这项工作是部分由美国国立卫生研究院的赞助资金（HL105114，HL088391，NS066652和HL068878到YEC），美国心脏协会（National科学家开发0835237N到JZ）。 YEC的财政支持，因为心血管内科赋予弗雷德里克Huetwell教授在密歇根大学医学中心（UMMC）。这项工作动用Center支持Core Services中的平移科学和治疗（CAMTraST）在UMMC先进典型。

所有的动物维护，保养和使用程序是在美国密歇根大学的动物的使用和护理审查和批准由大学委员会。 1。 ZFN设计选型<ol><li>提供感兴趣的基因（ 如兔ApoCIII）的基因组序列信息，给制造商。选择ZFN（次）基于所述的ZFN分数的实验。 </li></ol> 2。兔胚胎收集<ol><li> Superovulate性成熟（6-18个月）新西兰白（NZW）雌兔皮下注射（SC）注射FSH两次/天，3毫克的前两个注射，5毫克为未来两针和6毫克的剂量在过去的两针。 </li><li>第一次注射FSH诱导排卵后管理一个单一的静脉注射（IV）注射200 IU的人绒毛膜促性腺激素在72小时。交配的雌性超排用公兔注射hCG后。 </li><li> Euthani泽的超排供体兔用戊巴比妥钠（150毫克/千克，ⅳ）在16〜18小时后的人工授精（psi）的。 </li><li>通过精心搜罗了整个输卵管和卵巢的结构恢复输卵管壶腹。 </li><li>冲洗每个输卵管与HEPES 10毫升操纵介质缓冲TCM 199补充有10％胎牛血清，通过从输卵管的子宫端注入介质。收集的冲洗介质在输卵管卵巢端开口至培养皿。 </li><li>观察和识别回收胚胎在冲洗介质，在操作液洗胚胎三次，让他们在操纵媒体在38.5℃空气中，并观察它们在立体显微镜下受精的发生。准备注射的原核阶段的胚胎19-21ħ磅</li></ol> 3。 ZFN mRNA的制备<ol><li>重悬在无RNA酶的0.1X TE（1毫摩尔Tris-CL pH值之前，纯化所得的mRNA8.0，0.1 mM EDTA）中。测量的浓度，并在-80℃下与mRNA储存直至使用。 </li><li>准备编码ZFN对（4-10纳克/μl，以1 mMTris-CL，0.1毫米EDTA在pH值7.5），分装成10微升小瓶的mRNA。它们存储在-80ºC，直到准备使用。注射前，解冻RNA小瓶（次），并保持在冰上。 </li></ol> 4。胚胎显微注射<ol><li>通过将操作介质的5微升滴在具有媒体室中取出一个1井室幻灯片准备显微注射的平台。顶盖采用矿物油和地方媒体上滴加热显微镜载物台。 </li><li>将持有吸管（120微米直径）进入显微操纵器的支架臂，并将其放置到操作介质的下降。 </li><li>通过在微量牵引装置加热和拉硼硅玻璃毛细管制造注射微量。加载mRNA表达在注射针被注入。 </li><li>打开连接到一个微量压缩空气供给。 </li><li>将注射针插入注射座，把它放在显微操纵器并定位到操作介质的下降。 </li><li>设置的微量，但有以下建议参数:holdP = 2磅，InjP = 20磅，ClearP = 60磅，Injtime = 1秒，这将导致在1-5 2pl的注射体积。 </li><li>轻轻拍打它靠在固定吸管打开注射针的尖端。 </li><li>转移的胚胎（30-50）连接到平台上的微操作压降。用固定吸管捕捉胚胎和对准注射针，胚胎和沿x轴的保持吸管。 </li><li>在400倍放大，通过胚胎推进注射针。要小心避免细胞核。