CN105296537A - 一种基于睾丸内注射的基因定点编辑技术 - Google Patents
一种基于睾丸内注射的基因定点编辑技术 Download PDFInfo
- Publication number
- CN105296537A CN105296537A CN201510914035.0A CN201510914035A CN105296537A CN 105296537 A CN105296537 A CN 105296537A CN 201510914035 A CN201510914035 A CN 201510914035A CN 105296537 A CN105296537 A CN 105296537A
- Authority
- CN
- China
- Prior art keywords
- injection
- testis
- animal
- gene
- liquid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明公开了一种简便高效的基因定点编辑方法，涉及到转基因动物的制备、基因功能研究及生物医学领域。睾丸注射法是利用精子具有主动结合、转运、整合外源DNA的能力而发展的一种精子介导基因转移方法。本发明方法是优化了动物睾丸注射法，将利用成簇规律间隔短回文重复序列系统（CRISPR/Cas9）构建的载体通过睾丸多点注射或曲细精管显微注射，使之与精子染色体进行整合，实现基因的删除、替换、插入等目的。然后通过自然交配、人工授精等多种途径获得转基因动物后代。该方法充分利用了CRISPR/Cas9基因编辑系统的优势，结合现有的转基因动物技术，在大幅度提高基因修饰动物的获得效率的同时，避开了体外细胞操作的繁琐和对昂贵精密仪器设备的要求。
Description
技术领域
本发明涉及一种通过小鼠睾丸注射进行基因定点编辑的技术，属于动物遗传育种领域。
背景技术
转基因动物是指在基因组内稳定地整合以实验方法导入的外源基因，且外源基因能稳定遗传给后代的遗传工程动物。由于转基因动物在发育生物学和遗传学基础研究、培育动物新品种、大规模生产药用蛋白一直生产再植器官等方面有着广泛的应用前景，成为当前研究的热点。
精子具有主动结合、转运、整合外源DNA的能力，并在受精时导入卵母细胞，获得转基因动物。精子介导基因转移（Sperm-mediatedgenetransfer,SMGT）是目前获得转基因动物简单而高效的方法之一。睾丸注射法作为SMGT方法的一种，其是将外源目的基因直接注入动物睾丸内，和精原干细胞（SSCs）染色体进行整合，获得基因得到修饰或编辑的精子，然后通过自然交配、人工授精、胞浆内注射等多种途径使外源目的基因进入胚胎，获得转基因动物。此法避免了传统的精子介导基因转移方法的精子与外源DNA共孵育时精浆的抑制作用，无需复杂的体外处理过程，而且耗费小，操作简便，是近年来颇受关注的一种方法。
基因编辑技术是近年来发展起来的可以对基因组完成精确修饰的一种技术，可以完成基因定点InDel突变、敲入、多位点同时突变和小片段缺失等，可在基因组水平上进行精确的基因编辑。成簇规律间隔短回文重复序列系统（Clusteredregularlyinterspacedshortpalindromicrepeats，associatedRNAguidedendonucleaseCas，CRISPR/Cas9）是第三代人工核酸内切酶技术，与锌指核酸内切酶（ZincFingerNucleases，ZFNs）和类转录激活因子效应物核酸酶（TranscriptionActivator-likeEffectorNucleases，TALENs）一样可用于各种复杂基因组的编辑。目前该技术成功应用于人类细胞治疗遗传缺陷疾病，由于其突变效率高，制作简单及成本低的特点，被认为是一种具有广阔应用前景的基因定点改造分子工具。
目前基因编辑技术主要是把人为设计的质粒载体通过显微注射的方法注入胚胎，有繁琐的体外细胞操作过程，对昂贵精密仪器设备也有严格要求。本发明克服了细胞培养的技术难题和成本高昂的难题，充分利用了CRISPR/Cas9基因编辑系统的优势，结合现有的转基因动物技术，大幅度提高基因修饰动物的获得的效率。
发明内容
本发明方法重点是优化了小鼠睾丸注射法，将CRISPR/Cas9构建的载体通过睾丸内多点注射或曲细精管显微注射，使之与精子染色体进行整合，实现基因的删除、替换、插入等目的。主要包括以下步骤：
一、睾丸注射液的制备
1、载体的构建与检测根据目的基因序列设计用于特异性靶向目的基因的sgRNA，根据设计出的sgRNA序列合成一对序列互补的双链寡核苷酸序列；将双链寡核苷酸序列与线性化的CRISPR/Cas9质粒连接，转化提取带有特异性靶向目的基因的sgRNA的表达载体（根据需要，载体可以加入合适的表达标签）。
2、睾丸注射液
（1）注射转染A液：无菌、无血清的DMEM培养液与脂质体Lipofectamine?2000按照9：1的比例进行混合；
（2）注射转染B液：无菌、无血清的DMEM培养液与CRISPR/Cas9表达质粒混合，每mlDMEM培养液中加入45μg表达质粒；
（3）注射转染C液：1%台盼蓝溶液；
（4）将等体积的注射转染A液和B液混合，室温下作用30分钟后，继续加入注射转染C液，体积为注射转染A液的1/10，即获得睾丸注射液。
二、动物的选择和管理
成年雄性动物，睾丸发育正常。在睾丸注射前保定动物，全身麻醉。
三、睾丸内注射
用手术刀于阴囊开一小口，将单侧睾丸通过开口裸露于体外，用微量注射器吸取相应剂量的试剂，穿过睾丸白膜刺入睾丸实质5-6mm，将睾丸注射液缓缓注射入睾丸。
可睾丸内多点注射，也可进行曲细精管显微注射。对另一侧睾丸进行相同操作。
第一次注射后，隔24小时再重复操作一次。
四、注射效果的检测与动物的应用
第二次注射5天后，采集小鼠精液，提取精子DNA，设计引物，进行PCR和SouthernBlotting实验，检测注射效果和外源基因编辑效果。阳性小鼠通过自然交配、人工授精、胞浆内注射等多种途径使外源目的基因进入胚胎，大量、高效的获得转基因小鼠。
具体实施方式
在本发明中所使用的术语，除非有另外的说明，一般具有本领域普通技术人员通常理解的含义。
