Where is cre lt

Yang Du 1. Kristbjorn Orri Gudmundsson 1 ,. Access enabled via: An Institution. PDF Full text Related articles. Abstract Long-term hematopoietic stem cells LT-HSCs have the ability to self-renew and differentiate into all blood cell lineages. Understanding the genetic networks that regulate LT-HSC function in the adult bone marrow requires inducible gene targeting … more. Citations 2 Recent citations: Bjorg Gudmundsdottir et al. Related articles Based on techniques. Gress , , Springer Protocols.

Vosshenrich , , Springer Protocols. See more. Smith , , Springer Protocols. Davis , , Springer Protocols. Annu Rev Cell Dev Biol — Rossi L et al Less is more: unveiling the functional core of hematopoietic stem cells through knockout mice. However, in the present study we have employed a sensitive reporter-based approach to demonstrate that Pf4-Cre also recombines in a significant proportion of both fetal liver and bone marrow hematopoietic stem cells HSCs , including the most primitive fraction containing the long-term repopulating HSCs.

Finally, we show for the first time that Pf4 transcripts are present in adult HSCs and primitive hematopoietic progenitor cells. These results have fundamental implications for the use of the Pf4-Cre mouse model and for our understanding of a possible role for Pf4 in the development of the hematopoietic lineage.

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K , Tenovus Scotland K. K and T. W is a BHF chair. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.

Cre-loxP technology allows the deletion or expression of genes to be regulated temporally and spatially in animals [1] , [2] , [3] , [4]. The nature of a specific promoter used to drive Cre recombinase expression determines the tissue specificity and timing of expression.

Several tissue-specific Cre deleter strains have been developed in order to study the effects of conditional gene expression or deletion in a specific tissue or organ system [2] , [5] , [6]. The recent arrival of a megakaryocyte specific Cre driven by the platelet factor 4 promoter Pf4-Cre [7] has significantly impacted on platelet and megakaryocyte studies [8] , [9] , [10]. This mouse model contributed towards the confirmation of the role of platelets in the separation of the lymphatic and vasculature systems [8] , [11].

However, there are reports that endogenous Pf4 is expressed in cells outside of the megakaryocyte lineage, for example osteoclasts and monocytes [12] , [13] , suggesting that the Pf4 promoter may not be specific to the platelet-megakaryocyte lineage.

Here we performed lineage-tracing studies revealing that Pf4-Cre marks a portion of fetal liver and bone marrow hematopoietic stem cells HSCs and their downstream progeny. Consistent with this, we show that Pf4 transcripts are present in HSCs and primitive hematopoietic cells.

Taken together, these results provide genetic evidence that Pf4-Cre does not recombine solely in the megakaryocyte lineage but in addition in a large proportion of stem and progenitor cells and all their progeny.

Therefore, our results imply that hematopoietic phenotypes of conditional mutations generated using the Pf4-Cre deleter must be interpreted with caution. These observations additionally indicate a possible role for PF4 in haematopoietic stem or progenitor function, which merits future investigation. All experiments were carried out according to UK Home Office regulations.

Cells were stained as previously described [15] , [16]. Platelets and megakaryocytes were isolated from the peripheral blood and the bone marrow according to standard procedures [17] , [18]. BetaMicroglobulin was used as the housekeeping control. Mouse femur and tibia were removed and placed either in ice-cold formalin for overnight fixation quick fix or room-temperature for long fixation, and then processed into paraffin blocks.

Tissue sections were then cut, and processed for RFP immunohistochemistry. The antigen is retrieved by boiling slides in citrate buffer pH6 for 4 min in a microwave. The slides were visualized for 2 min before termination with dH 2 O. The slides were counterstained with Meyers Haematoxylin before mounting. First we confirmed efficient Pf4-Cre-mediated recombination in platelets and megakaryocytes. This result is lower than that seen by Tiedt et al but this is likely to be due to the presence of immature megakaryocytes which have not yet undergone recombination and also those that will need time to switch on RFP expression.

However, our data indicate that all mature platelet producing megakaryocytic have undergone recombination as expected. This expression of RFP outside the megakaryocyte lineage was confirmed by immunostaining for RFP expression in bone marrow histology samples Fig. Images are representative of 2 independent experiments. On average Our data indicated that Therefore, RFP expression in the stem cell and progenitor compartments is not due to contamination from a platelet or megakaryocyte population.

These immunophenotypic analyses demonstrated that Pf4-Cre exhibits its activity in the most primitive compartment of the adult hematopoietic differentiation hierarchy. Immunophenotypic analyses of fetal liver from These data indicate that the Pf4-Cre transgene is activated in fetal liver HSCs during embryonic development.

Fetal liver from day E The presence of RFP in HSCs and their progeny raised a question of whether the endogenous Pf4 gene might also be expressed in the most primitive hematopoietic cells.

These analyses revealed that endogenous Pf4 transcripts are present in all tested populations of the LSK compartment Fig. However, Tiedt et al [7] and Bertozzi et al [8] have inferred the specificity of the Pf4-Cre to the megakaryocyte lineage through the use of histology and immunostaining. We believe there are technical reasons why these results differ to our results.