Generation and characterization of a TRIM21 overexpressing mouse line

总结/摘要 蛋白质的特异性去除对于卵母细胞/卵子/胚胎生物学的研究至关重要，但通常 用于蛋白质消耗的方法通常由于蛋白质稳定性和/或补偿性而具有局限性。 机制等这个项目的目标是生成和表征鼠标线，包含 卵母细胞特异性的组成型表达TRIM 21基因。TRIM 21是一种细胞内抗体， 受体和结合细胞内抗体的E3泛素连接酶。然后它调动 蛋白酶体与抗体结合的蛋白质结合，其中感兴趣的蛋白质通过蛋白酶体降解。 蛋白酶体途径该过程发生得非常迅速，因此是急性炎症的特异性方法。 消耗大量稳定的蛋白质。虽然TRIM 21在不同的组织中表达不同程度， 不同的组织，它需要在卵母细胞和卵子中过度表达。这是通过 显微注射编码TRIM 21的RNA，随后注射对TRIM 21特异的抗体。 要降解的蛋白质。如果有一只老鼠， 在卵母细胞和卵中表达TRIM 21蛋白，使得分离的未成熟卵母细胞或成熟卵 只需要注射一次抗体，而不是两次，先注射RNA，再注射抗体。 我们计划制造的小鼠将在卵母细胞中特异性表达TRIM 21;然而， 可以将小鼠培育成表达其他组织特异性克雷斯的小鼠，因此这种小鼠不仅将受益于 其他研究卵母细胞和卵子的实验室，以及更广泛的科学界。目标1将 使用CRISPR/Cas9产生这只小鼠。靶向载体将含有CAG启动子， 通过LoxP-STOP-LoxP序列，然后是用HA标记的TRIM 21序列。该构建体 将被引导到Rosa 26位点，这是一个被广泛用于过度表达的安全港位点。 基因.将建立者小鼠与Zp 3-Cre小鼠一起繁殖，以将CAG启动子直接放置在Zp 3-Cre小鼠的前面。 TRIM 21，以获得含有HA标签的TRIM 21在卵母细胞中的组成型过表达， 鸡蛋目的2将通过进行测试来表征TRIM 21敲入小鼠，以确认卵母细胞 并且卵可靠地表达功能性TRIM 21。这只老鼠将有助于研究相对较短的- 活细胞，如卵母细胞和卵子，将是非常宝贵的卵母细胞成熟的研究， 受精它还可以用于产生其他组织特异性敲入，因此将是一种通用的方法。 这对其他实验室来说也是一个很好的工具。

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.