一旦细胞膜被刺穿，按下脚踏板注入。注射后，拔出针头，松开胚胎。重复此过程，所有剩余的EMbryos。 </li><li>在胚胎培养基，它由Earle氏平衡盐溶液（EBSS），补充有非必需氨基酸（NEAA），必需氨基酸（EAA），1mM的L-谷氨酰胺，0.4mM的丙酮酸钠，和10％的洗注射的胚胎三次FBS。 </li><li>在5％CO 2的空气中孵育胚胎在38.5℃下进行1-2小时，然后才可手术转移到同步收件人雌性NZW兔（参见步骤5.1）的输卵管中。可选地，培养的胚胎直到他们达到胚泡期它们被用于体外检验或测定之前。 </li></ol> 5。胚胎移植<ol><li>选择一个女性NZW兔5-12个月大，健康状况良好，体重约。 4.0至5.0千克活性物质受体动物。管理促性腺激素释放激素（GnRH）注射液肌肉注射（IM，50微克/毫升，0.3毫升/动物）给收件人在同一时间点与注射hCG供体动物（S）的（见步骤2.2）。 </LI&gt; <li>注射完成后，准​​备收件人兔子胚胎移植（促性腺激素释放激素注射后一般16-24小时）。麻醉兔10-40毫克/千克氯胺酮（IM），和/或蒸发的异氟烷（2-4％），通过吸入给药。通过无菌眼药膏的应用过度干燥保护兔子的眼睛。观察动物，直到它完全是无意识的， 即 。不响应脚蹬反射;挤压垫脚是用来检查缺乏踏板反射是足够的麻醉的指示。 </li><li>刮胡子兔子的腹部，洗净用消毒擦洗，其次是消毒液，擦拭用无菌纱布海绵剃光区域。 </li><li>在无菌条件下进行手术，记录收​​件人兔子大约每15分钟在整个手术生命体征。 </li><li>转让10-25胚胎1受体动物，这取决于胚胎的可用数目。 </li><li>在本月底手术过程中，关闭肌肉层涂层可吸收缝线。缝合采用可吸收缝线无论是表皮下缝合或使用nonabsorable缝合（ 如单丝，尼龙或类似）单纯间断缝合皮肤皮肤。 </li><li>保持动物温暖和监视它，直到它的胸骨斜卧为止。 </li><li>每天监测动物。按照兽医的指示来照顾动物。辖美洛昔康（SC 1.0毫克/千克），以减轻疼痛和/或减少发热每日邮报操作，如果确定有必要的兽医。 </li><li>约10-14天手术后取出nonabsorable皮肤缝线。 </li></ol> 6。检测的ZFN诱导基因中断<ol><li> 在体外验证，在体外培养的胚胎进行基因分型。开发了以桑椹/胚泡阶段于5μlNP40溶液（1％NP40加1微克/毫升蛋白酶K在Taq缓冲液裂解单个胚胎）为0.5-1小时，在55℃，并在95℃下10分钟</li><li>对于后代的基因分型，收集耳部皮肤样本新生儿包，提取基因组DNA，并用它们作为PCR测序模板。适用佳柏不停地收集后，取样面积止血和减少不适。 </li><li>从步骤6.2或6.3使用裂解如使用LA-Taq酶和ZFN靶位点的PCR引物对，分别位于上游（5&#39;-TGAGGCCGGGAAGGGAGCAGTCG-3&#39;）和下游（5&#39;-GCCAGGCCCACCCACGGAACAGC-3&#39;）模板进行PCR 。 </li><li>纯化PCR产物并用测序引物（5&#39;-TCTGCACGCTTGGGGCTGGAG-3&#39;）序列它们。 </li><li>找出突变体样品通过寻找周围的靶向位点序列图双曲线。标签的那些双曲线为"积极"。 </li><li>克隆"正"PCR产物为TA克隆载体，拿起10-20克隆，序列的插入，并确认周围的T突变argeting网站。 </li></ol>

