Gene Targeting Using Modular Zinc Finger Nulceases

使用模块化锌指核酸酶进行基因靶向

• 批准号: 8234884
• 负责人: HOWARD I SIROTKIN
• 金额: $ 7.85万
• 依托单位: STATE UNIVERSITY NEW YORK STONY BROOK
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-04-01 至 2015-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8234884
• 关键词: Alleles; Animal Model; Binding; Binding Sites; Biological Models; Biological Process; Biomedical Research; Cell Culture System; Chimeric Proteins; Computer Assisted; DNA Binding Domain; DNA Double Strand Break; Effectiveness; Embryo; Engineering; Exonuclease; Gene Targeting; Genes; Genetic; Germ-Line Mutation; Goals; Human Genome Project; Laboratories; Lesion; Mammalian Cell; Mammals; Mediating; Methods; Molecular; Monitor; Mutate; Mutation; Plants; Procedures; Protocols documentation; Reporting; Research Personnel; System; Technology; Testing; Zebrafish; Zinc Fingers; biological systems; cost; design; gene function; interest; loss of function; novel; nuclease; research study; tool

DESCRIPTION (Provided by Applicant): One fundamental method to investigate biological processes is by loss-of-function studies. The goal of these efforts is to determine the activity of a gene by disrupting that gene and monitoring the effects on a biological system. Recently, gene targeting technology has been developed using zinc finger nucleases (ZFNs) as a method to mutate specific genes. The approach involves engineering zinc finger DNA binding domains to recognize specific target sequences. These domains can then be tethered to the FOK1 exonuclease domains to generate a fusion protein that can induce double stranded DNA breaks near the zinc finger binding sites. ZFNs have been successfully used to disrupted genes in a variety of systems ranging from plants to mammals. Thus far, only a relatively small number of laboratories have reported successful efforts to employ ZFNs. The use of ZFNs has been curtailed by the difficulty in generating zinc finger domains that bind to specific target sequences, and by the high cost of commercially available ZFNs. In preliminary studies, computer aided "modular" design has been an effective means to engineer functional ZFNs. The experiments in this application evaluate the effectiveness of a strategy to use modular ZFN design to generate germ-line mutations using zebrafish as a model system. Zebrafish is a powerful system for genetic, cellular, and molecular studies, and the ability to routinely perform gene targeting studies would be a major advance. The methods in this application are broadly applicable to biomedical research in both cell culture systems and model organisms. RELEVANCE: The human genome project has identified thousands of novel genes, but the activity of many of these genes remains unknown. The ability to disrupt specific genes is a powerful means to determine the function of these genes. The experiments in this application evaluate the effectiveness of one means to accomplish this goal using model organisms and cell culture systems.

描述（由申请人提供）：研究生物学过程的一种基本方法是通过功能丧失研究。 这些努力的目的是通过破坏该基因并监测对生物系统的影响来确定基因的活性。 最近，使用锌指核酸酶（ZFN）作为突变特定基因的方法开发了基因靶向技术。 该方法涉及工程锌指DNA结合域以识别特定的靶序列。 然后，这些结构域可以将其束缚在FOK1核酸外切酶结构域中，以生成一种融合蛋白，该融合蛋白可以在锌指结合位点附近诱导双链DNA断裂。 ZFN已成功地用于破坏从植物到哺乳动物的各种系统中的基因。 到目前为止，只有相对少数实验室报告了成功地雇用ZFN的努力。 难以生成与特定目标序列结合以及市售ZFN的高成本的锌指域的困难，从而减少了ZFN的使用。 在初步研究中，计算机辅助的“模块化”设计是设计功能性ZFN的有效手段。 本应用程序中的实验评估了使用模块化ZFN设计策略的有效性，以使用斑马鱼作为模型系统生成种系突变。 斑马鱼是一种用于遗传，细胞和分子研究的强大系统，定期进行基因靶向研究的能力将是一个重大进展。 本应用中的方法广泛适用于细胞培养系统和模型生物的生物医学研究。 相关性：人类基因组项目已经鉴定出数千种新基因，但是许多这些基因的活性仍然未知。 破坏特定基因的能力是确定这些基因功能的强大手段。 本应用程序中的实验评估了一种手段使用模型生物和细胞培养系统实现此目标的有效性。

项目成果

CoRest1 regulates neurogenesis in a stage-dependent manner.

CoRest1 以阶段依赖性方式调节神经发生。

• DOI: 10.1002/dvdy.86
• 发表时间: 2019
• 期刊: Developmental dynamics : an official publication of the American Association of Anatomists
• 作者: Monestime,CamilliaM;Taibi,Andrew;Gates,KeithP;Jiang,Karen;Sirotkin,HowardI
• 通讯作者: Sirotkin,HowardI

Zebrafish sin3b mutants are viable but have size, skeletal, and locomotor defects.

• DOI: 10.1002/dvdy.24581
• 发表时间: 2017-11
• 期刊: Developmental dynamics : an official publication of the American Association of Anatomists
• 作者: Moravec CE;Yousef H;Kinney BA;Salerno-Eichenholz R;Monestime CM;Martin BL;Sirotkin HI
• 通讯作者: Sirotkin HI

Rest mutant zebrafish swim erratically and display atypical spatial preferences.

其余的突变斑马鱼游动不规律，并表现出非典型的空间偏好。

• DOI: 10.1016/j.bbr.2015.02.026
• 发表时间: 2015-05-01
• 期刊: BEHAVIOURAL BRAIN RESEARCH
• 影响因子: 2.7
• 作者: Moravec, Cara E.;Li, Edward;Maaswinkel, Hans;Kritzer, Mary F.;Weng, Wei;Sirotkin, Howard I.
• 通讯作者: Sirotkin, Howard I.