CN106191114A - 利用CRISPR‑Cas9系统敲除鱼类MC4R基因的育种方法 - Google Patents
利用CRISPR‑Cas9系统敲除鱼类MC4R基因的育种方法 Download PDFInfo
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Abstract
本发明提供一种利用CRISPR‑Cas9系统敲除鱼类MC4R基因的育种方法，包括MC4R基因打靶位点的确定、gRNA的制备、体外显微注射与敲除验证、MC4R基因敲除鱼的筛选、杂合鱼的培育等步骤。该育种方法适用于所有的经济鱼类，通过敲除鱼类的MC4R基因，从而实现鱼类的快速生长育种。与传统的育种方法相比，本发明方法具有准确性高、成本低、得到纯系时间短的特点。与转基因育种相比，由于基因敲除是使鱼本身的基因功能缺失，不会引入外来基因，因此，不存在转基因安全问题。
Description
技术领域
本发明涉及鱼类的分子育种领域，特别是涉及一种利用CRISPR-Cas9系统敲除鱼类MC4R基因的育种方法。
背景技术
黑素皮质素受体4(Melanocortin 4receptor，MC4R)属于G-蛋白偶联受体的家族，是下丘脑腹内侧核分泌的一类肽类物质，在调节能量动态平衡和肥胖症发生上具有重要作用。早在1997年，Huszar等通过对敲除MC4R基因的小鼠进行研究，发现敲除MC4R基因的小鼠出现遗传性肥胖，表现出多食、胰岛素分泌过多、肥胖等症状(Huszar D,Lynch C A,Fairchild-Huntress V,et al.Cell,1997,88(1):131-141.)。近年来一系列的研究结果表明，MC4R基因突变或多态性对动物生长性能有显著的影响。2009年，过表达抑制MC4R活性的AGRP，美国黑鲈表现出了摄食增加和体型变长的表征(Sanchez E,Rubio V C,Thompson D,et al.AJP:Regulatory,Integrative and Comparative Physiology,2009,296(5):R1293-R1306.)。2010年，Lampert发现MC4R的突变会引起硬骨鱼类生长速度增快，体型增大(Lampert K P,Schmidt C,Fischer P,et al.Curr Biol,2010,20(19):1729-1734.)。但是，目前还没有研究报道在鱼类中敲除MC4R基因是否能够用于提高水产动物的生长育种速度。
发明内容
鉴于以上所述现有技术的缺点，本发明的目的在于提供一种利用CRISPR-Cas9系统敲除鱼类MC4R基因的育种方法，用于解决现有技术中鱼类水产动物的生长速度较慢等问题。
为实现上述目的及其他相关目的，本发明第一方面提供一种利用CRISPR-Cas9系统敲除鱼类MC4R基因的育种方法，包括如下步骤：
1)MC4R基因打靶位点的确定：选择MC4R基因打靶位点序列；
2)gRNA的制备：设计含有MC4R基因打靶位点序列的上游引物以及与其匹配的下游引物，通过体外转录得到gRNA；
3)体外显微注射：鱼产卵后，收集受精卵，将gRNA和Cas9RNA注射进入鱼的受精卵，对注射的受精卵进行孵化培育；
4)MC4R基因敲除鱼的筛选：通过剪尾检测选出步骤3)中MC4R基因敲除阳性的鱼，这类鱼具有比野生鱼更快的生长速度。
进一步地，步骤1)中，所述MC4R基因打靶位点序列如SEQ ID NO.1所示。
进一步地，步骤2)中，上游引物序列如SEQ ID NO.2所示，下游引物序列如SEQ IDNO.3所示。
进一步地，步骤2)中，以含有gRNA骨架的质粒为模板进行体外转录，质粒序列如SEQ ID NO.4所示，通过RNA体外转录试剂盒转录为gRNA。
进一步地，步骤2)中，通过PCR扩增gRNA的DNA序列，PCR反应程序为：98℃5min；98℃10s，58℃30s，72℃20s共35个循环，72℃5min。
更进一步地，步骤2)中，以纯化后的产物为模板进行体外转录，转录体系为20μl，其中纯化后的产物600ng、T7转录酶1μl、反应缓冲液2μl、NTP混合物1μl、余量为无菌水，37℃孵育3h，转录产物经琼脂糖凝胶电泳检测后，通过RNA纯化试剂盒纯化回收，-80℃保存。
进一步地，步骤3)中，注射体系的总体积10μl，包括cas9RNA 300ng/μl、gRNA30ng/μl、酚红0.5μl、余量为无菌水。