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作者什么都没有透露。

在目前的工作，使用ZFN技术生成5 ApoCIII KO兔。以前生产GTT兔的只报告还使用了ZFN技术18。目前的工作证实，ZFN是有用的有效靶基因兔。 在本研究中，转基因率（正套/总出生）is23.8％（5/21），这相当于在兔子前面的ZFN报告（30.8％，16/52）18，和那些报道在其他动物物种，包括斑马鱼19，20的小鼠，大鼠21和17的猪使用ZFN的。这个速度实际上比许多传统的转基因兔生产的研究，这通常属于在5-20％范围内的比率较高。例如，在努力通过常规的DNA显微注射产品ApoCIII转基因兔，丁等人获得3个阳性创始人出54幼崽出生（5.6％）22。 与以前的研究结果，突变的靶向位点是可变的，包括缺失或插入含有不同数量的基础（从在五KO兔1-21）。根据创始人动物的特定序列的信息，可以预测，至少四个（ 即 ，那些含Δ1，1，1，或Δ20突变）将有ApoCIII功能丧失。动物的R-16与Δ21可能不显示功能的损失，因为这突变被预测的7个氨基酸仅引起的损失，但不是一个读码框移位。最后，表型测定是必要的，以确定是否这些创始人动物确实显示ApoCIII功能的丧失。 概无在本研究中所产生的五个KO兔含有等位基因修改。有趣的是，这也是在以前的ZFN兔子报告的情况。相反，当目标的ZFNα1，3 - 半乳糖转移酶分别适用于猪细胞中，针对单个等位基因从7-46％之间，大约有三分之一的突变产生单步双等位基因敲除23的频 ​​率。与此相一致，当TALEN应用于猪成纤维细胞中，被发现在总的阳性克隆14的约15-40％的双等位基因的修改。这是可能的故障，产生双等位基因KO兔在本研究中可以指示物种差异（ 即兔与猪），用于基于核酸酶基因打靶的这种能力。它是，但是，更有可能的是这仅仅是因为在这个项目中产生的KO兔子的数量相对较少。我们相信，ZFN能够产生bialleic突变兔，其应被视为ZFN基于GTT兔生产的另一个潜在的优势。还需要进一步的实验来证实ZFN兔这种能力。 总之，目前的工作证明是ZFN基于基因打靶的方法是有效的生产KO兔。具体而言，我们产生5 ApoCIII KO兔的23.8％的满意率转基因。这些动物被认为提供的是关于人体脂质代谢这种蛋白质的作用比相应的小鼠模型更有意义的信息。我们预测ZFN基于基因打靶，以及其他基于核酸的技术，如TALEN和RGEN，在nonmurine动物将显著促进新型动物模型中研究各种人类疾病的发展。 <img alt="图1" fo:content-width="5in" src="/files/ftp_upload/50957/50957fig1.jpg" width="500px" /> 图1。流程图生产中使用ZFN技术击倒兔的 。生产中使用ZFN技术KO兔开始与感兴趣的基因的选择。序列信息提供，ZFN集设计，和主旨ected以酵母为基础的内部验证。在检查点（CP）1（CP-1），只有那些通过内部验证（内部阳性对照的50％或更高）集将被提供给用户选择。如果没有设置通过CP-1，另外的序列可能需要设计有效的ZFN集。 体外胚胎验证其次，以确保所选择的ZFN集可以在选定的地点（CP-2）诱导突变。如果通过CP-2（ 体外胚胎&gt; = 10％的突变率）ZFN集（s）将只被使用。在失败的CP-2要求的ZFN集的重新设计。胚胎捐赠者将编写和原核阶段的胚胎会与验证ZFN套被显微注射。这些显微注射的胚胎将被转移到同步胚胎收件人。一个月后怀孕期，新生儿会进行基因分型（CP-3）。如果没有一个新生儿是积极的，额外的显微注射将被执行。这需要2-3个月从项目开始到CP-2，假设n过程中O故障。它从CP-2额外的2-3个月到CP-3。因此，有可能产生一个基因敲除兔子使用ZFN技术4-6个月的时间。 <a href="https://www.jove.com/files/ftp_upload/50957/50957fig1highres.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图2" fo:content-width="5in" src="/files/ftp_upload/50957/50957fig2.jpg" width="500px" /> 图2。 ZFN设计 。三ZFN集（集1，2和3），其中针对于外显子1或兔ApoCIII（A）外显子2不同的序列。所有三组经受酵母MEL-1报告基因分析，以确定ZFN活动。根据制造商的协议，即显示该制造商的内部阳性对照的&gt; 50％的活动的ZFN被视为用于在体外和体内的基因组编辑实验是有用的。该ZFN活动是为组1 224.2％，为组2 196.9％和集3（B）177.8％，因此设定1是在本研究中选择的。 <a href="https://www.jove.com/files/ftp_upload/50957/50957fig2highres.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图3" fo:content-width="5in" src="/files/ftp_upload/50957/50957fig3.jpg" width="500px" /> 图3 在体外胚胎ZFN的验证 。套装1的mRNA表达（5微克/毫升），显微注射至35原核阶段的胚胎兔（A）的细胞质。冲洗无胚胎显微注射治疗组（n = 31）作为对照组。对BL发展率为77.4％，对照组。在显微注射组，BL率为51.4％。 16酰胺酶进行PCR测序，8（50％）被确定为阳性，说明台1是一种有效的ZFN集以诱导突变的圆盾婷网站（B，apoc3wt.seq，黑盒装）ApoCIII兔。含突变BLS的是BL＃APOC-3，4，5，7，11，13，15，和17（B，红色下划线）。其余的BLS（＃APOC-2，6，9，10，12，14和16）不要在靶向部位有突变。 <a href="https://www.jove.com/files/ftp_upload/50957/50957fig3highres.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图4" fo:content-width="5in" src="/files/ftp_upload/50957/50957fig4.jpg" width="500px" /> 图4。生产ApoCIII KO兔 。一百四十五胚胎显微注射ZFN设为1的mRNA被转移到7假孕收件人兔（A）。新鲜刷新胚胎（n = 75人），而不ZFN显微注射转移到收件人（N = 6）作为对照组。按总期限套件/总胚胎实验组术语率为14.5％（21/145），而速度是29.3％（22/75），对照组（A组）。出出生的显微注射组，5（R 1，R9，R11，R12和R16）在21试剂盒鉴定的PCR测序（B）后作为阳性KO试剂盒。在目标站点上的插入缺失包括两个插入（+1两个R9和R11）和三个缺失（Δ1，Δ20，Δ21，R1，R12和R16，分别）。 <a href="https://www.jove.com/files/ftp_upload/50957/50957fig4highres.jpg" target="_blank">点击这里查看大图</a> 。

