The role of hippocampal PML in transgenic mouse circadian rhythm

• Autorzy: Olech-Kochanczyk G. , Kiryk-Jaskiewicz A. , Domanskyi A. , Konopka W. , Wilczynski G.M.
• Języki publikacji: EN

Abstrakty

EN

INTRODUCTION: Circadian clock is an evolutionarily well-conserved mechanisms in higher organisms. PML protein implicated in many important biological processes (response to DNA damage, cell division control) influences also circadian rhythm by regulating nuclear localization of Per2, a significant positive regulator of the clock transcriptional mechanisms. AIM(S): Our aim is to explain how overexpression or knock‑out of PML affect the oscillations of levels of proteins involved in circadian rhythm in hippocampus. METHOD(S): We generated transgenic mouse models with overexpression or knock-out of PML gene that are induced in specific time and location in brain. The project consists of two tasks: 1) generation of transgenic animals with overexpression of PML gene using AAV vectors encoding PML and mCherry reporter gene, and 2) generation of transgenic animals with knock-out of PML gene using AAV vectors encoding PML gene-targeting gRNAs and Cas9 (CRISPR/Cas9 system). RESULTS: We generated AAV vectors with PML-mCherry and microinjected them into the hippocampus of mice that were subject to behavioral tests (in the IntelliCage system). AAV PML-mCherry mice showed impairment in circadian activity 45 days after surgery and displayed proper spatial learning of cage corners with appetitive reinforcement and slower re-learning process. Next, we designed gRNAs for PML exon I and II and generated plasmid vectors containing gRNAs for the PML gene and tdTomato reporter. Using both these vectors and vectors containing Cas9 endonuclease fused with GFP reporter we transfected NIH 3T3 cells to induce PML gene knock-out in vitro. Next, using T7E1 endonuclease we confirmed that knock-out of PML works. Afterwards, we generated AAV vectors encoding PML gene-targeting gRNAs and Cas9 endonuclease. Both vectors were injected into the hippocampus. CONCLUSIONS: Thus far research on PML function in the circadian rhythm was usually performed in vitro. Our tools enable us to study the role of PML in mammalian molecular clock mechanisms in vivo.

• Wydawca: -
• Czasopismo: Acta Neurobiologiae Experimentalis
• Rocznik: 2017
• Tom: 77
• Numer: Suppl.1
• Opis fizyczny: p.137

Twórcy

autor

Olech-Kochanczyk G.

• Nencki Instiute of Experimental Biology Polish Academy of Sciences, Warsaw, Poland

autor

Kiryk-Jaskiewicz A.

• Nencki Instiute of Experimental Biology Polish Academy of Sciences, Warsaw, Poland

autor

Domanskyi A.

• Institute of Biotechnology, University of Helsinki, Helsinki, Finland

autor

Konopka W.

• Nencki Instiute of Experimental Biology Polish Academy of Sciences, Warsaw, Poland

autor

Wilczynski G.M.

• Nencki Instiute of Experimental Biology Polish Academy of Sciences, Warsaw, Poland