进一步地，步骤4)中，通过剪尾检测选出步骤3)中MC4R基因敲除阳性的鱼，命名为P0代，P0代与野生型鱼杂交，得到F1代杂合体，筛选出MC4R基因敲除阳性的鱼。
更进一步地，步骤4)中，得到F1代杂合体后，通过测序与野生型鱼比较，选出两种品系，品系1缺失8个碱基，品系2缺失4个碱基，均引起基因的移码突变，选择F1代相同品系的鱼进行自交，得到F2代，通过剪尾检测，挑选得到MC4R基因敲除阳性的纯系鱼，纯系鱼具有比野生型鱼更快的生长速度。
更进一步地，步骤4)中，从F2代中通过剪尾检测挑选出MC4R基因敲除阳性的鱼后，使其与野生型鱼杂交，得到快速生长的杂合鱼。
进一步地，步骤3)中，还包括敲除验证，具体为：选取经过培育的受精卵并分别提取DNA，在MC4R基因打靶位点两侧设计引物，扩增片段大小为500bp左右。引物序列如SEQ IDNO.5和SEQ ID NO.6所示，对DNA进行扩增，利用T7核酸内切酶I确定打靶效率，随后亚克隆到测序载体中送样测序并同野生型鱼的基因序列进行比较验证，确认靶基因的有效敲除。
进一步地，步骤3)中，采用T7E1验证碱基突变的反应体系为：PCR产物8.5μl、缓冲液1μl，混合均匀后，在PCR仪中退火。其中，缓冲液的具体组成为：50mM NaCl、10mM Tris-HCl、10mM MgCl2、1mM DTT，pH 7.9(25℃)。
进一步地，步骤3)中，退火程序为：95℃5min，94℃2sec，-0.1℃/cycle,200times，75℃1sec，-0.1℃/cycle,600times，16℃2min，退火完成后，加入T7E1酶，37℃孵育30min。
进一步地，步骤3)中，采用T7核酸内切酶I验证碱基突变的反应体系中缓冲液的具体组成为：50mM NaCl、10mM Tris-HCl、10mM MgCl2、1mM DTT，pH 7.9。
如上所述，本发明的利用CRISPR-Cas9系统敲除鱼类MC4R基因的育种方法，具有以下有益效果：利用生物技术上最新基因敲除技术CRISPR-Cas9系统，对MC4R进行基因敲除，实现水产动物的快速生长育种。本发明为首次在鱼类动物中敲除MC4R基因，该育种方法适用于所有的经济鱼类，通过敲除鱼类的MC4R基因，从而实现鱼类的快速生长育种。与传统的育种方法相比，本专利方法具有准确性高、成本低、得到纯系时间短的特点。与转基因育种相比，由于基因敲除是使鱼本身的基因功能缺失，不会引入外来基因，因此，不存在转基因安全问题。
附图说明
图1显示为本实施例中斑马鱼受精卵注射后，T7E1验证打靶结果。
图2显示为本实施例中注射后MC4R基因测序比对图。
图3显示为本实施例中基因敲除鱼的筛选流程图。
图4显示为本实施例中F1代与野生型斑马鱼生长对比图(左为雄鱼，右为雌鱼；上排为F1代斑马鱼，下排为野生型斑马鱼)。
具体实施方式
以下通过特定的具体实例说明本发明的实施方式，本领域技术人员可由本说明书所阐明的内容轻易地了解本发明的其他优点与功效。本发明还可以通过另外不同的具体实施方式加以实施或应用，本说明书中的各项细节也可以基于不同观点与应用，在没有背离本发明的精神下进行各种修饰或改变。
本实施例以斑马鱼为例，当然，本发明也适用于其他经济鱼类，具体包括鲤鱼、鲫鱼、罗非鱼、草鱼、鲢鱼、鳙鱼、斑鳢、乌鳢等其他经济鱼类。
1、斑马鱼MC4R基因打靶位点的确定
首先，在基因数据库查找斑马鱼MC4R基因的基因序列，对比其氨基酸序列，找到其功能区域，将功能区域附近的基因序列输入到CRISPR-Cas9打靶位点设计网站上(http://zifit.partners.org/ZiFiT/)，根据网站给出的结果选择合适的打靶序列，尽量选择位于编码区前面的打靶序列。本发明选择的打靶位点序列如SEQ ID NO.1所示。当然，MC4R基因打靶位点的序列不限于SEQ ID NO.1所示的序列，其他能够敲除该MC4R基因的打靶序列也在本发明的保护范围之内。
2、gRNA的制备
本发明采用PCR的方法扩增出gRNA的转录模板。首先根据选定的打靶序列设计引物，上下游引物序列如SEQ ID NO.2和SEQ ID NO.3所示。以含有gRNA骨架的质粒pTarget为模板，质粒序列如SEQ ID NO.4所示。PCR反应程序为：98℃5min；98℃10s，58℃30s，72℃20s共35个循环，72℃5min。PCR产物经1％(质量体积分数，即所称取的琼脂糖粉的质量(g)比所加的缓冲液的体积(mL)即胶的浓度)琼脂糖凝胶电泳检测后，通过PCR产物纯化试剂盒回收产物，除去多余的盐离子、酶及引物等杂物。以纯化后的产物为模板进行体外转录，转录体系为20μl，其中纯化后的产物600ng，T7转录酶1μl，反应缓冲液2μl，NTP混合物1μl，加无菌水补足20μl。37℃孵育3h。转录产物经1％琼脂糖凝胶电泳检测后，通过RNA纯化试剂盒纯化回收，保存在-80℃冰箱中。反应缓冲液的具体组成为：50mM NaCl，10mM Tris-HCl，10mMMgCl2，1mM DTT(pH 7.9，温度25℃)。