载脂蛋白（APO）C-III（ApoCIII）是一种主要在肝脏中合成和小肠小O-糖基化的分泌蛋白。它位于血浆乳糜微粒（CM）的表面上，极低密度脂蛋白（VLDL）和高密度脂蛋白（HDL）。 ApoCIII已被公认为是心血管疾病1的一个危险因素。患者ApoCIII的遗传性缺陷具有低血浆甘油三酯（TG）水平，降低亚临床冠状动脉粥样硬化2,3。升高的血浆ApoCIII水平，另一方面，经常关联与胰岛素抵抗，肥胖，和高甘油三酯血症（HTG）4,5。 基因敲除（KO）是一个强有力的手段，研究了基因的功能。为符合人类突变人群的观察，ApoCIII基因在小鼠基因敲除导致减少血浆TG和保护餐后HTG 6。 ApoCIII这样的积极作用显现是独立的载脂蛋白E（ApoE基因）的，视为缺乏载脂蛋白E既与ApoCIII的小鼠也免受餐后高脂血症7。虽然这些ApoCIII KO小鼠模型提供了宝贵的信息ApoCIII在人体中的可能的功能，应该注意的是小鼠的脂蛋白的代谢是由人类在许多方面不同。例如，小鼠缺乏血浆胆固醇酯转移蛋白（CETP），参与VLDL，LDL的运输的主要酶，和高密度脂蛋白8。因此，有必要开发一种合适的动物模型，以进一步了解ApoCIII 在体内的生理作用。 兔子是一个经典模型动物物种9-11。它有一个短的酝酿期（30-31天），大窝产仔数（4-12/litter），可在室内设施方便地放置。相较于小鼠，兔在系统发育上更接近人类10。重要的是，像胡人，但不像老鼠，兔子是低密度脂蛋白丰富的哺乳动物和有大量的胆固醇酯转运蛋白水平10,12中。此外，他们很容易受到富含胆固醇的饮食引起动脉粥样硬化的病变相似，看到在人类动脉粥样硬化13。基于这些原因，我们推测ApoCIII KO兔子可以作为一个比他们的鼠标同行更好的模型来研究在人类脂质代谢和动脉粥样硬化ApoCIIIplay的角色。 生产基因靶向转基因（GTT）兔子一直是个挑战。这主要是由于缺乏生殖系传递的胚胎干细胞（ESC），并在兔体细胞核转移（SCNT）的效率极低。 ESC是主要的工具来生成GTT小鼠，而和体细胞核移植已成功地应用于产生KO动物物种缺乏生殖系传递的胚胎干细胞，包括猪，羊，牛，但不是兔子。最近锌指核酸酶（ZFN），陈scription活化剂-样效应核酸酶（TALEN）14，和RNA指导核酸内切酶（RGEN）15出现了基因组编辑作为有力手段。这些核酸酶，即所谓的"分子剪刀"，是有效率的产生双链断裂（DSB）在基因组中，可以导致靶基因的功能KO或用于DNA序列在基因组中的特定位点整合一些物种16。 2011年，首批适应ZFN技术，我们利用这些细胞进行体细胞核移植17后生成过氧化物酶体增殖物激活受体γ（PPARγ）KO猪细胞通过这种方法，成功地产生PPARγKO猪。同年，高效免疫球蛋白基因破坏和有针对性的更换兔也使用ZFN报18。 在本研究中，产生了5 ApoCIII KO兔证实通过PCR测序，用ZFN设为1（ 见图1 </sTRONG&gt;。对流程图的图示）。这些动物是新颖的资源来研究脂质代谢ApoCIII的角色。

