Establishment and characterization of mouse lines useful for endogenous protein degradation via an improved auxin-inducible degron system (AID2).
Makino-Itou H, Yamatani N, Okubo A, Kiso M, Ajima R, Kanemaki MT, Saga Y.
Development, Growth and Differentiation (2024) Sep;66(7):384-393. DOI:10.1111/dgd.12942
The development of new technologies opens new avenues in the research field. Currently, conditional gene knockout strategies are employed to examine temporal and spatial gene function. However, phenotypes are sometimes not observed because of the time required for depletion due to the long half-life of the target proteins. Protein knockdown using an improved auxin-inducible degron system, AID2, overcomes such difficulties owing to rapid and efficient target depletion. Here, we established several mouse lines useful for AID2-medicated protein knockdown, which include knock-in mouse lines in the ROSA26 locus; one expresses TIR1(F74G), and the other is the reporter expressing AID-mCherry. We also established a germ-cell-specific TIR1 line and confirmed the protein knockdown specificity. In addition, we introduced an AID tag to an endogenous protein, DCP2 via the CAS9-mediated gene editing method. We confirmed that the protein was effectively eliminated by TIR1(F74G), which resulted in the similar phenotype observed in knockout mouse within 20 h.
Figure: The germ-cell-specific TIR1 line was crossed with the reporter line TG-CAG-AID-mCherry. Immunofluorescence images of embryonic testes (E15.5) prepared from a pregnant CAG-AID-mCherrymother crossed with an Oct-dPE-TIR1(F74G)-FLAG male. TIR1(-) means sample without Oct-dPE-TIR1(F74G) and TIR1(+) means double transgenic sample. The mother was subjected to 5-Ph-IAA injection at E14.5. Antibodies used were anti-mCherry, anti-FLAG, and anti-E-cadherin (CDH). The scale bar indicates 100 mm.
