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。

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			<td height="21" style="height:21px;width:245px;">APOC3-ZFN</td>
			<td style="width:276px;">Sigma Aldrich</td>
			<td style="width:136px;">CSTZFNY-1KT</td>
			<td style="width:167px;">Target Gene: APOC3, Lot Number: 12201118MN</td>
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		<tr height="21">
			<td height="21" style="height:21px;">mMESSAGE kit</td>
			<td>Invitrogen</td>
			<td>AM1344M</td>
			<td>mRNA synthesis</td>
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		<tr height="21">
			<td height="21" style="height:21px;">MEGAclear Kit&#160;</td>
			<td>Invitrogen</td>
			<td style="width:136px;">AM1908M</td>
			<td>mRNA purification</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Follicle-stimulating hormone</td>
			<td>Bioniche Life Sciences</td>
			<td style="width:136px;">Folltropin-V</td>
			<td>Treating embryo donor rabbits</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Human chorionic gonadotropin</td>
			<td>Intervet</td>
			<td style="width:136px;">Chorulon</td>
			<td>Treating embryo donor rabbits</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gonadotropin-releasing hormone</td>
			<td>Prospecbio</td>
			<td>HOR-255</td>
			<td>Treating embryo recipient rabbits</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Earle&#39;s Balanced Salt Solution (EBSS)&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:136px;">SH30029.02</td>
			<td>Embryo culture, base medium</td>
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		<tr height="21">
			<td height="21" style="height:21px;">MEM (nonessential amino acid)</td>
			<td style="width:276px;">Sigma Aldrich</td>
			<td style="width:136px;">M7145</td>
			<td>Embryo culture, supplements</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BME AMINO ACIDS solution</td>
			<td style="width:276px;">Sigma Aldrich</td>
			<td style="width:136px;">B6766</td>
			<td>Embryo culture, supplements</td>
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			<td height="21" style="height:21px;">Glutamine</td>
			<td>Gibco</td>
			<td>25030-149</td>
			<td>Embryo culture, supplements</td>
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			<td height="21" style="height:21px;">Sodium pyruvate&#160;</td>
			<td>Gibco</td>
			<td>11360-070</td>
			<td>Embryo culture, supplements</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal bovine serum</td>
			<td style="width:276px;">Sigma Aldrich</td>
			<td>12003C</td>
			<td>Embryo culture, supplements</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES buffered TCM 199&#160;</td>
			<td>Gibco&#160;</td>
			<td style="width:136px;">12350039</td>
			<td>Embryo manipulation medium</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Incubator</td>
			<td style="width:276px;">Eppendorf&#160;</td>
			<td style="width:136px;">Galaxy 170</td>
			<td>Embryo culture equipment</td>
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			<td height="18" style="height:18px;">Micromanipulator&#160;</td>
			<td>Eppendorf&#160;</td>
			<td style="width:136px;">TransferMan NK 2</td>
			<td>Embryo manipulation equipment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micropipette puller</td>
			<td>Sutter Instruments Inc.</td>
			<td style="width:136px;">P-1000</td>
			<td>Embryo manipulation equipment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microinjector&#160;</td>
			<td>Tritech Research</td>
			<td style="width:136px;">MINJ-D&#160;</td>
			<td>Embryo manipulation equipment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Borosilicate glass capillary tubes&#160;</td>
			<td>World Precision Instruments, Inc.</td>
			<td style="width:136px;">TW100F-6</td>
			<td>Embryo manipulation supply</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Euthasol (pentobarbitol sodium)</td>
			<td style="width:276px;">Virbac AH, Inc.</td>
			<td style="width:136px;">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 ...