3、体外显微注射与敲除验证
体外显微注射：挑选性成熟的斑马鱼，2条雌鱼1条雄鱼，放入产卵盒，在产卵盒中间插入透明隔板，将雌雄鱼分开。将产卵盒置于26-29℃恒温室中黑暗过夜饲养，光周期为白昼14h，黑暗10h。次日上午，拔开透明隔板，保持安静的环境，让鱼自行追逐产卵。产卵后，收取受精卵，通过显微注射仪进行单细胞期胚胎注射。注射体系为：cas9 RNA终浓度300ng/μl，gRNA终浓度30ng/μl，酚红0.5μl，无菌水补足10μl。注射部位为受精卵的动物极。此时即可对注射的受精卵进行孵化培育，当然，也可以先对注射的受精卵进行敲除验证之后，再对敲除成功的受精卵进行孵化培育，提高筛选的效率。
敲除验证：注射后的第二天，在对照组与注射组的斑马鱼胚胎中各随机选取5个受精卵，分别提取DNA，在MC4R基因打靶位点两侧选择500bp左右的序列设计引物，引物序列如SEQ ID NO.5和SEQ ID NO.6所示。以此引物对斑马鱼DNA进行扩增。T7核酸内切酶I(T7E1)具有识别并切割不完全配对DNA、异源双链DNA特性，因此用该酶进行碱基突变验证，反应体系为：PCR产物8.5μl，缓冲液(50mM NaCl，10mM Tris-HCl，10mM MgCl2，1mM DTT(pH 7.9，25℃))1μl，混合均匀后，在PCR仪中退火。退火程序为：95℃5min，94℃2sec，-0.1℃/cycle,200times，75℃1sec，-0.1℃/cycle,600times，16℃2min。退火完成后，加入0.5μl T7E1酶，37℃孵育30min。孵育完成后通过2％琼脂糖凝胶电泳检测是否打靶成功，结果如图1所示。随后亚克隆到测序载体中送样测序并同野生型基因序列进行比较验证，确认靶基因的有效敲除，如图2所示。对敲除成功的受精卵进行孵化培育。
受精卵孵化培育的具体过程为：受精卵在培养皿中孵育至第5天，每天早晚喂食蛋黄，10天后开始喂食丰年虾幼虫，期间注意经常换水。幼鱼培育至一个月后，移到斑马鱼循环水养殖系统，光暗周期为14h光照，10h黑暗，水温为28℃。每天早晚喂食丰年虫幼虫两次，分别为7:00和17:00。
4、MC4R基因敲除斑马鱼的筛选
两个月后，对每条鱼进行剪尾检测，按照上步所述方法检测每条鱼是否有突变。选出检测基因敲除阳性的斑马鱼，命名为P0代。P0代鱼性成熟后与野生型斑马鱼杂交，得到F1代杂合体，此时，每条F1代鱼只有一种碱基突变情况，挑选出其中碱基缺失或增加非3的倍数的F1代，相同突变情况的F1代自交，其后代F2代中即可筛选出基因完全突变的纯系鱼。本实施例中，通过测序与野生型比较，选出两种品系，品系1缺失8个碱基，品系2缺失4个碱基，均引起基因的移码突变。F1代相同品系的鱼进行自交，得到F2代，通过剪尾检测，挑选出其中MC4R敲除阳性的纯系鱼，如图3所示。
选择上述两种品系的原因主要在于：同一个打靶位点打靶成功后，无论是碱基缺失还是增加，起作用的是错义突变，即碱基的增加或缺失导致mRNA密码子发生改变，从而导致氨基酸发生改变，使基因失活，因为在翻译过程中，一个密码子包含3个碱基，因此错义突变有两种情况，分别是3n+1和3n+2，这两种情况分别导致不同的移码突变，最终翻译出的氨基酸也不同，但是相对于野生型来说，这两个品系的基因都发生了突变，基因功能已都失活或丧失。
步骤3中注射的初代鱼，因为基因编辑是在其发育过程中进行的，所以这些鱼本身有可能只有身体上某个部位的基因碱基发生了缺失或增加，而且会有很多种缺失情况，第一步与野生型杂交，是为了选择出性腺中的基因被敲除了的个体，只有这类个体，才能将其基因突变传递给下一代，而且使得后代的突变情况单一化，这样再通过F1代之间的杂交，就能获得整个基因组都被敲除了的纯系鱼。
