Trim-Away

Trim-Away is a highly effective method for rapidly and directly degrading the target protein without requiring modification of the genome³³. The remarkable speed of Trim-Away means that phenotypes can be observed immediately following the degradation of the protein of interest at any stage of a particular biological process. Since the duration for mouse oocyte finishes the transition from prophase to anaphase takes only a few hours, the best way for acute protein degradation is Trim-away, which avoids the compensation effect after long-term gene editing in CKO mice. However, effective of antibodies and off-targets are the obvious limitation for Trim-Away used in oocyte³³. Thus, an alternative antibody is needed to test the availability and off-targets of Trim-Away.

Purified Trim21 mRNA was mixed with an indicated antibody, stored at −80 °C and used within one month. The final concentration of Trim21 mRNA was 1 mg/ml, and the final concentrations of anti-CENP-F (Abcam ab5) and anti-DRP1 (Abcam ab180769) were 1.25 mg/ml and 1.50 mg/ml, respectively. For the GV stage Trim-Away, oocytes were injected with 5 pl Trim21 mRNA/antibody mix and maintained in the medium containing milrinone for 3 h and then washed out for further development. For pro-MI stage Trim-Away, oocytes matured for 2 h after NEBD were injected with 5 pl Trim21 mRNA/antibody mix.