下面结合具体实施例，并参照数据，进一步详细地阐述本发明。应理解，实施例仅用于说明本发明而不应当也不会用于限制本发明的范围。此外应当理解，在阅读了本发明讲授的内容之后，本领域技术人员可以对本发明做各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。以下实施例中未详细描述的各种过程和方法是本领域中公知的常规方法。
1、载体的构建与检测
（1）基因靶点选择：利用软件设计基因的sgRNA序列，由20个碱基构成，sgRNA前面为转录起始信号，后面为PAM序列（NGG）。
（2）寡核苷酸链sgRNA退火，形成核苷酸双链。
（3）酶切CRISPR/Cas9表达质粒，使其线性化。
（4）CRISPR/Cas9-sgRNA表达载体与核苷酸双链在连接酶的作用下进行连接。
（5）连接后产物转化至感受态细胞，扩大培养后挑选阳性克隆，测序鉴定及扩大培养抽提表达质粒。
2、睾丸注射液的制备
（1）注射转染A液：无菌、无血清的DMEM培养液与脂质体Lipofectamine?2000按照9：1的比例进行混合；
（2）注射转染B液：无菌、无血清的DMEM培养液与CRISPR/Cas9表达质粒混合，每mlDMEM培养液中加入45μg表达质粒；
（3）注射转染C液：1%台盼蓝溶液；
（4）将等体积的注射转染A液和B液混合，室温下作用30分钟后，继续加入注射转染C液，体积为注射转染A液的1/10，即获得睾丸注射液。
3、动物的选择和管理
成年雄性大鼠，睾丸发育正常。在睾丸注射前保定动物，全身麻醉。
4、睾丸内注射
用手术刀于阴囊开一小口，将单侧睾丸通过开口裸露于体外，用微量注射器吸取相应剂量的试剂，穿过睾丸白膜刺入睾丸实质5-6mm，将睾丸注射液缓缓注射入睾丸。
可睾丸内多点注射，也可进行曲细精管显微注射。对另一侧睾丸进行相同操作。
第一次注射后，隔24小时再重复操作一次。
5、注射效果的检测
第二次注射5天后，采集小鼠精液，提取精子DNA，设计引物，进行PCR和SouthernBlotting实验，检测注射效果和外源基因编辑效果。
6、睾丸内注射雄性小鼠的应用
第二次注射5天后，经检测精子为阳性小鼠通过自然交配、人工授精、胞浆内注射等多种途径使外源目的基因进入胚胎，大量、高效的获得转基因小鼠。
Claims (3)
1.一种基于小鼠睾丸内注射的基因定点编辑技术，其特征包括如下步骤：
一、睾丸注射液的制备
1、载体的构建与检测根据目的基因序列设计用于特异性靶向目的基因的sgRNA，根据设计出的sgRNA序列合成一对序列互补的双链寡核苷酸序列；将双链寡核苷酸序列与线性化的CRISPR/Cas9质粒连接，转化提取带有特异性靶向目的基因的sgRNA的表达载体（根据需要，载体可以加入合适的表达标签）；
2、睾丸注射液
（1）注射转染A液：无菌、无血清的DMEM培养液与脂质体Lipofectamine?2000按照9：1的比例进行混合；
（2）注射转染B液：无菌、无血清的DMEM培养液与CRISPR/Cas9表达质粒混合，每mlDMEM培养液中加入45μg表达质粒；
（3）注射转染C液：1%台盼蓝溶液；
（4）将等体积的注射转染A液和B液混合，室温下作用30分钟后，继续加入注射转染C液，体积为注射转染A液的1/10，即获得睾丸注射液；
二、动物的选择和管理
成年雄性动物，睾丸发育正常；
在睾丸注射前保定动物，全身麻醉；
三、睾丸内注射
用手术刀于阴囊开一小口，将单侧睾丸通过开口裸露于体外，用微量注射器吸取相应剂量的试剂，穿过睾丸白膜刺入睾丸实质5-6mm，将睾丸注射液缓缓注射入睾丸；
可睾丸内多点注射，也可进行曲细精管显微注射；
对另一侧睾丸进行相同操作；
第一次注射后，隔24小时再重复操作一次；
四、注射效果的检测与动物的应用
第二次注射5天后，采集小鼠精液，提取精子DNA，设计引物，进行PCR和SouthernBlotting实验，检测注射效果和外源基因编辑效果；
阳性小鼠通过自然交配、人工授精、胞浆内注射等多种途径使外源目的基因进入胚胎，大量、高效的获得转基因小鼠。
2.根据权利要求1所述的制备转基因动物的技术，包括睾丸内注射与基因定点编辑技术相结合。
3.根据权利要求1所述的制备转基因动物的技术，优化的小鼠睾丸内多点注射方法。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510914035.0A CN105296537A (zh) | 2015-12-12 | 2015-12-12 | 一种基于睾丸内注射的基因定点编辑技术 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510914035.0A CN105296537A (zh) | 2015-12-12 | 2015-12-12 | 一种基于睾丸内注射的基因定点编辑技术 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN105296537A true CN105296537A (zh) | 2016-02-03 |
Family
ID=55194362
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510914035.0A Pending CN105296537A (zh) | 2015-12-12 | 2015-12-12 | 一种基于睾丸内注射的基因定点编辑技术 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105296537A (zh) |
Cited By (26)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9526784B2 (en) | 2013-09-06 | 2016-12-27 | President And Fellows Of Harvard College | Delivery system for functional nucleases |
| US10077453B2 (en) | 2014-07-30 | 2018-09-18 | President And Fellows Of Harvard College | CAS9 proteins including ligand-dependent inteins |