将纯系的MC4R基因敲除的鱼与野生型斑马鱼杂交，即可得到100％的具有较大生长优势的杂合鱼。分别培育该杂合斑马鱼、野生型斑马鱼：在同一个养殖系统，相同养殖密度下(1g/L，每升水养殖1g重量的斑马鱼)，每日喂食相同数量的食物，6个月成年斑马鱼杂合雄鱼体长为3.3±0.2cm，体重为0.52±0.05g，野生型雄鱼体长为3.0±0.1cm，体重为0.45±0.04g，杂合雄鱼比野生型雄鱼的体长增加10％，体重增加了15.6％。杂合雌鱼体长为3.7±0.3cm，体重为0.74±0.05g，野生型雌鱼体长为3.2±0.1cm，体重为0.51±0.05g，杂合雌鱼比野生型雌鱼的体长增加15.6％，体重增加了45.1％。本实施例获得纯系鱼所需时间为：3*达到性成熟所需的时间，例如，斑马鱼3个月可性成熟，所以得到纯系斑马鱼的时间为3*3＝9个月，时间较短。
综上所述，本发明的利用CRISPR-Cas9系统敲除鱼类MC4R基因的育种方法，利用生物技术上最新基因敲除技术CRISPR-Cas9系统，对MC4R进行基因敲除，实现水产动物的快速生长育种。本发明为首次在鱼类动物中敲除MC4R基因，该育种方法适用于所有的经济鱼类，通过敲除鱼类的MC4R基因，从而实现鱼类的快速生长育种。与传统的育种方法相比，本专利方法具有准确性高、成本低、得到纯系时间短的特点。与转基因育种相比，由于基因敲除是使鱼本身的基因功能缺失，不会引入外来基因，因此，不存在转基因安全问题。
上述实施例仅例示性说明本发明的原理及其功效，而非用于限制本发明。任何熟悉此技术的人士皆可在不违背本发明的精神及范畴下，对上述实施例进行修饰或改变。因此，举凡所属技术领域中具有通常知识者在未脱离本发明所揭示的精神与技术思想下所完成的一切等效修饰或改变，仍应由本发明的权利要求所涵盖。
Claims (10)
1.一种利用CRISPR-Cas9系统敲除鱼类MC4R基因的育种方法，其特征在于，包括如下步骤：
1)MC4R基因打靶位点的确定：选择MC4R基因打靶位点序列；
2)gRNA的制备：设计含有MC4R基因打靶位点序列的上游引物以及与其匹配的下游引物，通过体外转录得到gRNA；
3)体外显微注射：鱼产卵后，收集受精卵，将gRNA和Cas9RNA注射进入鱼的受精卵，对注射的受精卵进行孵化培育；
4)MC4R基因敲除鱼的筛选：通过剪尾检测选出步骤3)中MC4R基因敲除阳性的鱼。
2.根据权利要求1所述的利用CRISPR-Cas9系统敲除鱼类MC4R基因的育种方法，其特征在于：步骤1)中，所述MC4R基因打靶位点序列如SEQ ID NO.1所示。
3.根据权利要求1所述的利用CRISPR-Cas9系统敲除鱼类MC4R基因促进鱼类快速生长的育种方法，其特征在于：步骤2)中，上游引物序列如SEQ ID NO.2所示，下游引物序列如SEQ ID NO.3所示。
4.根据权利要求1所述的利用CRISPR-Cas9系统敲除鱼类MC4R基因的育种方法，其特征在于：步骤2)中，以含有gRNA骨架的质粒为模板进行体外转录，质粒序列如SEQ ID NO.4所示。
5.根据权利要求1所述的利用CRISPR-Cas9系统敲除鱼类MC4R基因的育种方法，其特征在于：步骤2)中，通过PCR扩增gRNA的DNA序列，PCR反应程序为：98℃ 5min；98℃ 10s，58℃30s，72℃ 20s共35个循环，72℃ 5min。
6.根据权利要求1所述的利用CRISPR-Cas9系统敲除鱼类MC4R基因的育种方法，其特征在于：步骤3)中，注射体系的总体积10μl，包括cas9RNA 300ng/μl、gRNA 30ng/μl、酚红0.5μl、余量为无菌水。
7.根据权利要求1所述的利用CRISPR-Cas9系统敲除鱼类MC4R基因的育种方法，其特征在于：步骤4)中，通过剪尾检测选出步骤3)中MC4R基因基因敲除阳性的鱼后，命名为P0代，P0代与野生型鱼杂交，得到F1代杂合体，筛选出MC4R基因敲除阳性的鱼。
8.根据权利要求7所述的利用CRISPR-Cas9系统敲除鱼类MC4R基因的育种方法，其特征在于：步骤4)中，得到F1代杂合体后，通过测序与野生型鱼比较，选出两种品系，品系1缺失8个碱基，品系2缺失4个碱基，均引起基因的移码突变，选择F1代相同品系的鱼进行自交，得到F2代，通过剪尾检测，挑选得到MC4R基因敲除阳性的纯系鱼。
9.根据权利要求8所述的利用CRISPR-Cas9系统敲除鱼类MC4R基因的育种方法，其特征在于：步骤4)中，从F2代中通过剪尾检测挑选出MC4R基因敲除阳性的鱼后，使其与野生型鱼杂交，得到快速生长的杂合鱼。
