<li>用细钳抓住其中一个子宫角, 从腹部缩回组织。将子宫与 mesometrium 和脂肪组织分开。在输卵管和卵巢之间进行切割, 并在输卵管和子宫的交界处 (包括子宫的一小部分) 附近进行第二次切割。将两个输卵管放在同一个冲洗盘中。</li>
	</ol>
</li>
	<li>冲洗和收集受精卵<ol>
<li>解剖显微镜下, 用 #5 的钳夹住子宫角的一侧, 将自制的针头 (图 3) 插入漏斗端的输卵管腔内, 用 300-400 µL 的 HECM-9 培养基冲洗输卵管。</li>
		<li>立即收集受精卵, 在处理盘中用 HECM-9 介质冲洗两次。在这个发展阶段, 所有的受精卵都应该从卵丘细胞中剥脱。</li>
		<li>将受精卵放入孵化器 (37.5 ° c, 10% CO2, 5% O2, 85% N2和100% 湿度) 后立即收集, 以尽量减少暴露于光线下。</li>
	</ol>
</li>
</ol>5. PN 注射
<ol><li>在整个 PN 注射过程中, 在冰上解冻 Cas9/sgRNA RNP, 并在冰上保持复合体。</li>
	<li>在注射实验前立即用 Cas9/sgRNA RNP 溶液加载注射针。将注射针的后端放入含有 Cas9/sgRNA RNP 溶液的管内, 并允许 Cas9/sgRNA RNP 溶液通过毛细管作用填充针。</li>
	<li>准备注射碟和针头。将持有者的吸管与矿物油填充到3毫米的支架针的弯曲处。将支架放在微量中, 将针的尖端水平放在盘子的底部, 然后用吸管将针的尖端填充到中间。设置注射针在 10-15 角度对面的持有人。</li>
	<li>PN 注射液<ol>
<li>确定一个合子, 并应用一个良好的吸力与持有人针。理想情况下, 男性和女性核在相同的焦距范围内, 并与保持架平行。</li>
		<li>穿透透明和男性核 (3 点的位置) 与注射针。</li>
		<li>一旦原膨胀, 将注射针迅速取出, 防止细胞核附着在针头上, 避免损伤原。</li>
	</ol>
</li>
</ol>6. 受精卵转移到 Pseudopregnant 仓鼠
<ol><li>麻醉 pseudopregnant 仓鼠 (与上述输精管结扎术中描述的相同), 并将其放置在腹卧床的外科悬垂或干纸巾上。</li>
	<li>将眼部润滑剂应用于动物眼睛, 防止眼部干燥, 用布或纸遮住头部以保护眼睛免受光线的影响。准备身体的侧面, 剃须在身体两侧的4厘米 x 4 厘米的皮肤面积, 然后应用3次聚维酮碘磨砂和3倍70% 乙醇磨砂。使2厘米垂直切口2厘米尾端到最后一个肋骨 (如图 4a, 4b) 在动物背部的两侧。</li>
	<li>用钳子抓住脂肪垫, 轻轻地拉, 直到输卵管和子宫可见。用止血夹住脂肪垫, 并在脊柱上反射脂肪垫背 (图 4c)。定位生殖道, 使子宫管可以穿透30针的胚胎移植 (图 4d)。</li>
	<li>用 HECM-9 在新菜中冲洗注射受精卵。使用新的玻璃吸管 (直径: 〜 150-200 µm) 负载 10-15 受精卵。将受精卵排列成一连串的珍珠, 接着是几个气泡。将吸管插入6.3 步的开放穿透管中, 轻轻吹入吸管, 将受精卵转移至输卵管。继续吹, 直到第一个气泡被释放到输卵管 (图 4e)。</li>
	<li>释放脂肪垫并将组织返回腹部。在相同的过程中, 将相同数量 (10-15) 的注射胚胎转移到另一个输卵管。</li>
	<li>缝合肌肉组织和皮肤在2层。用盐酸丁丙诺啡 (0.5 毫克/千克) 皮下注射作为术后镇痛。把仓鼠归还笼子, 把笼子放在暖和的垫子上, 以便恢复。当动物完全从麻醉中恢复时, 将笼子交还给动物室。术后镇痛药六小时后再给你。</li>
</ol>