<table><tbody><tr><td>APOC3-ZFN</td><td>Sigma Aldrich</td><td>CSTZFNY-1KT</td><td>Target Gene: APOC3, Lot Number: 12201118MN</td></tr><tr><td>mMESSAGE kit</td><td>Invitrogen</td><td>AM1344M</td><td>mRNA synthesis</td></tr><tr><td>MEGAclear Kit&amp;nbsp;</td><td>Invitrogen</td><td>AM1908M</td><td>mRNA purification</td></tr><tr><td>Follicle-stimulating hormone</td><td>Bioniche Life Sciences</td><td>Folltropin-V</td><td>Treating embryo donor rabbits</td></tr><tr><td>Human chorionic gonadotropin</td><td>Intervet</td><td>Chorulon</td><td>Treating embryo donor rabbits</td></tr><tr><td>Gonadotropin-releasing hormone</td><td>Prospecbio</td><td>HOR-255</td><td>Treating embryo recipient rabbits</td></tr><tr><td>Earle&#039;s Balanced Salt Solution (EBSS)&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>SH30029.02</td><td>Embryo culture, base medium</td></tr><tr><td>MEM (nonessential amino acid)</td><td>Sigma Aldrich</td><td>M7145</td><td>Embryo culture, supplements</td></tr><tr><td>BME AMINO ACIDS solution</td><td>Sigma Aldrich</td><td>B6766</td><td>Embryo culture, supplements</td></tr><tr><td>Glutamine</td><td>Gibco</td><td>25030-149</td><td>Embryo culture, supplements</td></tr><tr><td>Sodium pyruvate&amp;nbsp;</td><td>Gibco</td><td>11360-070</td><td>Embryo culture, supplements</td></tr><tr><td>Fetal bovine serum</td><td>Sigma Aldrich</td><td>12003C</td><td>Embryo culture, supplements</td></tr><tr><td>HEPES buffered TCM 199&amp;nbsp;</td><td>Gibco</td><td>12350039</td><td>Embryo manipulation medium</td></tr><tr><td>Incubator</td><td>Eppendorf</td><td>Galaxy 170</td><td>Embryo culture equipment</td></tr><tr><td>Micromanipulator&amp;nbsp;</td><td>Eppendorf</td><td>TransferMan NK 2</td><td>Embryo manipulation equipment</td></tr><tr><td>Micropipette puller</td><td>Sutter Instruments Inc.</td><td>P-1000</td><td>Embryo manipulation equipment</td></tr><tr><td>Microinjector&amp;nbsp;</td><td>Tritech Research</td><td>MINJ-D</td><td>Embryo manipulation equipment</td></tr><tr><td>Borosilicate glass capillary tubes&amp;nbsp;</td><td>World Precision Instruments, Inc.</td><td>TW100F-6</td><td>Embryo manipulation supply</td></tr><tr><td>Euthasol (pentobarbitol sodium)</td><td>Virbac AH, Inc.</td><td>ANADA#200-071</td><td>Euthanization of embryo donor rabbits</td></tr></tbody></table>

production of apolipoprotein c-iii knockout rabbits using zinc finger nucleases

载脂蛋白（APO）C-III（ApoCIII）位于血浆乳糜微粒（CM）的表面上，极低密度脂蛋白（VLDL）和高密度脂蛋白（HDL）。人们已经认识到，高水平的血浆ApoCIII constitutea风险因子的心血管疾病（CVD）。升高的血浆ApoCIII水平往往关联与胰岛素抵抗，肥胖，高甘油三酯血症和。上的ApoCIIIin脂质代谢和心血管疾病的作用非常宝贵的知识已经从转基因小鼠模型，包括ApoCIII敲除（KO）小鼠获得的，但是，应该注意的是脂蛋白的小鼠的代谢是由人类在许多方面不同。它不知道到现在为止升高血浆ApoCIII是否是直接动脉粥样硬化。我们致力于发展ApoCIII KO兔的基础上，兔子可以作为reasonablemodelfor研究人类脂质代谢和动脉粥样硬化的假设本研究。锌指核酸酶（ZFN）靶向套鸭兔oCIIIgene经受体外验证之前，胚胎显微注射。该基因被注入到35兔原核期胚胎的细胞质，并评估了突变率在囊胚状态。分别测定了16囊胚，在目标网站满意50％的突变率（8/16）达到了，配套使用设置1的体内实验。接下来，我们145显微注射胚胎设为1的mRNA，并转移这些胚胎7收件人兔子。经过30天的妊娠期，21套出生，其中五人后的PCR测序分析证实为ApoCIII KO兔。劫动物率（＃KO包/总出生）为23.8％。整体生产效率为3.4％（5 kits/145胚胎转移）。目前的工作表明，ZFN是一个以生产KO兔高效的方法。这些ApoCIII KO兔子是新颖的资源来研究脂质代谢ApoCIII的角色。

在 ZFN对体外验证 十六ZFN对最初设计。组1的靶向破坏序列位于兔ApoCIII外显子2（ 图2A）。三对（组1，2，和3， 图2A）被选择，并进行酵母MEL-1报告基因分析来确定ZFN活性（ 图2B）。置1具有ZFN得分的224.2％，高于组2（196.9％）和组3（177.8％）的高，比选择阈值（内部阳性对照，即 50％）高得多。因此，设置1被选定为在体外验证实验。 一组特定的需要导致在10％或更高的胚胎一个阳性率通过在体外胚胎验证的基础上，内部验证标准。组1的mRNA表达（5微克/毫升），显微注射到35原核期兔胚胎（ 图3A &lt;的细胞质/ STRONG&gt;）。冲洗无胚胎显微注射治疗组（n = 31）作为对照组。开发十八胚胎显微注射组，以基本法舞台上D5，其中16进行测序，和八个（BL＃APOC-3，4，5，7，11，13，15，17， 如图3B所示 ，红色下划线）显示突变序列的靶向位点（黑盒装， 如图2B所示 ，最上面一排，apoC3wt.seq）。申请人在基本法率显微注射组（51.4％）的比对照组（77.4％），说明显微注射胚胎轻微受损发育能力低。在显微注射胚胎的正突变率是50.0％（8/16），比选择阈值要高得多（ 即 10％）。这些结果显示，组1是一种有效的ZFN集以诱导突变的兔ApoCIII的靶向位点。因此，设置1被选定为在体内生产ApoCIII KO兔。 生产ApoCIII KO兔 mRNA的øf ZFN集1被显微注射到兔原核期胚胎的细胞质中，并转移145微注射的胚胎至7假孕收件人兔（ 图4A）。新鲜刷新胚胎（n = 75人），而不ZFN显微注射转移到收件人（N = 6）作为对照组。妊娠后30天，21套出生的显微注射组中。 PCR测序确定了五个（APOC3-R1，R9，R11，R12和R16）为阳性KO包（ 图4B）。按总期限套件/总胚胎术语率为14.5％（21/145），而速度是29.3％（22/75），对照组。在KO率计算方法为总KO包/总期限为23.8％（5/21）。一KO试剂盒（载脂蛋白C-R16）死亡5天产后（20.0％，1/5）。该indelsat靶向位点包括两个插入和3缺失;它们areΔ1，1，1，Δ20和Δ21，对兔＃R1，R9，R11，R12，和R16，分别为（ 图4B）。 毕oinformatics分析来识别潜在的ZFN脱靶具有7个或更少的错配，以设置1序列，如先前描述的17。只有一个可能的预测脱靶部位，位于14号染色体上，被认定与7不匹配的基点。 PCR法检测到任何的创始人动物没有脱靶事件。

