The majority of genetically modified C57BL/6 mice contain congenic passenger DNA around the targeted gene locus as they were generated from 129-derived embryonic stem cells (ESCs) with subsequent backcrossing to the C57BL/6 genetic background. When studying the role of atypical chemokine receptor 4 (ACKR4) in the immune system, we realized that the two available Ackr4-deficient mouse strains (Ackr4-/- and Ackr4GFP/GFP ) show profoundly different phenotypes: Compared to wild-type and Ackr4GFP/GFP mice, Ackr4-/- mice show a strong accumulation of plasma blasts in mesenteric lymph node and spleen as well as increased B cell proliferation after in vitro activation. This phenotype was maintained after further backcrossing to C57BL/6 mice and was even present in heterozygous Ackr4+/- animals, suggesting that a gene variant on the targeted chromosome might cause this phenotype. Exome sequencing revealed that a region of approximately 20 Mbp around the Ackr4 locus on chromosome 9 still originates from the 129 background based on high variant density observed. In activated Ackr4-/- and Ackr4GFP/GFP B cells, transcripts of genes around the Ackr4 locus were equally deregulated compared to C57BL/6 B cells, whereas increased expression of IL-6 was selectively observed in B cells of Ackr4-/- mice. Because the gene encoding for IL-6 is placed on chromosome 5 these findings suggest that passenger DNA around the Ackr4 locus has an indirect effect on B cell activation and IL-6 production. Results of the present study should not only lead to the reinterpretation of data from earlier studies using Ackr4-/- mice but should remind the scientific community about the limitations of mouse models using mice created by gene-targeting of nonsyngeneic ESCs.