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该公司是一家专门为生物医学研究和农业应用创造基因工程动物的生物技术企业, 在这方面有经济利益。

为了更好地利用黄金叙利亚仓鼠的潜力作为人类疾病的模型, 我们开发了一个 PN 注射协议, 以提供一个 CRISPR/Cas9 复合体为目标的仓鼠基因组。PN 注入协议优化了几个关键变量, 包括胚培养基、温度和光的波长13。还有几个仓鼠特定的动物处理程序, 需要遵循成功地进行基因打靶的仓鼠。例如, 性成熟的雌性仓鼠有稳定的4天的动情周期, 不能与外源激素同步 (例如PMSG)。因此, 准确地跟踪每个雌性的动情周期对于超数排卵和假的制备都是非常重要的。30-60 受精卵可以生产的女性, 如果她是成功的排。为了达到成功的超数排卵, 有必要考虑 species-specific 沉积脂肪。雌性仓鼠, 特别是那些超过12周的年龄, 往往有过量的腹部脂肪, 使注射器针必须正确定位, 以充分穿透脂肪垫时, 执行激素注射。我们已经发现, 穿透中途进入腹腔达到适当的距离激素分娩。我们总结了在表 4中对 PN 注射液、仓鼠处理和畜牧业的独特要求。
对于 PN 注射, 必须通过实验确定每一次注射的胚胎转移到注射盘的数量, 以避免延长注射时间。胚胎留在盘子里的时间越长, 他们经历曝光的几率就越大。我们建议将大约15胚胎移植到注射盘中进行每一次注射。这个数字在注射轮数和胚胎对光的耐受照射之间取得了良好的平衡。由于仓鼠胚胎的胞质和原膜均具有相当的柔韧性, 因此必须对注射针施加大量力, 以穿透原膜。在这段时间内, 核必须保持在焦距范围之内。
研究人员在进行胚胎移植时应考虑以下的手术问题。首先, 通过身体壁的切口与动物的身体大小相对应是很重要的。如果切口太小, 则可能导致受精卵从输卵管向腹部返回时的挤压。除了切口尺寸外, 通过处理相关的脂肪垫而非生殖组织, 可使输卵管受精卵的可能性最小化。至于切口大小, 也重要的是要考虑较大的切口可能会对动物造成更多的压力, 并表现出更高的并发症几率 (例如感染裂开)。其次, 为了减少出血量, 研究人员在进入腹部时应注意避免切割主要的血管, 因为过量失血会增加手术压力, 降低胚胎移植的成功率, 延长恢复时间。

金黄叙利亚仓鼠 (Mesocricetus 鲫) 是生物医学研究中使用最广泛的啮齿动物之一。根据美国农业部的数据, 2015年美国使用了大约10万只仓鼠, 这代表了动物福利法 (http://www.aphis.usda.gov) 所涵盖的物种在实验室动物总数中的13%。2017年3月10日)。
在研究一些人类疾病时, 仓鼠比其他啮齿动物提供了一些优势。例如, nitrosobis (2-代丙基) 胺 (防喷) 诱导的胰胆管癌的组织病理学与人胰腺肿瘤相似, 而防喷治疗主要诱发大鼠甲状腺肿瘤和肺、肝肿瘤鼠标1。由于仓鼠是唯一发现支持复制腺的小鼠, 它们也是测试 adenovirus-based 溶载体和 anti-adenovirus 药物的选择模型2,3,4。另一个例子, 其中仓鼠模型提供了比老鼠和老鼠的优势是在高脂血症的研究。人和仓鼠在脂质代谢通路上具有很大的相似性, 两个物种携带胆固醇酯转运蛋白 (CETP) 的基因编码, 这在脂代谢中起着中心作用, 而 CETP 在小鼠和大鼠中则不存在5。此外, 在感染埃博拉病毒6后, 仓鼠发展出血性疾病更能代表人类的表现。仓鼠也是研究动脉粥样硬化的选择模型7, 口腔癌的8, 和炎症病9。最近, 它也已经证明, 仓鼠是高度敏感的安第斯山脉病毒感染和发展汉坦病毒性肺综合征样疾病, 提供了唯一的啮齿动物模型的安第斯山脉感染10。
为了解决新的基因动物模型无法满足的需要, 研究人类疾病, 没有可靠的小鼠模型, 我们最近成功地将 CRISPR/Cas9 系统应用到仓鼠, 并产生了几行基因工程仓鼠11。仓鼠受精卵对环境环境非常敏感, 因此在其他物种中开发的 PN 注射协议是不合适的。因此, 我们开发了一种用于仓鼠的 PN 注射协议, 它适用于处理仓鼠胚胎的特殊要求体外。在这里, 我们描述了详细的 PN 注射过程中使用的 CRISPR/Cas9 系统和相应的步骤, 从单一指南 RNA (sgRNA) 的准备转移到接受雌性注射胚胎。

