National Avian Research Facility
National Avian Research Facility

RUNX1 reporter lines

RUNX1-eGFP and RUNX1-Cre

Summary & Utility

The RUNX1-eGFP line is a transgenic reporter chicken line with Runt-related transcription factor 1 (RUNX1) promoter/enhancer driven eGFP expression and is based upon the P1 promoter and the +23 enhancer1 of the chicken RUNX1 locus. The RUNX1-eGFP line enables the visualisation of chicken hematopoietic cells and haematopoietic cell progenitor populations2. 

Similar to the CSF1R reporter lines the RUNX1-eGFP line can be used to visualise chicken lymphoid tissues by whole mount fluorescence microscopy (Figure 1). In conjunction with the CSF2R staining3, the RUNX1-eGFP line can also be used to simultaneously visualise and distinguish avian granulocytes (Basophils, Eosinophils and Heterophils) and monocytes by flow cytometry (Figure 2). Degranulation of heterophils can also be quantified. 

 

RUNX1 eGFP
Figure 1. RUNX1-eGFP reporter transgene visualisation of chicken lymphoid tissues. A) Whole mount view of the thymus from an embryonic day 14 RUNX1-eGFP embryo. Scale bar = 200 µm. B) Higher magnification of the thymus in A). Scale bar = 50 µm. C) Whole mount view of the ileum from a 16-week-old RUNX1-eGFP hen. Lymphoid follicles (green spots) can be easily visualized due to high level expression of the RUNX1-eGFP transgene in immune cells. Scale bar = 200 µm.
RUNX1 eGFP granulocytes
Figure 2. Differentiation and identification of chicken granulocytes on the basis of CSF2-Fc-647 staining. Binding of recombinant CSF2-647 identifies blood cells that express the Colony stimulating factor 2 receptor (CSF2R; also known as GM-CSF receptor (GM-CSFR)). Histochemical staining (Eosin-Toluidine Blue) of FACS sorted cell populations enables these GM-CSFR+ subsets to be identified as basophils, monocytes, eosinophils and heterophils. While eosinophils and heterophils express similar levels of GM-CSFR, they can be readily differentiated from each other on the basis of RUNX1-eGFP expression levels, granularity (SCC) and cell size (FSC) by flow cytometric analysis.

 

RUNX1-Cre line

The RUNX1-Cre line is a transgenic reporter chicken line with chicken RUNX1 promoter/enhancer driven Cre recombinase expression. The RUNX1-Cre line uses the same promoter/enhancer as the RUNX1-eGFP line to drive Cre recombinase expression. This line of birds has also been crossed to the Chameleon line to enable cell lineage tracing and clonal analysis of selected cell populations, such as haematopoietic cells post-hatch, skeletal muscle cells and epithelial cells (Figure 3).

 

RUNX1 Cre
Figure 3. Visualisation of selected cell populations in tissue from a two year old RUNX1-Cre x Chameleon chicken. A) Caecal tonsil follicular epithelium. Clonal populations of epithelial cells express the same fluorescent protein. Scale bar = 50 µm. B) Fluorescent labelling of macrophages in the iris. Scale bar = 100 µm. C) Fluorescent labelling of skeletal muscle (breast) myotubes. Scale bar = 200 µm.
 

Line origin

The generation of the RUNX1-eGFP and RUNX1-Cre lines was funded by the Medical Research Council (MR/L018160/1). These RUNX1 lines were created by the use of lentiviral gene transfer. The RUNX1 control elements utilised in the construction of the transgene consist of the P1 promoter and the +23 enhancer1. The cell lineage specificity of reporter gene expression was initially confirmed by transfecting chick embryonic blood cells with the RUNX1-eGFP construct to report Runt-related transcription factor 1 gene activity. These lines were developed by Dr Adam Balic and Professor Helen Sang.

To reference this line for publications please contact Dr Adam Balic.

 

Publications

  1. Bee, T., Ashley, E. L., Bickley, S. R., Jarratt, A., Li, P. S., Sloane-Stanley, J., Göttgens, B. & de Bruijn, M. F. The mouse Runx1 +23 hematopoietic stem cell enhancer confers hematopoietic specificity to both Runx1 promoters. Blood. 113(21):5121-4 (2009).
  2. Davey, M. G., Balic, A., Rainger, J., Sang, H. M. & McGrew, M. J. Illuminating the chicken model through genetic modification. Int. J. Dev. Biol. 62, 257–264 (2018).
  3. Wu, Z., Hu, T., Chintoan-Uta, C., Macdonald, J., Stevens, M. P., Sang, H., et al. Development of novel reagents to chicken FLT3, XCR1 and CSF2R for the identification and characterization of avian conventional dendritic cells. Immunology 165, 171–194. (2022).