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

Research

JoVE Journal

Medicine

18.0K 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

Isolating and Using Sections of Bovine Mesenteric Artery and Vein as a Bioassay to Test for Vasoactivity in the Small Intestine

Mammalian gastrointestinal systems are constantly exposed to compounds (desirable and undesirable) that can have an effect on blood flow to and from that system. Changes in blood flow to the small intestine can result in effects on the absorptive functions of the organ. Particular interest in toxins liberated from feedstuffs through fermentative and digestive processes has developed in ruminants as an area where productive efficiencies could be improved. The video associated with this article describes an in vitro bioassay developed to screen compounds for vasoactivity in isolated cross-sections of bovine mesenteric artery and vein using a multimyograph. Once the blood vessels are mounted and equilibrated in the myograph, the bioassay itself can be used: as a screening tool to evaluate the contractile response or vasoactivity of compounds of interest; determine the presence of receptor types by pharmacologically targeting receptors with specific agonists; determine the role of a receptor with the presence of one or more antagonists; or determine potential interactions of compounds of interest with antagonists. Through all of this, data are collected real-time, tissue collected from a single animal can be exposed to a large number of different experimental treatments (an in vitro advantage), and represents vasculature on either side of the capillary bed to provide an accurate picture of what could be happening in the afferent and efferent blood supply supporting the small intestine.

The small intestine is frequently exposed to toxins that can influence blood flow and negatively impact nutrient absorption. Using a multimyograph and mesenteric artery and vein isolates, compounds or toxins of interest can be screened for vasoactivity.

隔离和使用的牛肠系膜动脉和静脉部分，作为生物测定在小肠测试的Vasoactivity

Mammalian gastrointestinal systems are constantly exposed to compounds (desirable and undesirable) that can have an effect on blood flow to and from that ...

科研

医学

A Step by Step Protocol for Subretinal Surgery in Rabbits

Age related macular degeneration (AMD), retinitis pigmentosa, and other RPE related diseases are the most common causes for irreversible loss of vision in adults in industrially developed countries. RPE transplantation appears to be a promising therapy, as it may replace dysfunctional RPE, restore its function, and thereby vision.
Here we describe a method for transplanting a cultured RPE monolayer on a scaffold into the subretinal space (SRS) of rabbits. After vitrectomy xenotransplants were delivered into the SRS using a custom made shooter consisting of a 20-gauge metallic nozzle with a polytetrafluoroethylene (PTFE) coated plunger. The current technique evolved in over 150 rabbit surgeries over 6 years. Post-operative follow-up can be obtained using non-invasive and repetitive in vivo imaging such as spectral domain optical coherence tomography (SD-OCT) followed by perfusion-fixed histology.
The method has well-defined steps for easy learning and high success rate. Rabbits are considered a large eye animal model useful in preclinical studies for clinical translation. In this context rabbits are a cost-efficient and perhaps convenient alternative to other large eye animal models.

Retinal pigment epithelium (RPE) replacement strategies and gene-based therapy are considered for several retinal degenerative conditions. For clinical translation, large eye animal models are required to study surgical techniques applicable in patients. Here we present a rabbit model for subretinal surgery geared towards RPE transplantation, which is versatile and cost-efficient.

一种用于兔视网膜下手术分步协议

Age related macular degeneration (AMD), retinitis pigmentosa, and other RPE related diseases are the most common causes for irreversible loss of vision in ...

A High Throughput, Multiplexed and Targeted Proteomic CSF Assay to Quantify Neurodegenerative Biomarkers and Apolipoprotein E Isoforms Status

Many neurodegenerative diseases are&#160;still lacking effective treatments. Reliable biomarkers for identifying and classifying these diseases will be important in the development of future novel therapies. Often&#160;potential new biomarkers do not make it&#160;into the clinic&#160;due to limitations&#160;in their development and&#160;high costs.&#160;However,&#160;targeted proteomics using Multiple Reaction Monitoring Liquid Chromatography-tandem/Mass Spectrometry (MRM LC-MS/MS), specifically using triple quadrupole mass spectrometers, is one method that can be used to rapidly evaluate and validate biomarkers&#160;for clinical translation into diagnostic laboratories. Traditionally, this platform has been used extensively for measurement of small molecules&#160;in clinical laboratories, but it is the potential to analyze proteins, that makes it an attractive alternative to ELISA (Enzyme-Linked Immunosorbent Assay)-based methods. We describe here how targeted proteomics can be used to measure multiplexed markers of dementia, including the detection and quantitation of the known risk factor apolipoprotein E isoform 4 (ApoE4).
In order to make the assay suitable for translation, it is designed to be rapid, simple, highly specific and cost effective. To achieve this, every step in the development of the assay must be optimized for the individual proteins and tissues they are analyzed in. This method describes a typical workflow including various tips and tricks to developing a targeted proteomics MRM LC-MS/MS for translation.
The method development is optimized using custom synthesized versions of tryptic quantotypic peptides, which calibrate&#160;the MS for detection and then spiked into CSF to determine correct identification of the endogenous peptide in the chromatographic separation prior to analysis in the MS. To achieve absolute quantitation, stable isotope-labeled internal standard versions of the peptides with short amino acid sequence tags and containing a trypsin cleavage site, are included in the assay.