<table><tbody><tr><td>Cas9</td><td>Invitrogen</td><td>B25640</td><td>1 &amp;mu;g/&amp;mu;L (~6.1 &amp;mu;M)</td></tr><tr><td>GeneArtTM Precision Synthesis Kit</td><td>Invitrogen</td><td>A29377</td><td>For sgRNA synthesis</td></tr><tr><td>Albumin from human serum</td><td>Sigma</td><td>A1653</td><td>For cultivation medium</td></tr><tr><td>Illuminator</td><td>Nikon</td><td>NI-150</td><td>For embryo transfer</td></tr><tr><td>Incubator</td><td>New Brunswick</td><td>Galaxy 14S</td><td>For embryo cultivation</td></tr><tr><td>Microforge</td><td>Narishige</td><td>PB-7</td><td>For making injection needles</td></tr><tr><td>Microscope</td><td>Nikon</td><td>ECLIPSE Ti-S</td><td>For microinjection</td></tr><tr><td>Microscope</td><td>invitrogen</td><td>SMZ745T</td><td>For embryo transfer</td></tr><tr><td>Mineral oil</td><td>Sigma</td><td>M1840</td><td>Keep in dark</td></tr><tr><td>PMSG</td><td>Sigma</td><td>G4877-2000IU</td><td>For superovulation</td></tr></tbody></table>

production of genetically engineered golden syrian hamsters by pronuclear injection of the crispr/cas9 complex

在小鼠体内首次建立了原 (PN) 注射技术, 将国外的遗传物质引入到单细胞阶段胚胎的核中。引入的遗传物质可以融入胚胎基因组, 并在移植后的胚胎转移到寄养母亲之后, 利用外来的遗传信息产生转基因动物。在小鼠成功后, PN 注射液已成功应用于其他许多动物品种。近年来, PN 注射成功地引入了基因修饰的试剂, 如 CRISPR/Cas9 系统, 在几个实验室和家畜物种中实现 site-specific 的基因修饰。除了掌握特殊的显微注射技术, 通过 PN 注射液生产转基因动物外, 研究人员还必须了解目标物种的繁殖生理和行为, 因为每个物种都呈现出独特挑战.例如, 金黄叙利亚仓鼠胚胎有独特的处理要求在体外, 因此 PN 注射技术是不可能的在这个种类直到最近突破由我们的小组。通过我们的物种改良 PN 注射协议, 我们成功地生产了几个基因敲除 (KO) 和敲 (基) 仓鼠, 已成功地用于人类疾病模型。在这里, 我们描述了 PN 注射程序, 交付 CRISPR/Cas9 复合物的受精卵的仓鼠, 胚胎处理条件, 胚胎移植程序, 和畜牧业所需的生产转基因仓鼠。