Watch this Scientific Journal Video about Production of Apolipoprotein C-III Knockout Rabbits using Zinc Finger Nucleases at JoVE.com

Production of Apolipoprotein C-III Knockout Rabbits using Zinc Finger Nucleases

在基因靶向工具最近发展使得生产淘汰赛（KO）的兔子可能。在目前的工作中，我们使用生成的锌指核酸酶（ZFN）五，载脂蛋白（APO）C-III KO兔。这项工作表明，ZFN是一个以生产KO兔高效的方法。

生产用锌指核酸载脂蛋白C-III淘汰赛兔

Apolipoprotein (Apo) C-III (ApoCIII) resides on the surface of plasma chylomicron (CM), very low density lipoprotein (VLDL) and high density lipoproteins ...

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JoVE Journal

Medicine

18.1K 次观看.  University of Michigan Medical Center. 在基因靶向工具最近发展使得生产淘汰赛（KO）的兔子可能。在目前的工作中，我们使用生成的锌指核酸酶（ZFN）五，载脂蛋白（APO）C-III KO兔。这项工作表明，ZFN是一个以生产KO兔高效的方法。 

视频: Production of Apolipoprotein C-III Knockout Rabbits using Zinc Finger Nucleases

Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells

The CRISPR/Cas9 genome engineering system has revolutionized biology by allowing for precise genome editing with little effort. Guided by a single guide RNA (sgRNA) that confers specificity, the Cas9 protein cleaves both DNA strands at the targeted locus. The DNA break can trigger either non-homologous end joining (NHEJ) or homology directed repair (HDR). NHEJ can introduce small deletions or insertions which lead to frame-shift mutations, while HDR allows for larger and more precise perturbations. Here, we present protocols for generating knockout cell lines by coupling established CRISPR/Cas9 methods with two options for downstream selection/screening. The NHEJ approach uses a single sgRNA cut site and selection-independent screening, where protein production is assessed by dot immunoblot in a high-throughput manner. The HDR approach uses two sgRNA cut sites that span the gene of interest. Together with a provided HDR template, this method can achieve deletion of tens of kb, aided by the inserted selectable resistance marker. The appropriate applications and advantages of each method are discussed.

Recent advances in the ability to genetically manipulate somatic cell lines hold great potential for basic and applied research. Here, we present two approaches for CRISPR/Cas9 generated knockout production and screening in mammalian cell lines, with and without the use of selectable markers.

选择依赖和独立生成CRISPR / Cas9介导的哺乳动物细胞基因敲除

The CRISPR/Cas9 genome engineering system has revolutionized biology by allowing for precise genome editing with little effort. Guided by a single guide ...

科研

遗传学

Efficient Production and Identification of CRISPR/Cas9-generated Gene Knockouts in the Model System Danio rerio

Characterization of the clustered, regularly interspaced, short, palindromic repeat (CRISPR) system of Streptococcus pyogenes has enabled the development of a customizable platform to rapidly generate gene modifications in a wide variety of organisms, including zebrafish. CRISPR-based genome editing uses a single guide RNA (sgRNA) to target a CRISPR-associated (Cas) endonuclease to a genomic DNA (gDNA) target of interest, where the Cas endonuclease generates a double-strand break (DSB). Repair of DSBs by error-prone mechanisms lead to insertions and/or deletions (indels). This can cause frameshift mutations that often introduce a premature stop codon within the coding sequence, thus creating a protein-null allele. CRISPR-based genome engineering requires only a few molecular components and is easily introduced into zebrafish embryos by microinjection. This protocol describes the methods used to generate CRISPR reagents for zebrafish microinjection and to identify fish exhibiting germline transmission of CRISPR-modified genes. These methods include in vitro transcription of sgRNAs, microinjection of CRISPR reagents, identification of indels induced at the target site using a PCR-based method called a heteroduplex mobility assay (HMA), and characterization of the indels using both a low throughput and a powerful next-generation sequencing (NGS)-based approach that can analyze multiple PCR products collected from heterozygous fish. This protocol is streamlined to minimize both the number of fish required and the types of equipment needed to perform the analyses. Furthermore, this protocol is designed to be amenable for use by laboratory personal of all levels of experience including undergraduates, enabling this powerful tool to be economically employed by any research group interested in performing CRISPR-based genomic modification in zebrafish.