| US10113163B2 (en) | 2016-08-03 | 2018-10-30 | President And Fellows Of Harvard College | Adenosine nucleobase editors and uses thereof |
| US10167457B2 (en) | 2015-10-23 | 2019-01-01 | President And Fellows Of Harvard College | Nucleobase editors and uses thereof |
| US10227581B2 (en) | 2013-08-22 | 2019-03-12 | President And Fellows Of Harvard College | Engineered transcription activator-like effector (TALE) domains and uses thereof |
| US10323236B2 (en) | 2011-07-22 | 2019-06-18 | President And Fellows Of Harvard College | Evaluation and improvement of nuclease cleavage specificity |
| 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 |
| US10597679B2 (en) | 2013-09-06 | 2020-03-24 | President And Fellows Of Harvard College | Switchable Cas9 nucleases and uses thereof |
| CN111110865A (zh) * | 2019-11-27 | 2020-05-08 | 哈尔滨医科大学 | 一种腺相关病毒双重载体基因治疗系统及其在治疗黏多糖贮积症ⅱ型中的应用 |
| CN111269936A (zh) * | 2017-12-29 | 2020-06-12 | 浙江省农业科学院 | 质粒载体组、dna组和外源基因导入的基因编辑方法 |
| US10745677B2 (en) | 2016-12-23 | 2020-08-18 | President And Fellows Of Harvard College | Editing of CCR5 receptor gene to protect against HIV infection |
| US10858639B2 (en) | 2013-09-06 | 2020-12-08 | President And Fellows Of Harvard College | CAS9 variants and uses thereof |
| US11268082B2 (en) | 2017-03-23 | 2022-03-08 | President And Fellows Of Harvard College | Nucleobase editors comprising nucleic acid programmable DNA binding proteins |
| US11306324B2 (en) | 2016-10-14 | 2022-04-19 | President And Fellows Of Harvard College | AAV delivery of nucleobase editors |
| US11319532B2 (en) | 2017-08-30 | 2022-05-03 | President And Fellows Of Harvard College | High efficiency base editors comprising Gam |
| US11447770B1 (en) | 2019-03-19 | 2022-09-20 | The Broad Institute, Inc. | Methods and compositions for prime editing nucleotide sequences |
| US11542496B2 (en) | 2017-03-10 | 2023-01-03 | President And Fellows Of Harvard College | Cytosine to guanine base editor |
| US11542509B2 (en) | 2016-08-24 | 2023-01-03 | President And Fellows Of Harvard College | Incorporation of unnatural amino acids into proteins using base editing |
| 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 |
| US12084663B2 (en) | 2022-11-14 | 2024-09-10 | President And Fellows Of Harvard College | Incorporation of unnatural amino acids into proteins using base editing |
-
2015
- 2015-12-12 CN CN201510914035.0A patent/CN105296537A/zh active Pending
Cited By (50)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US12006520B2 (en) | 2011-07-22 | 2024-06-11 | President And Fellows Of Harvard College | Evaluation and improvement of nuclease cleavage specificity |
