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    • The CD and CD alleles differ by

    2018-11-08

    The CD45.1 and CD45.2 HMBA Linker Supplier differ by only five amino acids within the extracellular domain (Zebedee et al., 1991), resulting in epitope changes that permit specific recognition by monoclonal antibodies (Shen, 1981). The majority of the commonly used mouse strains express the CD45.2 allele. Backcrossing of mice expressing the CD45.1 allele (SJL) into the C57BL/6 background (CD45.2) has resulted in the development of the mouse strain B6.SJL-PtprcaPepcb/Boy (B6.SJL). As the mice have been backcrossed over many generations, they have been termed congenic, with the presumption that they differ only at the CD45 locus. Table 1 contains a description of the nomenclature for the mouse strains described in this article. A competitive transplant is typically performed by transplanting an equal number of CD45.1 and CD45.2 donor cells into the background of CD45.1, CD45.2, or heterozygous (CD45.1/2) mice. As the recipient mice are given a myeloablative dose of γ-irradiation prior to the transplant, there are few (usually less than 5%, mostly T lymphocytes) residual host cells, and thus the more-readily available and less expensive CD45.2 mice are generally used as transplant recipients (Figure 1A). The advantage of using F1 CD45.1/2 heterozygote recipient mice is that the host residual cells are doubly stained by both CD45.1 and CD45.2 monoclonal antibodies, and thus can be distinguished from the transplanted cells. Hundreds of competitive transplants have been reported in the literature. Most of these are performed to test the impact of a genetically modified test gene. Unfortunately, the WT congenic B6.SJL (CD45.1) HSPCs have an inherent disadvantage over WT C57BL/6 (CD45.2) HSPCs, with reported defects in homing efficiency, reduction in transplantable long-term hematopoietic stem cells, and a cell-intrinsic engraftment defect as demonstrated by Waterstrat et al. (2010) and us (Figure 1B). This advantage is particularly marked if C57BL/6J mice are used as recipients and is mildly attenuated when B6.SJL mice are used as recipients, suggesting that B6.SJL cells may be impaired in their ability to interact with C57BL/6J stromal cells (Waterstrat et al., 2010). This lack of competitive balance between the two putatively congenic strains has called into question the interpretation of prior results concerning the impact of modified genes and has created a conundrum for the field. The molecular basis for the inherent advantage of C57BL/6J (CD45.2) HSPCs over B6.SJL (CD45.1) is not known. The CD45 gene is inherited within a locus that comprises almost 40 megabases and more than 300 predicted genes (Waterstrat et al., 2010). The basis for the discordant HSPC function of the two genotypes is presumably within this 40 Mb region and may include immunologically or functionally active gene products. Several international consortia, including KOMP (Knock-Out Mouse Program) and EUCOMM (European Conditional Mouse Mutagenesis Program), have ongoing programs to generate new strains in the uniform C57BL/6N background (Sacca et al., 2013). Thus, the ideal control for HSPC characterization of these new strains is a functionally equivalent C57BL/6N mouse with the minimal change in the CD45 gene to render it detectable by a CD45.1 antibody. Here, we define the specific epitope difference between the CD45.1 and CD45.2 alleles and describe the creation and testing of a CD45.1 knockin point mutation in the background of the most commonly used C57BL/6N mouse strain. Our data demonstrate that, contrary to the B6.SJL strain, HSPC derived from this mouse are functionally equivalent. We therefore propose that this mouse represents the ideal competitor for competitive HSPC transplant, reducing the number of controls required when working in the C57BL/6N background; we have named it CD45.1STEM (single targeted exon mutation).
    Results
    Discussion Our data demonstrate that, by introducing a single point mutation into the genetic background of a C57BL/6N mouse, we could generate a knockin strain that is identifiable using the commercially available CD45.1 antibody (clone A20). HSPCs from this knockin strain, named C57BL/6N-CD45.1STEM (single targeted exon mutation) are equivalent in competitive transplant assays when compared with HSPCs from either C57BL/6N or C57BL/6J mice.