所述协议在生产转基因仓鼠中的效率取决于以下两个关键步骤的结果: 受赠雌性的活产率和预期基因修饰的活幼崽数。活产率是胚胎质量的直接结果, 是个体进行 PN 注射和胚胎移植过程的技术。为了确保纵胚胎的发育潜能不会受到损害 , 在对仓鼠胚胎进行的体外处理过程中需要非常小心。我们定期获得 60-80% 的 pseudopregnant 受赠女性的活产率。表 3演示了从RAG1挖空实验 (pseudopregnant 雌性) 和STAT2淘汰赛试验 (使用了真正的怀孕的黑人女性) 的实时出生率. 在这种情况下, 出生率计算为产金幼崽的凋落物百分比。
由 PN 注射胚胎产生的转基因幼崽的百分比取决于 sgRNA 在引入 indels 和成功注射 Cas9/sgRNA 均一到核的效率。我们发现, sgRNA 效率在基因目标 (未发表的观察) 之间有差异。如表 3所示, 为STAT2设计的 sgRNA 导致了基因打靶效率为 88.9%, 而对RAG1的 sgRNA 则只有28.6% 的效率。图 5提供了从RAG1基因打靶中 pcr 限制片段长度多态性 (pcr-RFLP) 检测幼犬的基因分型结果的示例。重要的是要注意, 一些 indels 可能发生在限制酶识别序列之外, 例如 PCR-RFLP 可能低估了基因靶向效率。亚将 pcr 产物转化为 TA 克隆载体, 其次为 pcr 插入的桑格测序, 是准确测定基因靶向效率和揭示 indels 性质的必要条件。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56263/56263fig1.jpg"> 图 1: 雄性仓鼠输精管结扎术 a)和b)使2厘米垂直切口开始1.5 厘米颅骨到包皮延长 cranially;c)用两对镊子将输精管与相关的睾丸静脉和动脉分开;d)用镊子抓住输精管, 形成1厘米循环。加热第二套镊子, 并在烧灼两端同时切除循环。<a href="//cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56263/56263fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56263/56263fig2.jpg"> 图 2: 动情周期。 a) 1 天观察到的阴道分泌物是不透明、黄色和粘稠的b) 4 天的阴道分泌物是透明和黏稠的。<a href="//cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56263/56263fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56263/56263fig3.jpg"> 图 3: 准备用于胚胎隔离的针。 a)沿红色线断开一个30口径的针, 并将笔尖抛光, 直到它平整光滑。b)使 30-40 °角在〜 3 mm 从顶端。<a href="//cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56263/56263fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56263/56263fig4.jpg"> 图 4: 受精卵转移。 a)和b)使2厘米长的垂直切口从2厘米尾线开始到最后一个肋骨 (红线);c)用止血夹紧脂肪垫, 并反射组织背;d)调整输卵管的位置, 并用30口径针穿透开口;e)在胚胎移植的玻璃吸管中排列受精卵作为珍珠链, 并将其从穿透的打开中转移到输卵管。Bar=1,000 µm.<a href="//cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56263/56263fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56263/56263fig5.jpg"> 图 5。RFLP 单窝的创始人动物的 pcr 检测与RAG1 Indels.M: 1 Kb 加梯子。野生型。ID 1、3和7显示未修剪的带区与RAG1 indels 一致。<a href="//cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56263/56263fig5large.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>体重 (g)</td>
			<td>PMSG</td>
			<td>PMSG (ul)</td>
		</tr>
<tr>
<td>< 110</td>
			<td>10</td>
			<td>200</td>
		</tr>
<tr>
<td>110-135</td>
			<td>15</td>
			<td>300</td>
		</tr>
<tr>
<td>135-160</td>
			<td>20</td>
			<td>400</td>
		</tr>
<tr>
<td>160-185</td>
			<td>25</td>
			<td>500</td>
		</tr>
<tr>
<td>> 185</td>
			<td>30</td>
			<td>600</td>
		</tr>
<tr>
<td colspan="3">PMSG, 怀孕母马的血清 gonadatropin</td>
		</tr>
</tbody></table>表1。PMSG 与身体重量对应
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>鼠</td>
			<td>1天</td>
			<td>2天</td>
			<td>3天</td>
			<td>4天</td>
			<td>5天</td>
		</tr>
<tr>
<td>捐助</td>
			<td>PMSG (9-12 am)</td>
			<td></td>
			<td></td>
			<td>交配 (6-8 pm)</td>
			<td>受精卵隔离 (12-1 pm)</td>
		</tr>
<tr>
<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td>PN 注射液 (1-3 pm)</td>
		</tr>
<tr>
<td>收件人</td>
			<td></td>
			<td></td>
			<td></td>
			<td>交配 (6-8 pm)</td>
			<td>胚胎移植 (3-5 pm)</td>
		</tr>
</tbody></table>表2。捐助者/受援国准备时间安排
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>基因</td>
			<td>笑胚胎 (幼崽)</td>
			<td>笑凋落物 (收件人)</td>
			<td>笑阳性幼崽 (%)</td>
		</tr>
<tr>
<td>STAT2</td>
			<td>229 (54)</td>
			<td>14 (19) *</td>
			<td>48 (88.9)</td>
		</tr>
<tr>
<td>RAG1</td>
			<td>77 (21)</td>
			<td>3 (3)</td>
			<td>6 (28.6)</td>
		</tr>
<tr>
<td colspan="4">* 用雌鸟与育雄交配;被表明的凋落物的数量是仅那些被生产的金黄幼崽。</td>
		</tr>
</tbody></table>表3。Cas9/sgRNA 系统对金叙利亚仓鼠基因打靶效率的研究
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>条件/处理</td>
			<td>要求</td>
			<td>评论</td>
		</tr>
<tr>
<td>环境灯</td>
			<td>关闭所有环境灯</td>
			<td>仓鼠胚胎对光线敏感, 特别是对光的冷却</td>
		</tr>
<tr>
<td>光在体外处理</td>
			<td>PN 注射少于20分钟</td>
			<td>每回合操作15胚胎以减少光照射</td>
		</tr>
<tr>
<td rowspan="2">温度</td>
			<td>环境温度: 28±0.5 oC</td>
			<td rowspan="2">仓鼠胚胎对温度波动敏感</td>
		</tr>
<tr>
<td>处理温度: 37.5 oC</td>
		</tr>
<tr>
<td>中</td>
			<td>HECM-9</td>
			<td>不存储超过3天</td>
		</tr>
<tr>
<td>文化条件</td>
			<td>37.5 oC, 10% CO2, 5% o2和100% 湿度</td>
			<td>在孵化器中过夜</td>
		</tr>
<tr>
<td>胚胎移植后的饲养</td>
			<td>在截止日期前5天内不要更换笼子</td>
			<td>受干扰者将蚕食幼崽;提供足够的饲料/水</td>
		</tr>
</tbody></table>表4。仓鼠胚胎处理和饲养的独特要求
补充 1: 用 GeneArt 精密合成试剂盒合成 sgRNA.<a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56263/Supplement_1_sgRNA_synthesis.docx">请单击此处下载此文件.</a>
补充 2: 仓鼠胚培养 Medium-9 (HECM-9) 配方.<a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56263/Supplement_2_HECM-9_preparation.docx">请单击此处下载此文件.</a>