| US10323236B2 (en) | 2011-07-22 | 2019-06-18 | President And Fellows Of Harvard College | Evaluation and improvement of nuclease cleavage specificity |
| US11920181B2 (en) | 2013-08-09 | 2024-03-05 | President And Fellows Of Harvard College | Nuclease profiling system |
| US10954548B2 (en) | 2013-08-09 | 2021-03-23 | President And Fellows Of Harvard College | Nuclease profiling system |
| US10508298B2 (en) | 2013-08-09 | 2019-12-17 | President And Fellows Of Harvard College | Methods for identifying a target site of a CAS9 nuclease |
| US10227581B2 (en) | 2013-08-22 | 2019-03-12 | President And Fellows Of Harvard College | Engineered transcription activator-like effector (TALE) domains and uses thereof |
| US11046948B2 (en) | 2013-08-22 | 2021-06-29 | President And Fellows Of Harvard College | Engineered transcription activator-like effector (TALE) domains and uses thereof |
| US10858639B2 (en) | 2013-09-06 | 2020-12-08 | President And Fellows Of Harvard College | CAS9 variants and uses thereof |
| US11299755B2 (en) | 2013-09-06 | 2022-04-12 | President And Fellows Of Harvard College | Switchable CAS9 nucleases and uses thereof |
| US9526784B2 (en) | 2013-09-06 | 2016-12-27 | President And Fellows Of Harvard College | Delivery system for functional nucleases |
| US10597679B2 (en) | 2013-09-06 | 2020-03-24 | President And Fellows Of Harvard College | Switchable Cas9 nucleases and uses thereof |
| US9999671B2 (en) | 2013-09-06 | 2018-06-19 | President And Fellows Of Harvard College | Delivery of negatively charged proteins using cationic lipids |
| US9737604B2 (en) | 2013-09-06 | 2017-08-22 | President And Fellows Of Harvard College | Use of cationic lipids to deliver CAS9 |
| US10682410B2 (en) | 2013-09-06 | 2020-06-16 | President And Fellows Of Harvard College | Delivery system for functional nucleases |
| US10912833B2 (en) | 2013-09-06 | 2021-02-09 | President And Fellows Of Harvard College | Delivery of negatively charged proteins using cationic lipids |
| US11053481B2 (en) | 2013-12-12 | 2021-07-06 | President And Fellows Of Harvard College | Fusions of Cas9 domains and nucleic acid-editing domains |
| US10465176B2 (en) | 2013-12-12 | 2019-11-05 | President And Fellows Of Harvard College | Cas variants for gene editing |
| US11124782B2 (en) | 2013-12-12 | 2021-09-21 | President And Fellows Of Harvard College | Cas variants for gene editing |
| US10704062B2 (en) | 2014-07-30 | 2020-07-07 | President And Fellows Of Harvard College | CAS9 proteins including ligand-dependent inteins |
| US11578343B2 (en) | 2014-07-30 | 2023-02-14 | President And Fellows Of Harvard College | CAS9 proteins including ligand-dependent inteins |
| US10077453B2 (en) | 2014-07-30 | 2018-09-18 | President And Fellows Of Harvard College | CAS9 proteins including ligand-dependent inteins |