10.根据权利要求1所述的利用CRISPR-Cas9系统敲除鱼类MC4R基因的育种方法，其特征在于：步骤3)中，还包括敲除验证，具体为：选取经过培育的受精卵并分别提取DNA，在MC4R基因打靶位点两侧设计引物，引物序列如SEQ ID NO.5和SEQ ID NO.6所示，对DNA进行扩增，利用T7核酸内切酶I(T7E1)确定打靶效率，随后亚克隆到测序载体中送样测序并同野生型鱼的基因序列进行比较验证，确认靶基因的有效敲除。
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9999671B2 (en) | 2013-09-06 | 2018-06-19 | President And Fellows Of Harvard College | Delivery of negatively charged proteins using cationic lipids |
| CN108192927A (zh) * | 2017-12-29 | 2018-06-22 | 上海海洋大学 | 一种瓯江彩鲤的基因编辑与过表达操作方法 |
| US10077453B2 (en) | 2014-07-30 | 2018-09-18 | President And Fellows Of Harvard College | CAS9 proteins including ligand-dependent inteins |
| CN108624619A (zh) * | 2018-02-28 | 2018-10-09 | 西南大学 | 罗非鱼pgr/mstn基因纯合敲除雌性完全不育品系及构建方法 |
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| US10465176B2 (en) | 2013-12-12 | 2019-11-05 | President And Fellows Of Harvard College | Cas variants for gene editing |
| US10508298B2 (en) | 2013-08-09 | 2019-12-17 | President And Fellows Of Harvard College | Methods for identifying a target site of a CAS9 nuclease |
| CN110689924A (zh) * | 2018-07-04 | 2020-01-14 | 赛业（广州）生物科技有限公司 | 一种基于多种敲除类型的敲除策略筛选方法及系统 |
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| US11447770B1 (en) | 2019-03-19 | 2022-09-20 | The Broad Institute, Inc. | Methods and compositions for prime editing nucleotide sequences |
| US20220338453A1 (en) * | 2021-04-16 | 2022-10-27 | Auburn University | Engineered catfish and uses thereof |
| US11542509B2 (en) | 2016-08-24 | 2023-01-03 | President And Fellows Of Harvard College | Incorporation of unnatural amino acids into proteins using base editing |
| US11542496B2 (en) | 2017-03-10 | 2023-01-03 | President And Fellows Of Harvard College | Cytosine to guanine base editor |
| US11560566B2 (en) | 2017-05-12 | 2023-01-24 | President And Fellows Of Harvard College | Aptazyme-embedded guide RNAs for use with CRISPR-Cas9 in genome editing and transcriptional activation |
