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Generation of a C57BL/6J mouse strain expressing the CD45.1 epitope to improve hematopoietic stem cell engraftment and adoptive cell transfer experiments


Adoptive cell transfer between genetically identical hosts relies on the use of a congenic marker to distinguish the donor cells from the host cells. CD45, a glycoprotein expressed by all hematopoietic cells, is one of the main congenic markers used because its two isoforms, CD45.1 and CD45.2, can be discriminated by flow cytometry. 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, recent studies have identified genetic variations between these congenic strains and have notably highlighted a differential expression of cathepsin E (CTSE). The B6.SJL mouse presents a number of functional differences in hematopoietic stem cell engraftment potential and immune cell numbers compared with the B6 mouse. In this study, we showed that B6 and B6.SJL mice also differ in their CD8+ T cell compartment and CD8+ T cell responses to viral infection. We identified Ctse as the most differentially expressed gene between CD8+ T cells of B6 and B6.SJL and demonstrated that the differences reported between these two mouse strains are not due to CTSE. Finally, using CRISPR–Cas9 genome editing, we generated a CD45.1-expressing B6 mouse by inserting one nucleotide mutation (A904G) leading to an amino acid change (K302E) in the Ptprc gene of the B6 mouse. We showed that this new B6-Ptprcem(K302E)Jmar/J mouse resolves the experimental biases reported between the B6 and B6.SJL mouse lines and should thus represent the new gold standard for adoptive cell transfer experiments in B6.

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Fig. 1: B6.SJL and B6 mice show different CD8+ T cell compartment and antiviral response.
Fig. 2: Mutation of Ptprc by a CRISPR–Cas9 gene-editing approach to induce the expression of CD45.1 epitope in B6 mice.
Fig. 3: Mutation A but not mutation B is necessary for CD45.1 epitope expression.
Fig. 4: B6.mutA and B6 have similar hematopoietic cell compartment before and after transplantation.
Fig. 5: B6.mutA mice have a similar antiviral CD8 response to VV as B6 mice.
Fig. 6: Differential antiviral CD8+ T cell response and HSC engraftment capacity between B6 and B6.SJL mice is not due to CTSE.

Data availability

The mouse models will be available through EMMA ( once the paper is published. The microarray data have been deposited to the Gene Expression Omnibus ( under accession number GSE86601. Source data are provided with this paper.


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We acknowledge the contribution of AniRA-Cytometrie and AniRA-PBES, of the SFR BioSciences (UAR3444/CNRS, US8/Inserm, Ecole Normale Supérieure de Lyon, Université de Lyon). We acknowledge the contributions of the CELPHEDIA Infrastructure ( The project was funded by the intramural CIRI grant: AO-4-2018.

Author information

Authors and Affiliations



D.L., J.M., S.D. and S.M. designed the study. D.L., M.D. and S.D. performed the experiment. C.A., F.H. and M.T. produced the mouse models. D.L., J.M. and S.M. wrote the manuscript. B.C. and Y.L. provided helpful discussions. S.D., Y.L. and B.C. reviewed the manuscript.

Corresponding author

Correspondence to Jacqueline Marvel.

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The authors declare no competing interests.

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Lab Animal thanks Bonnie Dittel and Maegan Capitano for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Ctse mRNA expression levels in various immune cell types from Immgen database.

The Immunological Genome Project (ImmGen) provides an atlas of mRNA signatures across the immune system. The figure represents the Ctse mRNA levels from various immune cell types isolated from a mouse with a B6 or a B6.SJL background.

Extended Data Fig. 2 Analysis of potential off-target sites for mutation A insertion.

a, The potential off-target (OT) sites for mutA-guide RNA were determined using CRISPOR software. Weaker NAG or NGA PAM are also taken into account even if corresponding OT are less likely to happen. Potential OT loci were PCR-amplified and sequenced using genomic DNA from founder B6.mutA F1 mouse and compared to B6 control. b, Corresponding PCR gels are shown. O’GeneRuler 100 bp Plus DNA Ladder was used as a ladder (L). c, Sequence alignments of potential OT regions for B6.mutA and B6 mice. Sequence targeted by the guide RNA is in blue, PAM sequences are in orange (NGG, NGA or NAG) and nucleotide differences between B6 and B6.mutA are in red.

Source data

Extended Data Fig. 3 Sequencing trace data of potential off-target sites.

Chromatograms of sequenced PCR products for each potential mutA OT sites and associated double stranded sequences are represented for B6.mutA and B6 mice. Sequence targeted by the guide RNA is highlighted in blue and PAM sequence in orange. Nucleotide differences are highlighted with a red square.

Extended Data Fig. 4 B6 and B6.mutA mice have similar bone marrow reconstitution potential.

Irradiated B6/B6.SJL heterozygous mice received a 1:1 mixture of B6.mutA or B6.SJL and B6 bone marrow cells. The proportions of B and T cells within CD45.1 and CD45.2 donor cells was determined 17 weeks after transfer in the spleen by flow cytometry. Results are expressed as the median ± range (n = 5 mice/group, one of two representative experiments). The statistical significance of differences was determined by Mann-Whitney test (*P < 0.05, **P < 0,01).

Extended Data Fig. 5 Generation of B6.SJL.CTSE-KO mice.

B6.SJL.CTSE-KO mouse was generated by CRIPSR editing. A 83 bp deletion was made in exon 3 of the Ctse gene in B6.SJL mouse using two guide RNAs simultaneously. a, The targeted Ctse gene sequence is represented with guide RNA sequences highlighted in red, PAM sequences in blue and primer sequences in grey. b, DNA Sanger sequencing of the targeted Ctse gene region for both B6.SJL.CTSE-KO and B6.SJL.CTSE-WT mice (sequences and trace data, antisense strand).

Supplementary information

Reporting Summary

Supplementary Table 1

List of oligonucleotide sequences.

Source data

Source Data Extended Data Fig. 2

Unprocessed gels related to Extended Data Fig. 2.

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Laubreton, D., Djebali, S., Angleraux, C. et al. Generation of a C57BL/6J mouse strain expressing the CD45.1 epitope to improve hematopoietic stem cell engraftment and adoptive cell transfer experiments. Lab Anim 52, 324–331 (2023).

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