Efficient Production and Identification of CRISPR/Cas9-generated Gene Knockouts in the Model System Danio rerio

Targeted genome editing in the model system Danio rerio (zebrafish) has been greatly facilitated by the emergence of CRISPR-based approaches. Herein, we describe a streamlined, robust protocol for generation and identification of CRISPR-derived nonsense alleles that incorporates the heteroduplex mobility assay and identification of mutations using next-generation sequencing.

斑马斑马模型系统中 CRISPR/Cas9-generated 基因挖空的高效生产与鉴定

Characterization of the clustered, regularly interspaced, short, palindromic repeat (CRISPR) system of Streptococcus pyogenes has enabled the development ...

Culturing and Manipulation of O9-1 Neural Crest Cells

Neural crest cells (NCCs) are migrating multipotent stem cells that can differentiate into different cell types and give rise to multiple tissues and organs. The O9-1 cell line is derived from the endogenous mouse embryonic NCCs and maintains its multipotency. However, under specific culture conditions, O9-1 cells can differentiate into different cell types and be utilized in a wide range of research applications. Recently, with the combination of mouse studies and O9-1 cell studies, we have shown that the Hippo signaling pathway effectors Yap and Taz play important roles in neural crest-derived craniofacial development. Although the culturing process for O9-1 cells is more complicated than that used for other cell lines, the O9-1 cell line is a powerful model for investigating NCCs in vitro. Here, we present a protocol for culturing the O9-1 cell line to maintain its stemness, as well as protocols for differentiating O9-1 cells into different cell types, such as smooth muscle cells and osteoblasts. In addition, protocols are described for performing gene loss-of-function studies in O9-1 cells by using CRISPR-Cas9 deletion and small interfering RNA-mediated knockdown.

O9-1 is a multipotent mouse neural crest cell line. Here we describe detailed step-by-step protocols for culturing O9-1 cells, differentiating O9-1 cells into specific cell types, and genetically manipulating O9-1 cells by using siRNA-mediated knockdown or CRISPR-Cas9 genome editing.

O9-1 神经嵴细胞的培养与调控

Neural crest cells (NCCs) are migrating multipotent stem cells that can differentiate into different cell types and give rise to multiple tissues and ...

CRISPR/Cas9 Ribonucleoprotein-mediated Precise Gene Editing by Tube Electroporation

Gene editing nucleases, represented by CRISPR-associated protein 9 (Cas9), are becoming mainstream tools in biomedical research. Successful delivery of CRISPR/Cas9 elements into the target cells by transfection is a prerequisite for efficient gene editing. This protocol demonstrates that tube electroporation (TE) machine-mediated delivery of CRISPR/Cas9 ribonucleoprotein (RNP), along with single-stranded oligodeoxynucleotide (ssODN) donor templates to different types of mammalian cells, leads to robust precise gene editing events. First, TE was applied to deliver CRISPR/Cas9 RNP and ssODNs to induce disease-causing mutations in the interleukin 2 receptor subunit gamma (IL2RG) gene and sepiapterin reductase (SPR) gene in rabbit fibroblast cells. Precise mutation rates of 3.57%-20% were achieved as determined by bacterial TA cloning sequencing. The same strategy was then used in human iPSCs on several clinically relevant genes including epidermal growth factor receptor (EGFR), myosin binding protein C, cardiac (Mybpc3), and hemoglobin subunit beta (HBB). Consistently, highly precise mutation rates were achieved (11.65%-37.92%) as determined by deep sequencing (DeepSeq). The present work demonstrates that tube electroporation of CRISPR/Cas9 RNP represents an efficient transfection protocol for gene editing in mammalian cells.

Presented here is a protocol for efficient CRISPR/Cas9 ribonucleoprotein-mediated gene editing in mammalian cells using tube electroporation.

CRISPR/Cas9 核糖核酸蛋白介导的精确基因编辑通过管电穿孔

Gene editing nucleases, represented by CRISPR-associated protein 9 (Cas9), are becoming mainstream tools in biomedical research. Successful delivery of ...

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. ...