| US12043852B2 (en) | 2015-10-23 | 2024-07-23 | President And Fellows Of Harvard College | Evolved Cas9 proteins for gene editing |
| US11214780B2 (en) | 2015-10-23 | 2022-01-04 | President And Fellows Of Harvard College | Nucleobase editors and uses thereof |
| US10167457B2 (en) | 2015-10-23 | 2019-01-01 | President And Fellows Of Harvard College | Nucleobase editors and uses thereof |
| US11999947B2 (en) | 2016-08-03 | 2024-06-04 | President And Fellows Of Harvard College | Adenosine nucleobase editors and uses thereof |
| US10947530B2 (en) | 2016-08-03 | 2021-03-16 | President And Fellows Of Harvard College | Adenosine nucleobase editors and uses thereof |
| US10113163B2 (en) | 2016-08-03 | 2018-10-30 | President And Fellows Of Harvard College | Adenosine nucleobase editors and uses thereof |
| US11702651B2 (en) | 2016-08-03 | 2023-07-18 | President And Fellows Of Harvard College | Adenosine nucleobase editors and uses thereof |
| US11661590B2 (en) | 2016-08-09 | 2023-05-30 | President And Fellows Of Harvard College | Programmable CAS9-recombinase fusion proteins 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 |
| US11306324B2 (en) | 2016-10-14 | 2022-04-19 | President And Fellows Of Harvard College | AAV delivery of nucleobase editors |
| US11820969B2 (en) | 2016-12-23 | 2023-11-21 | President And Fellows Of Harvard College | Editing of CCR2 receptor gene to protect against HIV infection |
| US10745677B2 (en) | 2016-12-23 | 2020-08-18 | President And Fellows Of Harvard College | Editing of CCR5 receptor gene to protect against HIV infection |
| US11898179B2 (en) | 2017-03-09 | 2024-02-13 | President And Fellows Of Harvard College | Suppression of pain by gene editing |
| US11542496B2 (en) | 2017-03-10 | 2023-01-03 | President And Fellows Of Harvard College | Cytosine to guanine base editor |
| US11268082B2 (en) | 2017-03-23 | 2022-03-08 | President And Fellows Of Harvard College | Nucleobase editors comprising nucleic acid programmable DNA binding proteins |
| 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 |
| 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) |
| US11932884B2 (en) | 2017-08-30 | 2024-03-19 | President And Fellows Of Harvard College | High efficiency base editors comprising Gam |
| US11319532B2 (en) | 2017-08-30 | 2022-05-03 | President And Fellows Of Harvard College | High efficiency base editors comprising Gam |
| US11795443B2 (en) | 2017-10-16 | 2023-10-24 | The Broad Institute, Inc. | Uses of adenosine base editors |
| CN111269936B (zh) * | 2017-12-29 | 2022-07-12 | 浙江省农业科学院 | 质粒载体组、dna组和外源基因导入的基因编辑方法 |
