Presentation Details
| Characterization of the endothelial cell subpopulations marked by Tek and Cdh5 promoter driven Cre expression in different mouse strains David Svilar1, 2, Audrey Cleuren1, 3, David Siemieniak1, David Ginsburg1, 2, 4, 5. 1Life Sciences Institute, University of Michigan, Ann Arbor, MI, USA.2Department of Pediatrics, University of Michigan, Ann Arbor, MI, USA.3Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK, USA.4Department of Internal Medicine, University of Michigan, Ann Arbor, MI, USA.5Department of Human Genetics, University of Michigan, Ann Arbor, MI, USA |
Abstract
Background: The endothelium plays an important role in maintaining hemostasis, and mice are often used to study this endothelial cell (EC) contribution. While mice from the same inbred strain are genetically identical, the considerable genetic variation between strains can lead to observed differences in phenotypes. Furthermore, in order to specifically target ECs using a Cre-recombinase system, studies often employ either Tek-Cre (also known as Tie2) or Cdh5-Cre (also known as VE-cadherin) mice. Although both genes are considered to be generic EC markers, it is unknown whether they target potential distinct EC subpopulations. Objectives: Characterize expression profiles of ECs targeted by either Tek- or Cdh5-Cre in C57BL/6J and CAST/EiJ mice in order to get a better understanding of strain-specific differences in EC gene expression profiles, as well as potential differences in EC subpopulations targeted by either Tek or Cdh5-Cre. Methods: To evaluate the in vivo endothelial cell gene expression profiles, we utilized a Translating Ribosome Affinity Purification (TRAP) approach using the Ribotag mouse model, which selectively expresses the ribosomal protein RPL22 with a hemagglutinin (HA) tag in cells targeted by Cre-recombinase. This allows cell type-specific isolation of ribosomal complexes with their associated mRNA, that can then be isolated for downstream analyses to determine transcript abundance, as we have previously described. In addition to maintaining these mice on a C57BL/6J background, we also crossed them onto a genetically distinct strain within the Mus musculus species, CAST/EiJ. Results: We have previously shown that Tek-Cre on a C57BL/6J background results in efficient targeting of ECs across organs. To determine if there are differences in EC expression profiles between mouse strains, we outcrossed our original C57BL/6J Ribotag, Tek-Cre mice into CAST/EiJ. The resulting Tek-Cre positive offspring were used to perform TRAP on liver and kidney. To our surprise, the majority of samples (33 out of 36) did not show enrichment of EC-specific markers after immunoprecipitation based on the HA-tag. This prompted us to use a constitutively expressing Cdh5-Cre model to determine whether the lack of EC enrichment was unique to the use of Tek-Cre. Combining the Ribotag with Cdh5-Cre on either a C57BL/6J or the CAST/EiJ background resulted in robust enrichment of known endothelial cell markers, indicating specific targeting. Additionally, the Cdh5-Cre model enriched for Tek expression, suggesting some overlap of Tek and Cdh5 cells and that transient Tek expression in non-ECs may contribute to Tek-Cre mosaicism in CAST/EiJ. Conclusions: Our data suggests that either the presence of a constitutive Tek-Cre in the CAST/EiJ background leads to more frequent germline excision of our conditional Rpl22 allele as compared to this event occurring in C57BL/6J mice, or that the (transient) expression pattern of Tek is not limited to ECs in CAST/EiJ mice. Cdh5-Cre on the other hand, does provide specific targeting of the endothelial compartment in both C57Bl/6J and CAST/EiJ mice. Establishing this model allows us to compare EC gene expression profiles of C57BL/6J and CAST/EiJ mice, with future studies aimed at gaining insights into the phenotypic difference between ECs across mouse strains.
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