Generation and characterization of a TRIM21 overexpressing mouse line

TRIM21 过表达小鼠系的生成和表征

• 批准号: 9807181
• 负责人: LISA M MEHLMANN
• 金额: $ 8.2万
• 依托单位: UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-15 至 2021-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9807181
• 关键词: Acute; Antibodies; Antisense Oligonucleotides; Bacteria; Binding; Binding Proteins; Biology; CRISPR/Cas technology; Cells; Communities; Cultured Cells; Embryo; Fc Receptor; Fertilization; Financial compensation; Future; Generations; Genes; In Vitro; Injections; Knock-in Mouse; Knock-out; Knockout Mice; Knowledge; Messenger RNA; Metaphase; Methods; Mus; Neurons; Oocytes; Pathway interactions; Physiological Processes; Process; Proteins; RNA; RNA Interference; Research; SNAP23 gene; Site; Techniques; Testing; Time; Tissues; Transgenic Mice; Translations; Work; base; cell type; clinical development; design; egg; infertility treatment; interest; multicatalytic endopeptidase complex; oocyte maturation; overexpression; promoter; protein degradation; protein function; recruit; tool; ubiquitin-protein ligase; vector

SUMMARY/ABSTRACT Specific depletion of proteins is critical for the study of oocyte/egg/embryo biology, but commonly used methods for protein depletion often have limitations due to protein stability and/or compensatory mechanisms. The objective of this project is to generate and characterize a mouse line that contains an oocyte-specific, constitutively-expressing TRIM21 gene. TRIM21 is an intracellular antibody receptor and E3 ubiquitin ligase that binds to an antibody within the cell. It then recruits the proteasome to the antibody-bound protein, where the protein of interest is degraded through the proteasome pathway. This process occurs very rapidly and thus is a specific method for acutely depleting proteins that are abundant and stable. Although TRIM21 is expressed to varying degrees in diverse tissues, it needs to be overexpressed in oocytes and eggs. This is accomplished by microinjecting RNA encoding TRIM21, followed by a subsequent injection of an antibody specific for the protein to be degraded. It would be highly advantageous to have a mouse that constitutively expresses TRIM21 protein in oocytes and eggs, such that isolated immature oocytes or mature eggs would only need to be injected once, with antibody, rather than twice, with RNA and then antibody. The mouse we propose to make will express TRIM21 specifically in oocytes; however, the founder mice could be bred to mice expressing other tissue-specific Cres, so this mouse will benefit not only other labs studying oocytes and eggs, but the broader scientific community as well. Aim 1 will generate this mouse using CRISPR/Cas9. The targeting vector will contain a CAG promoter followed by a LoxP-STOP-LoxP sequence and then the TRIM21 sequence, tagged with HA. This construct will be guided into the Rosa26 locus, a safe harbor site that has been widely used for overexpressing genes. Founder mice will be bred with Zp3-Cre mice to place the CAG promoter directly in front of the TRIM21 to obtain constitutive overexpression of TRIM21, containing an HA tag, in oocytes and eggs. Aim 2 will characterize the TRIM21 knock-in mice by performing tests to confirm that oocytes and eggs reliably express functional TRIM21. This mouse will be useful for studies of relatively short- lived cells such as oocytes and eggs and will be invaluable for studies of oocyte maturation and fertilization. It can also be used to produce other tissue-specific knock-ins so will be a generally useful tool for other labs as well.

摘要/摘要 蛋白质的特异性耗竭对于研究卵母细胞/鸡蛋/胚胎生物学至关重要，但通常 蛋白质消耗的使用方法通常由于蛋白质稳定性和/或代偿性而具有局限性 机制。该项目的目的是生成和表征包含的鼠标线 卵母细胞特异性表达的TRIM21基因。 TRIM21是一种细胞内抗体 受体和E3泛素连接酶与细胞内的抗体结合。然后招募 抗体结合蛋白的蛋白酶体，其中感兴趣的蛋白质通过 蛋白酶体途径。这个过程非常迅速地发生，因此是一种急性的特定方法 耗尽丰富且稳定的蛋白质。尽管TRIM21在不同程度上表示 多种组织，需要在卵母细胞和卵中过表达。这是由 微注射编码TRIM21的RNA，然后随后注入特定于特异的抗体 要降解的蛋白质。拥有组成性的鼠标将非常有利 表达卵母细胞和卵中的TRIM21蛋白 只需要用抗体注射一次，而不是用RNA和抗体注射两次。 我们提出的小鼠将在卵母细胞中专门表达TRIM21。但是，创始人 可以将小鼠饲养到表达其他组织特异性CRE的小鼠中，因此该鼠标不仅会受益 其他研究卵母细胞和鸡蛋的实验室，但也是更广泛的科学界。目标1意志 使用CRISPR/CAS9生成此鼠标。靶向矢量将包含一个CAG启动子 通过loxp-stop-loxp序列，然后是用ha标记的trim21序列。这个结构 将引导到Rosa26基因座，这是一个安全港口，已广泛用于过表达 基因。创始人小鼠将用ZP3-CRE小鼠育成，将CAG启动子直接放在 trim21获得oocytes中包含ha标签的trim21的本构过表达 鸡蛋。 AIM 2将通过执行测试来确认卵母细胞来表征TRIM21敲入小鼠 卵可靠地表达功能性TRIM21。该小鼠将有助于研究相对较短的研究 生存的细胞，例如卵母细胞和卵，对于卵母细胞的成熟和 受精。它也可以用于生产其他组织特异性的敲击ins，因此通常是 其他实验室也有用的工具。