| First Author | Shen H | Year | 2021 |
| Journal | American Thoracic Society | Mgi Jnum | J:349974 |
| Mgi Id | MGI:7660797 | Doi | 10.1164/ajrccm-conference.2021.203.1_MeetingAbstracts.A1037 |
| Citation | Shen H, et al. (2021) Novel Transgenic Mouse Strain Gramd2CreERT2 for Investigation of Alveolar Epithelial Type I (AT1) Cell Plasticity and Phenotype. American Thoracic Society |
| abstractText | RATIONALE: Due to the limited specificity of âclassicalâ AT1 cell markers such as AQP5, PDPN, HOPX, and AGER, there is a need for identifying new AT1 cell markers and tools for characterizing AT1 cell phenotype. Recently, through transcriptome profiling or single-cell-RNA sequencing, respectively, we have identified and characterized two new AT1 cell markers, GRAMD2 and GPRC5A. We further generated a new transgenic mouse line, Gramd2CreERT2, and tested AT1 cell specificity of this strain. METHODS: We used CRISPR/Cas-assisted gene- targeting to knockin creERT2 and nuclear GFP (nGFP) into the stop codon of the endogenous Gramd2 gene. Because GFP protein could not be detected, we crossed the GD2CE (Gramd2 promoter-driving creERT2) mouse with the Cre-reporter mouse strain mTmG (membrane-targeted tandem dimer Tomato (mT) prior to Cre- mediated excision and membrane-targeted GFP (mG) after excision) to generate GD2CE;mTmG mice to evaluate lineage labeling by GD2CE by Western blotting, and double labeling immunofluorescence (IF) of tissues and isolated cells for GFP, AT1, AT2 and airway markers.RESULTS: By western blot, GFP is exclusively detected in the lung but not in other organs, indicating lung specificity. In lung sections, GFP colocalized with AT1 cell markers AQP5 or PDPN, but not with AT2 cell marker proSPC, indicating that in distal lung, GD2CE;mTmG lineage labels AT1 cells. However, the GFP positive signal was not detected in all AQP5 or PDPN positive AT1 cells, suggesting either incomplete recombination or heterogeneity of Gramd2 gene expression among AT1 cells. In the distal airway, very few GFP positive cells were detected and co-stained with CC10 (1.91 ± 0.36% of CC10+ cells were GFP and CC10 double-positive). We further quantified recombination efficiency by performing IF in cytospins of crude alveolar epithelial cell preparations. In cytospins, GFP colocalized with AQP5 (52.83 ± 3.42% of AQP5+ cells were GFP and AQP5 double-positive) and rarely with proSPC (0.56 ± 0.56% of SPC+ cells were GFP and SPC double-positive), confirming AT1 cell labeling in alveolar epithelium. Furthermore, we successfully utilized GD2CE;mTmG mice to sort GFP+ AT1 cells of high purity using a GFP antibody. 98.57 ± 0.79% of GFP+ cells were AQP5+, and 99.72 ± 0.19% of GFP+ cells were GPRC5A+. CONCLUSIONS: GD2CE mice label AT1 cells in distal lung with high specificity, with minimal labeling of airway cells. GD2CE mice are a novel tool for study of AT1 cell biology and will be useful for both isolation of AT1 cells and in vivo studies. Funding: NIH, Hastings Foundation |