We describe a high-throughput, multiplex, and targeted proteomic cerebrospinal fluid (CSF) assay developed with potential for clinical translation. The test can quantitate potential markers and risk factors for neurodegeneration, such as the apolipoprotein E variants (E2, E3 and E4), and measure their allelic expression.

高通量，复用和有针对性的蛋白质组学分析脑脊液量化神经退行性生物标志物和载脂蛋白E亚型状态

Many neurodegenerative diseases are&#160;still lacking effective treatments. Reliable biomarkers for identifying and classifying these diseases will be ...

Induction of Atherosclerotic Plaques Through Activation of Mineralocorticoid Receptors in Apolipoprotein E-deficient Mice

Atherosclerosis is due to a chronic inflammatory response affecting vascular endothelium and is promoted by several factors such as hypertension, dyslipidemia, and diabetes. To date, there is evidence to support a role for circulating aldosterone as a risk factor for the development of cardiovascular disease. Transgenic mouse models have been generated to study cellular and molecular processes leading to atherosclerosis. In this manuscript, we describe a protocol that takes advantage of continuous infusion of aldosterone in ApoE-/- mice and generates atherosclerotic plaques in the aortic root after 4 weeks of treatment. We, therefore, illustrate a method for quantification and characterization of atherosclerotic lesions at the aortic root level. The added value of aldosterone infusion is represented by the generation of atherosclerotic lesions rich in lipid and inflammatory cells after 4 weeks of treatment. We describe in detail the staining procedures to quantify lipid and macrophage content within the plaque. Notably, in this protocol, we perform heart tissue-embedding in OCT in order to preserve the antigenicity of cardiac tissue and facilitate detectability of antigens of interest. Analysis of the plaque phenotype represents a valid approach to study the pathophysiology of atherosclerosis development and to identify novel pharmacological targets for the development of anti-atherogenic drugs.

We describe a protocol to induce atherosclerosis in the aortic root of ApoE-/- mice fed with an atherogenic diet, through a continuous release of aldosterone. Methods to characterize plaque composition are also described.

载脂蛋白 E 缺乏小鼠盐皮质激素受体的活化诱导动脉粥样硬化斑块的研究

Atherosclerosis is due to a chronic inflammatory response affecting vascular endothelium and is promoted by several factors such as hypertension, ...

Preclinical Model of Hind Limb Ischemia in Diabetic Rabbits

Peripheral vascular disease is a widespread clinical problem that affects millions of patients worldwide. A major consequence of peripheral vascular disease is the development of ischemia. In severe cases, patients can develop critical limb ischemia in which they experience constant pain and an increased risk of limb amputation. Current therapies for peripheral ischemia include bypass surgery or percutaneous interventions such as angioplasty with stenting or atherectomy to restore blood flow. However, these treatments often fail to the continued progression of vascular disease or restenosis or are contraindicated due to the overall poor health of the patient. A promising potential approach to treat peripheral ischemia involves the induction of therapeutic neovascularization to allow the patient to develop collateral vasculature. This newly formed network alleviates peripheral ischemia by restoring perfusion to the affected area. The most frequently employed preclinical model for peripheral ischemia utilizes the creation of hind limb ischemia in healthy rabbits through femoral artery ligation. In the past, however, there has been a strong disconnect between the success of preclinical studies and the failure of clinical trials regarding treatments for peripheral ischemia. Healthy animals typically have robust vascular regeneration in response to surgically induced ischemia, in contrast to the reduced vascularity and regeneration in patients with chronic peripheral ischemia. Here, we describe an optimized animal model for peripheral ischemia in rabbits that includes hyperlipidemia and diabetes. This model has reduced collateral formation and blood pressure recovery in comparison to a model with a higher cholesterol diet. Thus, the model may provide better correlation with human patients with compromised angiogenesis from the common co-morbidities that accompany peripheral vascular disease.