Watch this Scientific Journal Video about Production of Genetically Engineered Golden Syrian Hamsters by Pronuclear Injection of the CRISPR/Cas9 Complex at JoVE.com

Production of Genetically Engineered Golden Syrian Hamsters by Pronuclear Injection of the CRISPR/Cas9 Complex

原 (PN) 注射的聚类定期 interspaced 短复发重复 (CRISPR) 和 CRISPR 相关 protein-9 核酸 (CRISPR/Cas9) 系统是一种高效的方法生产基因工程金黄叙利亚仓鼠。在此, 我们描述了详细的 PN 注射协议生产的基因剔除仓鼠与 CRISPR/Cas9 系统。

CRISPR/Cas9 复合原注射液生产基因工程金黄叙利亚仓鼠

The pronuclear (PN) injection technique was first established in mice to introduce foreign genetic materials into the pronuclei of one-cell stage embryos. ...

production-genetically-engineered-golden-syrian-hamsters-pronuclear

Universidad San Sebastian

Research

JoVE Journal

Bioengineering

12.9K 次观看.  Utah State University. 原 (PN) 注射的聚类定期 interspaced 短复发重复 (CRISPR) 和 CRISPR 相关 protein-9 核酸 (CRISPR/Cas9) 系统是一种高效的方法生产基因工程金黄叙利亚仓鼠。在此, 我们描述了详细的 PN 注射协议生产的基因剔除仓鼠与 CRISPR/Cas9 系统。

视频: Production of Genetically Engineered Golden Syrian Hamsters by Pronuclear Injection of the CRISPR/Cas9 Complex

CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy

As a promising genome editing platform, the CRISPR/Cas9 system has great potential for efficient genetic manipulation, especially for targeted integration of transgenes. However, due to the low efficiency of homologous recombination (HR) and various indel mutations of non-homologous end joining (NHEJ)-based strategies in non-dividing cells, in vivo genome editing remains a great challenge. Here, we describe a homology-mediated end joining (HMEJ)-based CRISPR/Cas9 system for efficient in vivo precise targeted integration. In this system, the targeted genome and the donor vector containing homology arms (~800 bp) flanked by single guide RNA (sgRNA) target sequences are cleaved by CRISPR/Cas9. This HMEJ-based strategy achieves efficient transgene integration in mouse zygotes, as well as in hepatocytes in vivo. Moreover, a HMEJ-based strategy offers an efficient approach for correction of fumarylacetoacetate hydrolase (Fah) mutation in the hepatocytes and rescues Fah-deficiency induced liver failure mice. Taken together, focusing on targeted integration, this HMEJ-based strategy provides a promising tool for a variety of applications, including generation of genetically modified animal models and targeted gene therapies.

CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy

The clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) system provides a promising tool for genetic engineering, and opens up the possibility of targeted integration of transgenes. We describe a homology-mediated end joining (HMEJ)-based strategy for efficient DNA targeted integration in vivo and targeted gene therapies using CRISPR/Cas9.

CRISPR/Cas9-mediated 目标集成在体内使用基于同源介导的端接连接策略

As a promising genome editing platform, the CRISPR/Cas9 system has great potential for efficient genetic manipulation, especially for targeted integration ...

科研

遗传学

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 ...

Lentiviral Mediated Production of Transgenic Mice: A Simple and Highly Efficient Method for Direct Study of Founders

For almost 40 years, pronuclear DNA injection represents the standard method to generate transgenic mice with random integration of transgenes. Such a routine procedure is widely utilized throughout the world and its main limitation resides in the poor efficacy of transgene integration, resulting in a low yield of founder animals. Only few percent of animals born after implantation of injected fertilized oocytes have integrated the transgene. In contrast, lentiviral vectors are powerful tools for integrative gene transfer and their use to transduce fertilized oocytes allows highly efficient production of founder transgenic mice with an average yield above 70%. Furthermore, any mouse strain can be used to produce transgenic animal and the penetrance of transgene expression is extremely high, above 80% with lentiviral mediated transgenesis compared to DNA microinjection. The size of the DNA fragment that can be cargo by the lentiviral vector is restricted to 10 kb and represents the major limitation of this method. Using a simple and easy to perform injection procedure beneath the zona pellucida of fertilized oocytes, more than 50 founder animals can be produced in a single session of microinjection. Such a method is highly adapted to perform, directly in founder animals, rapid gain and loss of function studies or to screen genomic DNA regions for their ability to control and regulate gene expression in vivo.

Here, we present a protocol to promote transgene integration and production of founder transgenic mice with high efficacy by a simple injection of a lentiviral vector in the perivitelline space of a fertilized oocyte.

慢病毒载体介导的转基因小鼠生产: 一种简单高效的创始人直接研究方法

For almost 40 years, pronuclear DNA injection represents the standard method to generate transgenic mice with random integration of transgenes. Such a ...

Generation of Genetically Modified Mice through the Microinjection of Oocytes

The use of genetically modified mice has significantly contributed to studies on both physiological and pathological in vivo processes. The pronuclear injection of DNA expression constructs into fertilized oocytes remains the most commonly used technique to generate transgenic mice for overexpression. With the introduction of CRISPR technology for gene targeting, pronuclear injection into fertilized oocytes has been extended to the generation of both knockout and knockin mice. This work describes the preparation of DNA for injection and the generation of CRISPR guides for gene targeting, with a particular emphasis on quality control. The genotyping procedures required for the identification of potential founders are critical. Innovative genotyping strategies that take advantage of the "multiplexing" capabilities of CRISPR are presented herein. Surgical procedures are also outlined. Together, the steps of the protocol will allow for the generation of genetically modified mice and for the subsequent establishment of mouse colonies for a plethora of research fields, including immunology, neuroscience, cancer, physiology, development, and others.

The microinjection of mouse oocytes is commonly used for both classic transgenesis (i.e., the random integration of transgenes) and CRISPR-mediated gene targeting. This protocol reviews the latest developments in microinjection, with a particular emphasis on quality control and genotyping strategies.

通过显微注射卵母细胞生成转基因小鼠

The use of genetically modified mice has significantly contributed to studies on both physiological and pathological in vivo processes. The pronuclear ...

