Generation of a C57Bl/6J mouse strain expressing the CD45.1 epitope to improve HSC engraftment and adoptive cell transfer experiments

Abstract Adoptive cell transfer between genetically identical hosts is a key tool in the study of the murine immune system. In most models, host and donor differ by a congenic marker, i.e. alleles of a gene coding for a surface protein expressed by immune cells and which allows to trace the donor cells using an epitope specific antibody (Ab). CD45, a glycoprotein expressed by all hematopoietic cells and encoded by the Ptprc gene, constitute one of the main congenic markers used. Its two isoforms, CD45.1 and CD45.2, can be discriminated by flow cytometry using the anti-CD45.1 (clone A20) and anti-CD45.2 (clone 104) Ab. As a consequence, C57BL/6J (B6; CD45.2) and B6.SJL-Ptprca Pepcb/BoyJ (B6.SJL; CD45.1) mice are widely used in adoptive cell transfer experiments, under the presumption that they differ only at the CD45 (Ptprc) locus. However, the B6.SJL congenic mouse was generated by crossing an SJL mouse with a B6 mouse, followed by serial back-crossing on B6 mice with the selection of CD45.1 expressing animals at each generation. Recent studies identified genetic variations between these congenic strains and highlighted, among other, a differential expression of cathepsin E (CTSE). B6.SJL mouse presents a number of functional differences in hematopoietic stem cell (HSC) engraftment-potential or immune cell numbers. We show that B6 and B6.SJL mice also differ in their CD8 T cells compartment and CD8 T cells responses to viral infection compared to B6 mouse. We identified Ctse as the most differentially expressed gene between CD8 T cells of B6 and B6.SJL demonstrate that the differences reported between these two mouse strains are not due to CTSE. In order to avoid biases associated with known or unknown polymorphisms found in congenic mice, we used CRISPR/Cas9 genome editing to introduce, in the Ptprc gene of B6 mouse (CD45.2 epitope), the point mutation(s) associated with its transformation into the CD45.1 epitope. We show that only one nucleotide mutation (A904G) leading to an amino acid change (K302E) is sufficient for the protein to be recognized by the anti-CD45.1 Ab. We show that this new B6-Ptprcem(K302E)Jmar/J mouse has similar immune compartment compared to B6 mouse and does not show any difference in HSC engraftment or CD8 antiviral responses. This new strain resolves the experimental biases reported between B6 and B6.SJL mouse lines and should thus represent the new gold standard for adoptive transfer experiments in B6.