| CN111269936A (zh) * | 2017-12-29 | 2020-06-12 | 浙江省农业科学院 | 质粒载体组、dna组和外源基因导入的基因编辑方法 |
| US11447770B1 (en) | 2019-03-19 | 2022-09-20 | The Broad Institute, Inc. | Methods and compositions for prime editing nucleotide sequences |
| US11795452B2 (en) | 2019-03-19 | 2023-10-24 | The Broad Institute, Inc. | Methods and compositions for prime editing nucleotide sequences |
| US11643652B2 (en) | 2019-03-19 | 2023-05-09 | The Broad Institute, Inc. | Methods and compositions for prime editing nucleotide sequences |
| CN111110865A (zh) * | 2019-11-27 | 2020-05-08 | 哈尔滨医科大学 | 一种腺相关病毒双重载体基因治疗系统及其在治疗黏多糖贮积症ⅱ型中的应用 |
| 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 |
| US12031126B2 (en) | 2020-05-08 | 2024-07-09 | The Broad Institute, Inc. | Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence |
| US12084663B2 (en) | 2022-11-14 | 2024-09-10 | President And Fellows Of Harvard College | Incorporation of unnatural amino acids into proteins using base editing |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105296537A (zh) | 一种基于睾丸内注射的基因定点编辑技术 | |
| CN105039339B (zh) | 一种以RNA介导的特异性敲除绵羊FecB基因的方法及其专用sgRNA | |
| Muggleton-Harris et al. | Cytoplasmic control of preimplantation development in vitro in the mouse | |
| Heo et al. | CRISPR/Cas9 nuclease-mediated gene knock-in in bovine-induced pluripotent cells | |
| CN104404036B (zh) | 基于CRISPR/Cas9技术的条件性基因敲除方法 | |
| CN104531705A (zh) | 利用CRISPR-Cas9系统敲除动物myostatin基因的方法 | |
| CN107446954A (zh) | 一种sd大鼠t细胞缺失遗传模型的制备方法 | |
| CN106244557A (zh) | 定点突变ApoE基因与LDLR基因的方法 | |
| Nishio et al. | Effects of voltage strength during electroporation on the development and quality of in vitro‐produced porcine embryos | |
| CN110484538A (zh) | 识别猪ROSA26基因的sgRNA及其编码DNA、基因编辑方法、试剂盒和应用 | |
| Zuo et al. | GOTI, a method to identify genome-wide off-target effects of genome editing in mouse embryos | |
| CN113881708B (zh) | 对动物受精卵进行电转染基因编辑的方法及其应用 | |
| CN102296090B (zh) | 一种基于体细胞核移植构建的牛体细胞克隆胚的处理方法 | |
| CN109234278A (zh) | 构建ApoC2基因敲除仓鼠模型的试剂盒和方法 | |
| CN107699571A (zh) | 一种猪生长抑素基因编辑位点及其应用 | |
| Namba et al. | GONAD: A new method for germline genome editing in mice and rats | |
| Kalds et al. | Generation of double-muscled sheep and goats by CRISPR/Cas9-mediated knockout of the myostatin gene | |
| CN109680011A (zh) | 一种利用CRISPR/Cas9系统敲除绵羊BMPR1B基因的方法 | |
| CN106282230A (zh) | 定点突变ldlr基因的方法 | |
| CN111088290B (zh) | 一种杜鹃素在基因编辑中的应用 | |
| Nagy et al. | Artificial gametes | |
| Trounson | A rapidly evolving revolution in stem cell biology and medicine | |
| CN106244556A (zh) | 定点突变ApoE基因的方法 | |
| CN105132426B (zh) | 一种以RNA介导的特异性敲除FGF5基因获得基因编辑绵羊的方法及其专用sgRNA | |
| CN105400808A (zh) | 利用生殖特异性启动子表达cre的重组酶载体 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20160203 |