Gp1ba-Cre or Pf4-Cre: pick your poison

In this issue of Blood, Nagy et al developed a novel Gp1ba-Cre mouse that uses the endogenous Gp1ba locus that encodes the glycoprotein Ibα (GPIbα) subunit of the GPib-IX receptor to drive Cre expression in murine megakaryocytes (MKs) for targeted ablation of floxed genes.¹ 

The rationale for these studies is to improve on the present gold standard: the platelet factor 4 (PF4)-Cre recombinase (Pf4-Cre) for creating a conditional knockout in MKs and platelets. In the Pf4-Cre mouse, the Cre construct was substituted into an ∼100-kb murine bacterial artificial chromosome (BAC) construct containing Cxcl4 and several other CXC genes.²  The Pf4-Cre mouse has been widely used to generate MK/platelet-targeted conditional knockout (KO) models to evaluate the mechanisms underlying platelet production and function, but there are several limitations with this model, including the additional copies of several CXC chemokines that may exert proinflammatory effects (see table for the pluses and minuses of the 2 constructs). Another limitation has come from the recognition that although PF4 expression may be high during megakaryopoiesis, it also is present in hematopoietic stem cells, and lymphoid- and myeloid-derived cells, especially in inflammatory states^3,4  (see table). Although the level of PF4 expression in leukocytes is likely to be lower than that seen in MKs, it has been shown that “ectopic” expression of Cre in the Pf4-Cre mice leads to the ablation of target genes in subpopulations of leukocytes and macrophages.⁵  Therefore, there is a need for an alternative deleter mouse strain that more specifically targets MKs. The authors selected the Gp1ba gene because it is highly expressed in MKs.⁶ 

The authors began their studies by measuring hematologic parameters in heterozygous knock-in (KI) mice (Gp1ba-Cre^+/KI) and found they had normal platelet counts, but a slight increase in platelet volume and reduction in surface GPIb-IX. Platelet function was preserved. Although mild, these platelet abnormalities may need to be accounted for when studying MK/platelet-specific expression using this new system (see table).

To determine whether Cre expression was restricted to the MK lineage, both Gp1ba-Cre^+/KI and Pf4-Cre^+/KI animals were crossed with mice that express membrane-targeted tandem dimer tomato (mT;tdTomato) and enhanced green fluorescent protein (mT;EGFP) prior to Cre-mediated recombination.⁷  Within 24 hours of Cre-mediated excision, affected cells begin to express mT;EGFP, followed by the more gradual loss of tdTomato fluorescence. The authors monitored the onset of EGFP expression and the loss of tdTomato expression to gain insight into the timing of Cre expression in different cellular populations. In both Gp1ba-Cre and Pf4-Cre mice, examination of the peripheral blood revealed that Cre-mediated recombination occurred in nearly all platelets and virtually no red cells. Importantly, in the bone marrow (BM), ∼95% of the MKs were EGFP⁺ in Pf4-Cre mice, only 74% of MKs in Gp1ba-Cre mice were EGFP⁺, suggesting gene ablation was more efficient in the Pf4-Cre mouse compared with the Gp1ba-Cre mouse. Leukocyte-EGFP expression diverged markedly between the 2: ∼2% of CD45⁺ cells in the Gp1ba-Cre mice were positive compared with ∼26% of CD45⁺ cells in Pf4-Cre mice. This difference was also seen in other myeloid/lymphoid lineages, showing that Cre recombinase is expressed in a variety of white cells in the Pf4-Cre system (see table).

These differences between the 2 constructs impacts the phenotype of conditional KO mice generated when Gp1ba-Cre or Pf4-Cre mice are crossed with floxed animals. For example, in studies with Csk and CD148 conditional KOs, compared with Gp1ba-Cre mice, Pf4-Cre mice had lower levels of Csk in platelets, a more severe thrombocytopenia and a greater increase in platelet volume. On the other hand, although Csk^fl/fl;Gp1ba-Cre^+/KI mice had normal white blood cell counts, they were significantly elevated in Csk^fl/fl;PF4-Cre^+/KI mice, likely as a consequence of gene deletion in lymphocytes. Similarly, conditional KOs of Shp1 generated on the Gp1ba-Cre background were healthy, whereas animals on the Pf4-Cre background developed a motheaten-like inflammatory phenotype, likely caused by gene ablation in leukocytes.

Although the article describes a new model for generating MK-specific KOs, it also provides potential new insights into the biology of PF4 and GPIbα. PF4-driven Cre expression occurs broadly outside of the MK lineage, although the relative amount of PF4 expression in leukocyte subsets vs MKs needs to be explored. The implications of these studies to PF4 biology must be viewed with caution as other factors besides the specificity of Cre expression may have contributed to the differences observed between the Pf4-Cre and GP1ba-Cre animals. Unlike the GP1ba-Cre KI construct that is embedded in the endogenous Gp1ba locus, the Pf4-Cre mouse was generated using a BAC construct in an unknown insertion site that may influence expression from the Cxcl4 locus. Moreover, the Pf4-Cre mouse contains 4 additional CXC chemokine genes that may alter leukocyte biology²  and modify PF4 expression (see table).

The authors also conclude that PF4 is expressed earlier than GPIbα in megakaryopoiesis. This conclusion must be tempered by the above discussion concerning the differences in the KI constructs and by the BAC PF4-driven Cre being more strongly expressed, leading to a brighter EGFP signal and to more efficient tdTomato extinction. Additionally, Cre-recombinase expression occurs when MKs are undergoing polyploidy change. The stronger PF4-driven Cre may be able to efficiently KO multiple copies of a gene in a polyploidic, maturing MK, whereas the Gp1ba promoter may be unable to disable all copies.

In summary, Nagy et al report important new insights into PF4 and Gp1ba-driven Cre-expression patterns in hematopoiesis, supporting that PF4 is sufficiently expressed in non-MK lineages to affect KO phenotype. Conclusions regarding physiologic PF4 expression need to be tempered by Pf4-Cre involvement in a BAC KI. Gp1ba-Cre expresses Cre during megakaryopoiesis at lower efficiency, but with fewer hematopoietic off-target effects. The Gp1ba-Cre^+/KI mice have only a single intact Gp1ba gene, leading to a mild macrothrombocytopenia that may color some MK/platelet studies. Thus, as outlined (see table), each construct has its pluses and minuses. Understanding the limitations of both systems is crucial to interpreting outcome in MK-specific KO studies. Limitations in these models do not overlap. Concurrent studies with the 2 deleter systems may improve insights into platelet biology.