Characterization ofPoldip2knockout mice: avoiding incorrect gene targeting

Abstract

AbstractPOLDIP2 is a multifunctional protein whose roles are only partially understood. Our laboratory previously reported physiological studies performed using a mouse gene trap model, which suffered from two limitations: perinatal lethality in homozygotes and constitutivePoldip2inactivation. To overcome these limitations, we developed a new conditional floxedPoldip2model. The first part of the present study shows that our initial floxed mice were affected by an unexpected mutation, which was not readily detected by Southern blotting and traditional PCR. It consisted of a 305 kb duplication aroundPoldip2with retention of the wild type allele and could be traced back to the original targeted ES cell clone. We offer simple suggestions to rapidly detect similar accidents, which may affect genome editing using both traditional and CRISPR-based methods. In the second part of the present study, correctly targeted floxedPoldip2mice were generated and used to produce a new constitutive knockout line by crossing with a Cre deleter. In contrast to the gene trap model, many homozygous knockout mice were viable, in spite of having no POLDIP2 expression. To further characterize the effects ofPoldip2ablation in the vasculature, an RNA-seq experiment was performed in constitutive knockout carotid arteries. Results support the involvement of POLDIP2 in multiple cellular processes and provide new opportunities for future in-depth study of its functions.