Characterization of Poldip2 knockout mice: Avoiding incorrect gene targeting

• Type of Material: Article
• Authors: Bernard Lassegue, Emory University; Sandeep Kumar, Emory University; Rohan Mandavilli, Emory University; Keke Wang, Emory University; Michelle Tsai, Emory University; Dong-Won Kang, Emory University; Catherine Demos, Emory University; Marina S. Hernandes, Emory University; Alejandra San Martin, Emory University; W. Robert Taylor, Emory University; Hanjoong Jo, Emory University; Kathy Griendling, Emory University
• Language: English
• Date: 2021-12-20
• Publisher: Public Library of Science (PLoS)
• Publication Version: Final Published Version
• Copyright Statement: © 2021 Lassègue et al
• License: Creative Commons Attribution 4.0 International
• Final Published Version (URL): http://dx.doi.org/10.1371/journal.pone.0247261
• Title of Journal or Parent Work: PLoS One
• Volume: 16
• Issue: 12
• Grant/Funding Information: This research was funded by National Institutes of Health grants HL95070 and HL152167 to K.G., HL119798, HL095070, and HL139757 to H.J., who was also supported by the Wallace H. Coulter Distinguished Faculty Chair Professorship.
• Research Categories: Health Sciences, Medicine and Surgery; Biology, Genetics

Abstract

POLDIP2 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 three limitations: perinatal lethality in homozygotes, constitutive Poldip2 inactivation and inadvertent downregulation of the adjacent Tmem199 gene. To overcome these limitations, we developed a new conditional floxed Poldip2 model. 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 around Poldip2 with 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 floxed Poldip2 mice 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 of Poldip2 ablation in the vasculature, RNA-seq and RT-qPCR experiments were performed in constitutive knockout arteries. Results show that POLDIP2 inactivation affects multiple cellular processes and provide new opportunities for future in-depth study of its functions.