| US11661590B2 (en) | 2016-08-09 | 2023-05-30 | President And Fellows Of Harvard College | Programmable CAS9-recombinase fusion proteins and uses thereof |
| US11732274B2 (en) | 2017-07-28 | 2023-08-22 | President And Fellows Of Harvard College | Methods and compositions for evolving base editors using phage-assisted continuous evolution (PACE) |
| US11795443B2 (en) | 2017-10-16 | 2023-10-24 | The Broad Institute, Inc. | Uses of adenosine base editors |
| US11898179B2 (en) | 2017-03-09 | 2024-02-13 | President And Fellows Of Harvard College | Suppression of pain by gene editing |
| US11912985B2 (en) | 2020-05-08 | 2024-02-27 | The Broad Institute, Inc. | Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence |
| CN117947033A (zh) * | 2024-02-22 | 2024-04-30 | 中国水产科学研究院长江水产研究所 | 一种3个gRNA联用的高效鲢bmp6基因敲除方法及应用 |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104232669A (zh) * | 2014-08-25 | 2014-12-24 | 中国水产科学研究院黑龙江水产研究所 | 一种采用基因敲除法构建基于鱼类CRISPR/Cas9系统的载体及其构建方法 |
| CN105594664A (zh) * | 2016-02-16 | 2016-05-25 | 湖南师范大学 | 一种stat1a基因缺失型斑马鱼 |
| CN105647969A (zh) * | 2016-02-16 | 2016-06-08 | 湖南师范大学 | 一种基因敲除选育stat1a基因缺失型斑马鱼的方法 |
-
2016
- 2016-07-29 CN CN201610618743.4A patent/CN106191114B/zh active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104232669A (zh) * | 2014-08-25 | 2014-12-24 | 中国水产科学研究院黑龙江水产研究所 | 一种采用基因敲除法构建基于鱼类CRISPR/Cas9系统的载体及其构建方法 |
| CN105594664A (zh) * | 2016-02-16 | 2016-05-25 | 湖南师范大学 | 一种stat1a基因缺失型斑马鱼 |
| CN105647969A (zh) * | 2016-02-16 | 2016-06-08 | 湖南师范大学 | 一种基因敲除选育stat1a基因缺失型斑马鱼的方法 |
Non-Patent Citations (3)
| Title |
|---|
| KARIM KHALIL等: "ALTERING MELANOCORTIN-4 RECEPTOR ACTIVITY IN CHANNEL CATFISH (Ictalurus punctatus) USING CRISPR/Cas9 SYSTEM", 《AQUACULTURE 2016》 * |
| SHAO-LIN XIE等: "A novel technique based on in vitro oocyte injection to improve CRISPR/Cas9 gene editing in zebrafish", 《SCIENTIFIC REPORTS》 * |
| 仇雪梅等: "虹鳟MC4R基因的PCR扩增及其应用", 《生物技术通报》 * |
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