ABSTRACTS – ORAL PRESENTATIONS - AMCA, spol. s r.o.

62. GREEN FLUORESCENCE PROTEIN (GFP) MICE IN HEMATOPOIETIC STEM CELL
TRANSPLANTATION EXPERIMENTS: THE POSSIBLE LIMITATIONS IN CHIMERISM
QUANTIFICATION
Filipp Savvulidi, Katarina Forgacova, Emanuel Necas, Ludek Sefc
Charles University in Prague, 1 st Faculty of Medicine, Prague, Czech Republic,
immunophi@gmail.com
Transgenic mice expressing enhanced Green Fluorescent Protein (GFP) under the
direction of the human ubiqutin C promoter (C57BL/6-Tg(UBC-GFP)30Scha/J mice) are
frequently used in hematopoietic stem cell (HSC) transplantation experiments. GFP is
expressed in the leukocytes, platelets, and erythrocytes of these mice. Bone marrow
cells are transplanted into congenic C57BL/6 recipients and chimerism of white blood
cells based on GFP expression is determined in lysed peripheral blood of recipient at
different intervals after transplantation. Another congenic mouse transplantation model
uses functionally identical isoforms of CD45 antigen - Ly5.1 (CD45.1) or Ly5.2 (CD45.2).
GFP and their congenic C57BL/6 recipients both express just Ly5.2 isoform.
In order to compare the accuracy of both models, as well as to establish a new model
for tracking of mutual competition of three hematopoietic tissues together, we cotransplanted
GFP(Ly5.2) and Ly5.1 bone marrow cells to sublethally irradiated wild-type
C57BL/6 (Ly5.2) recipients. We expected to distinguish GFP + Ly5.1 - Ly5.2 + , GFP - Ly5.1 -
Ly5.2 + and GFP - Ly5.1 + Ly5.2 - populations representing all three mouse strains involved.
Surprisingly, we found also GFP + Ly5.1 + Ly5.2 - cells (5-10%) which were not doublets nor
cells of any used strain. These cells were detected in lower number also in bone marrow
of transplanted mice which was not lysed in contrary to peripheral blood.
In order to explain the presence of evidently false, Ly5.1/GFP double-positive cells in
our observation, we incubated non-GFP Ly5.1 peripheral blood cells with supernatant
from lysed GFP blood. A significant portion of Ly5.1 cells gained GFP positivity (Fig. 1B).
Filtering of GFP supernatant with 0.22 µm filter abolished its GFP staining capacity (Fig. 1C).
We conclude that fragments of GFP cells after ammonium chloride lysis, presumably of
erythrocyte ghosts, attach at the surface of Ly5.1 cells generating artificial Ly5.1/GFP
double-positive particles. This cellular debris is relatively large in size (larger than 0.22µm).
Our results demonstrate that using a standard GFP - C57BL/6 congenic transplantation
model, a GFP derived chimerism can be overestimated by flow cytometry detection.
Acknowledgements
We thank M. Molik for his excellent technical help.
This work was supported by the Ministry of Education, Youth and Sports of the Czech
Republic (project PRVOUK-P24/LF1/3), and by Charles University in Prague (grant SVV-
2012-264507)
Legend to Fig.1: Peripheral
blood cells, singlets gated. (A)
Ly5.1 cells stained with anti-
Ly5.1 Ab; (B) Ly5.1 cells co-
Analytical Cytometry VII 79