A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys

Polycystic kidney disease (PKD) is one of the most common causes of end-stage kidney disease, a devastating disease for which there is no cure. The molecular mechanisms leading to cyst formation in PKD remain somewhat unclear, but many genes are thought to be involved. Wnt5a is a non-canonical glycoprotein that regulates a wide range of developmental processes. Wnt5a works through the planar cell polarity (PCP) pathway that regulates oriented cell division during renal tubular cell elongation. Defects of the PCP pathway have been found to cause kidney cyst formation. Our paper describes a method for developing a zebrafish cystic kidney disease model by knockdown of the wnt5a gene with wnt5a antisense morpholino (MO) oligonucleotides. Tg(wt1b:GFP) transgenic zebrafish were used to visualize kidney structure and kidney cysts following wnt5a knockdown. Two distinct antisense MOs (AUG - and splice-site) were used and both resulted in curly tail down phenotype and cyst formation after wnt5a knockdown. Injection of mouse Wnt5a mRNA, resistant to the MOs due to a difference in primary base pair structure, rescued the abnormal phenotype, demonstrating that the phenotype was not due to &#8220;off-target&#8221; effects of the morpholino. This work supports the validity of using a zebrafish model to study wnt5a function in the kidney.

A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys

We describe a method of generating a possible zebrafish model of polycystic kidney disease. We used Tg(wt1b:GFP) fish to visualize kidney structure. Knockdown of wnt5a was by morpholino injection. Pronephric cyst formation after wnt5a knockdown was observed in this GFP transgenic zebrafish.

多囊肾一种可能的斑马鱼型号:击倒<em&gt; Wnt5a的</em&gt;原因在斑马鱼肾囊肿

Polycystic kidney disease (PKD) is one of the most common causes of end-stage kidney disease, a devastating disease for which there is no cure. The ...

医学

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 ...

发育生物学

Genome Editing with CompoZr Custom Zinc Finger Nucleases (ZFNs)

Genome editing is a powerful technique that can be used to elucidate gene function and the genetic basis of disease. Traditional gene editing methods such as chemical-based mutagenesis or random integration of DNA sequences confer indiscriminate genetic changes in an overall inefficient manner and require incorporation of undesirable synthetic sequences or use of aberrant culture conditions, potentially confusing biological study. By contrast, transient ZFN expression in a cell can facilitate precise, heritable gene editing in a highly efficient manner without the need for administration of chemicals or integration of synthetic transgenes.
Zinc finger nucleases (ZFNs) are enzymes which bind and cut distinct sequences of double-stranded DNA (dsDNA). A functional CompoZr ZFN unit consists of two individual monomeric proteins that bind a DNA "half-site" of approximately 15-18 nucleotides (see Figure 1). When two ZFN monomers "home" to their adjacent target sites the DNA-cleavage domains dimerize and create a double-strand break (DSB) in the DNA.1 Introduction of ZFN-mediated DSBs in the genome lays a foundation for highly efficient genome editing. Imperfect repair of DSBs in a cell via the non-homologous end-joining (NHEJ) DNA repair pathway can result in small insertions and deletions (indels). Creation of indels within the gene coding sequence of a cell can result in frameshift and subsequent functional knockout of a gene locus at high efficiency.2 While this protocol describes the use of ZFNs to create a gene knockout, integration of transgenes may also be conducted via homology-directed repair at the ZFN cut site.
The CompoZr Custom ZFN Service represents a systematic, comprehensive, and well-characterized approach to targeted gene editing for the scientific community with ZFN technology. Sigma scientists work closely with investigators to 1) perform due diligence analysis including analysis of relevant gene structure, biology, and model system pursuant to the project goals, 2) apply this knowledge to develop a sound targeting strategy, 3) then design, build, and functionally validate ZFNs for activity in a relevant cell line. The investigator receives positive control genomic DNA and primers, and ready-to-use ZFN reagents supplied in both plasmid DNA and in-vitro transcribed mRNA format. These reagents may then be delivered for transient expression in the investigator&#x2019;s cell line or cell type of choice. Samples are then tested for gene editing at the locus of interest by standard molecular biology techniques including PCR amplification, enzymatic digest, and electrophoresis. After positive signal for gene editing is detected in the initial population, cells are single-cell cloned and genotyped for identification of mutant clones/alleles.

Genome Editing with CompoZr Custom Zinc Finger Nucleases ZFNs

The CompoZr Custom Zinc-Finger Nuclease (ZFN) Service enables precise genome editing in any organism or cell line at any locus defined by the user. This article describes the process for the design, manufacture, validation and implementation of the CompoZr Custom ZFN Service.

基因组编辑与的CompoZr自锌指核酸酶（ZFNs）

Genome editing is a powerful technique that can be used to elucidate gene function and the genetic basis of disease. Traditional gene editing methods such ...

生物学

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

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

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

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

TGF-β介质到中微过渡（末端MT）和使用CRISPR/Cas9基因编辑对末端效应器的功能评估

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

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 ...