We describe a surgical procedure used to induce peripheral ischemia in rabbits with hyperlipidemia and diabetes. This surgery acts as a preclinical model for conditions experienced in peripheral artery disease in patients. Angiography is also described as a means to measure the extent of introduced ischemia and recovery of perfusion.

糖尿病家兔欣德肢体缺血的临床前模型

Peripheral vascular disease is a widespread clinical problem that affects millions of patients worldwide. A major consequence of peripheral vascular ...

Establishment of a Severe Dry Eye Model Using Complete Dacryoadenectomy in Rabbits

Dry eye disease (DED) is a complex disease with multiple etiologies and variable symptoms, having ocular surface inflammation as its key pathophysiologic step. Despite advances in our understanding of DED, significant knowledge gaps remain. Advances are limited in part due to the lack of informative animal models. The authors recently reported on a method of DED induced by injecting all orbital lacrimal gland (LG) tissues with the lectin concanavalin A. Here, we report a novel model of aqueous-deficient DED based on the surgical resection of all orbital LG (dacryoadenectomy) tissues. Both methods use rabbits because of their similarity to human eyes in terms of the size and structure of the ocular surface. One week after removal of the nictitating membrane, the orbital superior LG was surgically removed under anesthesia, followed by removal of the palpebral superior LG, and finally removal of the inferior LG. Dacryoadenectomy induced severe DED, evidenced by a marked reduction in the tear break up time test and the Schirmer&#39;s tear test, and significantly increased tear osmolarity and rose bengal staining. Dacryoadenectomy-induced DED lasted at least eight weeks. There were no complications and animals tolerated the procedure well. The technique can be mastered relatively easily by those with adequate surgical experience and appreciation of the relevant rabbit anatomy. Since this model recapitulates the features of human aqueous-deficient DED, it is suitable for studies of ocular surface homeostasis, DED, and candidate therapeutics.

A novel approach is presented to induce chronic dry eye disease in rabbits by surgically removing all orbital lacrimal glands. This method, distinct from those previously reported, produces a stable, reproducible model of aqueous deficient dry eye well suited to study tear physiology and pathophysiology and ocular therapeutics.

在兔子中使用完全的白眼切除术建立严重干眼模型

Dry eye disease (DED) is a complex disease with multiple etiologies and variable symptoms, having ocular surface inflammation as its key pathophysiologic ...

Blind Endotracheal Intubation in Neonatal Rabbits

The newborn rabbit is a useful animal model for various pathologies and procedures. Airway management of the rabbit is complex due to its anatomical characteristics, which is further complicated in the case of the newborn. Of the different methods of advanced airway management, endotracheal intubation is less aggressive than tracheostomy, and is more feasible than supraglottic management given the lack of supraglottic devices of such a small size. As direct glottis visualization is very difficult in animals this size, this blind intubation model is presented as an effective alternative, especially for experiments requiring prolonged anesthesia. Using this method, we performed blind intubations with a 90% success rate.

We describe a technique of endotracheal intubation in newborn rabbits after esophageal catheterization with a gastric tube.

新生儿兔子的盲内窥管插管

The newborn rabbit is a useful animal model for various pathologies and procedures. Airway management of the rabbit is complex due to its anatomical ...