发育生物学

Use of Freeze-thawed Embryos for High-efficiency Production of Genetically Modified Mice

The use of genetically modified (GM) mice has become crucial for understanding gene function and deciphering the underlying mechanisms of human diseases. The CRISPR/Cas9 system allows researchers to modify the genome with unprecedented efficiency, fidelity, and simplicity. Harnessing this technology, researchers are seeking a rapid, efficient, and easy protocol for generating GM mice. Here we introduce an improved method for cryopreservation of one-cell embryos that leads to a higher developmental rate of the freeze-thawed embryos. By combining it with optimized electroporation conditions, this protocol allows for the generation of knockout and knock-in mice with high efficiency and low mosaic rates within a short time. Furthermore, we show a step-by-step explanation of our optimized protocol, covering CRISPR reagent preparation, in vitro fertilization, cryopreservation and thawing of one-cell embryos, electroporation of CRISPR reagents, mouse generation, and genotyping of the founders. Using this protocol, researchers should be able to prepare GM mice with unparalleled ease, speed, and efficiency.

Here, we present a modified method for cryopreservation of one-cell embryos as well as a protocol that couples the use of freeze-thawed embryos and electroporation for the efficient generation of genetically modified mice.

使用冷冻胚胎进行转基因小鼠高效生产

The use of genetically modified (GM) mice has become crucial for understanding gene function and deciphering the underlying mechanisms of human diseases. ...

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 ...

Zygote Microinjection for Creating Gene Cassette Knock-in and Flox Alleles in Mice

CRISPR-Cas technology has enabled the rapid and effortless generation of genetically modified mice. Specifically, mice and point mutant mice are readily produced by electroporation of CRISPR factors (and single-stranded oligo DNA donors) into the zygote. In contrast, gene cassette (&#62;1 kb) knock-in and floxed mice are mainly generated by microinjection of CRISPR factors and double-stranded DNA donors into zygotes. Genome editing technologies have also increased the flexibility of genetically modified mice production. It is now possible to introduce the intended mutations in the target genomic regions in a number of beneficial inbred mouse strains. Our team has produced over 200 gene cassette knock-in mouse lines, and over 110 floxed mouse lines by zygote microinjection of CRISPR-Cas9 following requests from several countries, including Japan. Some of these genome editing used BALB/c, C3H/HeJ, and C57BL/6N inbred strains, however most used C57BL/6J. Unlike the electroporation method, genome editing by zygote microinjection in various inbred strains of mice is not that easy. However, gene cassette knock-in and floxed mice on single inbred genetic backgrounds are as critical as genetic humanized, fluorescent reporter, and conditional knockout mouse models. Therefore, this article presents the protocol for the zygote microinjection of CRISPR factors and double-stranded DNA donors in C57BL/6J mice for generating gene cassette knock-in and floxed mice. This article exclusively focuses on nuclear injection rather than cytoplasmic injection. In addition to zygote microinjection, we outline the timeline for the production process and peripheral techniques such as induction of superovulation and embryo transfer.

The present protocol describes zygote microinjection of CRISPR-Cas9 and donor DNA to efficiently produce gene cassette knock-in and floxed mice.

受精卵显微注射用于在小鼠中产生基因盒敲入和絮状等位基因

CRISPR-Cas technology has enabled the rapid and effortless generation of genetically modified mice. Specifically, mice and point mutant mice are readily ...

Efficient Genome Editing of Mice by CRISPR Electroporation of Zygotes

With exceptional efficiency, accuracy, and ease, the CRISPR/Cas9 system has significantly improved genome editing in cell culture and lab animal experiments. When generating animal models, the electroporation of zygotes offers higher efficiency, simplicity, cost, and throughput as an alternative to the gold standard method of microinjection. Electroporation is also gentler, with higher viability, and reliably delivers Cas9/single-guide RNA (sgRNA) ribonucleoproteins (RNPs) into the zygotes of common laboratory mouse strains (e.g., C57BL/6J and C57BL/6N) that approaches 100% delivery efficiency. This technique enables insertion/deletion (indels) mutations, point mutations, the deletion of whole genes or exons, and small insertions in the range of 100-200 bp to insert LoxP or short tags like FLAG, HA, or V5. While constantly being improved, here we present the current state of CRISPR-EZ in a protocol that includes sgRNA production through in vitro transcription, embryo processing, RNP assembly, electroporation, and the genotyping of preimplantation embryos. A graduate-level researcher with minimal experience manipulating embryos can obtain genetically edited embryos in less than 1 week using this protocol. Here, we offer a straightforward, low-cost, efficient, high-capacity method that could be used with mouse embryos.

