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.
Methods for acutely degrading stable and abundant proteins are greatly lacking and are required for definitive studies of oocytes and eggs. The proposed project will generate and characterize a transgenic mouse line that constitutively expresses the protein, TRIM21, in oocytes and eggs, which permits rapid and complete protein degradation when expressed in conjunction with a specific antibody. This research will advance knowledge of crucial physiological processes in oocytes and eggs, which will establish a basis for future clinical developments, especially in treatment of infertility.