Multi-system Monitoring for Identification of Seizures, Arrhythmias and Apnea in Conscious Restrained Rabbits

Patients with ion channelopathies are at a high risk of developing seizures and fatal cardiac arrhythmias. There is a higher prevalence of heart disease and arrhythmias in people with epilepsy (i.e., epileptic heart.) Additionally, cardiac and autonomic disturbances have been reported surrounding seizures. 1:1,000 epilepsy patients/year die of sudden unexpected death in epilepsy (SUDEP). The mechanisms for SUDEP remain incompletely understood. Electroencephalograms (EEG) and electrocardiograms (ECG) are two techniques routinely used in the clinical setting to detect and study the substrates/triggers for seizures and arrhythmias. While many studies and descriptions of this methodology are in rodents, their cardiac electrical activity differs significantly from humans. This article provides a description of a non-invasive method for recording simultaneous video-EEG-ECG-oximetry-capnography in conscious rabbits. As cardiac electrical function is similar in rabbits and humans, rabbits provide an excellent model of translational diagnostic and therapeutic studies. In addition to outlining the methodology for data acquisition, we discuss the analytical approaches for examining neuro-cardiac electrical function and pathology in rabbits. This includes arrhythmia detection, spectral analysis of EEG and a seizure scale developed for restrained rabbits.

Using simultaneous video-EEG-ECG-oximetry-capnography, we developed a methodology to evaluate the susceptibility of rabbit models to develop provoked arrhythmias and seizures. This novel recording system establishes a platform to test the efficacy and safety of therapeutics and can capture the complex cascade of multi-system events that culminate in sudden death.

意识约束兔子中癫痫发作、心律失常和呼吸暂停的多系统监测

Patients with ion channelopathies are at a high risk of developing seizures and fatal cardiac arrhythmias. There is a higher prevalence of heart disease ...

Three-Dimensional Finger Motion Tracking during Needling: A Solution for the Kinematic Analysis of Acupuncture Manipulation

Three-dimensional (3D) motion tracking has been used in many fields, such as the researches of sport and medical skills. This experiment aimed to use 3D motion tracking technology to measure the kinematic parameters of the joints of fingers during acupuncture manipulation (AM) and establish three technical indicators "amplitude, velocity and time". This method can reflect the operation characteristics of AM and provide quantitative parameters along three axes of multiple finger joints. The current evidence shows that the method has great potential for future applications such as the study of the dose-effect relationship of acupuncture, teaching, and learning of AM, and the measurement and preservation of famous acupuncturists' AM.

This experimental method describes a solution for the kinematic analysis of acupuncture manipulation with three-dimensional finger motion tracking technology.

针刺过程中的三维手指运动跟踪：针灸操作运动学分析的解决方案

Three-dimensional (3D) motion tracking has been used in many fields, such as the researches of sport and medical skills. This experiment aimed to use 3D ...

A Modified Technique for Arteriovenous Fistula Construction in Rabbits

Juxta-anastomotic stenosis is a challenging problem that often causes non-maturation and decreases the patency of an arteriovenous fistula (AVF). Injury to the veins and arteries during the operation and hemodynamic changes can lead to intimal hyperplasia, leading to juxta-anastomotic stenosis. To reduce injury to the veins and arteries during the operation, this study proposes a new modified no-touch technique (MNTT) for AVF construction that can decrease the rate of juxta-anastomotic stenosis and improve the AVF patency. To unravel the hemodynamic changes and mechanisms of the MNTT, this study presented an AVF procedure using this technique. Although this procedure is technically challenging, 94.4% procedural success was achieved after adequate training. Ultimately, 13 out of 34 rabbits had a functional AVF 4 weeks after the surgery, leading to a 38.2% AVF patency rate. However, at 4 weeks, the survival rate was 86.1%. Ultrasonography showed active blood flow through AVF anastomosis. Furthermore, the spiral laminar flow was observed in the vein and artery near the anastomosis, suggesting that this technique may improve the hemodynamics of the AVF. On histological observation, significant venous intimal hyperplasia was observed at the AVF anastomosis, whereas no significant intimal hyperplasia was observed at the proximal external jugular vein (EJV) of the anastomosis. This technique will improve the understanding of the mechanisms underlying the use of MNTT for AVF construction and provide technical support for the further optimization of the surgical approach in AVF construction.

The present protocol proposes the creation of an arteriovenous fistula in rabbits using a modified no-touch technique. The technique involves the side-to-side anastomosis of the common carotid artery and external jugular vein without the dissection of the perivenous tissues or cutting off the artery.

一种改良的兔动静脉瘘构建技术

Juxta-anastomotic stenosis is a challenging problem that often causes non-maturation and decreases the patency of an arteriovenous fistula (AVF). Injury ...