Here, we describe a simple technique intended for the efficient generation of genetically modified mice called CRISPR RNP Electroporation of Zygotes (CRISPR-EZ). This method delivers editing reagents by electroporation into embryos at an efficiency approaching 100%. This protocol is effective for point mutations, small genomic insertions, and deletions in mammalian embryos.

通过受精卵的CRISPR电穿孔对小鼠进行高效基因组编辑

With exceptional efficiency, accuracy, and ease, the CRISPR/Cas9 system has significantly improved genome editing in cell culture and lab animal ...

High-throughput Parallel Sequencing to Measure Fitness of Leptospira interrogans Transposon Insertion Mutants During Golden Syrian Hamster Infection

In this manuscript, we describe a transposon sequencing (Tn-Seq) technique to identify and quantify Leptospira interrogans mutants altered in fitness during infection of Golden Syrian hamsters. Tn-Seq combines random transposon mutagenesis with the power of high-throughput sequencing technology. Animals are challenged with a pool of transposon mutants (input pool), followed by harvesting of blood and tissues a few days later to identify and quantify the number of mutants in each organ (output pools). The output pools are compared to the input pool to evaluate the in vivo fitness of each mutant. This approach enables screening of a large pool of mutants in a limited number of animals. With minor modifications, this protocol can be performed with any animal model of leptospirosis, reservoir host models such as rats and acute infection models such as hamsters, as well as in vitro studies. Tn-Seq provides a powerful tool to screen for mutants with in vivo and in vitro fitness defects.

High-throughput Parallel Sequencing to Measure Fitness of Leptospira interrogans Transposon Insertion Mutants During Golden Syrian Hamster Infection

We describe here a technique that combines transposon mutagenesis with high-throughput sequencing to identify and quantify transposon leptospiral mutants in tissues after a challenge of hamsters. This protocol can be used to screen mutants for survival and dissemination in animals and can also be applied to in vitro studies.

高通量并行测序在金黄叙利亚仓鼠感染期间测量钩端螺旋体螺旋体转插入突变体的适应性

In this manuscript, we describe a transposon sequencing (Tn-Seq) technique to identify and quantify Leptospira interrogans mutants altered in fitness ...

免疫与感染

Removal of an Internal Translational Start Site from mRNA While Retaining Expression of the Full-Length Protein

The CRISPR-Cas9 gene-editing system, based on genome repair mechanisms, enables the generation of gene-modified mouse models more quickly and easily relative to traditional homologous recombination. The CRISPR-Cas9 system is particularly attractive when a single-point mutation is desired. The gap junction protein, Connexin 43 (Cx43), is encoded by gene Gja1, which has a single coding exon and cannot be spliced. However, Gja1 produces not only full-length Cx43 protein but up to six N-terminus truncated isoforms by a process known as internal translation, the result of ribosomal translation initiation at internal AUG (Methionine) start sites. GJA1-20k is the most commonly generated truncated isoform of Cx43 initiated at the AUG codon at position 213 of Gja1 mRNA. Because residue 213 occurs at the end of the last transmembrane domain of Cx43, GJA1-20k is effectively the 20 kDa C-terminus tail of Cx43 as an independent protein. Previous investigators identified, in cells, that a critical role of GJA1-20k is to facilitate trafficking of full-length Cx43 gap junction hemichannels to the plasma membrane. To examine this phenomenon in vivo, a mutant mouse with a Gja1 point-mutation was generated that replaces the ATG (Methionine) at residue 213 with TTA (Leucine, M213L mutation). The result of M213L is that Gja1 mRNA and full-length Cx43 are still generated, yet the translation of Gja1-20k is significantly reduced. This report focuses on choosing the restriction enzyme site to develop a one amino acid mutated (Gja1M213L/M213L) mouse model. This protocol describes genetically modified mice by the CRISPR-Cas9 system and rapid genotyping by combining PCR and restriction enzyme treatments.

The present protocol describes a single M213L mutation in Gja1 that retains full-length Connexin43 generation but prevents translation of the smaller GJA1-20k internally translated isoform.

从mRNA中去除内部翻译起始位点，同时保留全长蛋白的表达

The CRISPR-Cas9 gene-editing system, based on genome repair mechanisms, enables the generation of gene-modified mouse models more quickly and easily ...