Stable Overexpression of miRNAs in Bone Marrow-Derived Murine Mast Cells Using Lentiviral Expression Vectors

Abstract

MicroRNAs (miRNAs) constitute a class of molecules regulating gene expression in many different cell types, including cells of the mammalian immune system. Indeed, changes in miRNA expression patterns have been implicated in various physiological and pathological processes. Mast cells (MCs) are hematopoietic cells that originate in the bone marrow and migrate into the tissues, where they mature and reside. They have an important immunoregulatory and effector role in IgE-associated allergic disorders, as well as in certain innate and adaptive immune responses. An effective way to explore the functions of miRNAs in murine MCs includes the modification of miRNA expression in primary bone marrow-derived mast cells (BMMCs), followed by the analysis of the phenotypic consequences of such perturbation. In this chapter, we describe how to differentiate BMMCs and transduce them with lentiviruses. As an example, we expressed miR-221 and miR-222, which showed stable expression in BMMCs and acted as post-transcriptional regulators of c-Kit expression.

Two Functionally Distinct Subsets of Mast Cells Discriminated By IL-2–Independent CD25 Activities

“…To investigate this possibility further, we reconstituted CD25 expression in CD25KO mast cells by lentiviral transduction. As we previously reported (15,33), efficiency of BMMC transduction was routinely 40-50%, as assessed by expression of surface CD25 (Fig. 4D).…”

“…Murine bone marrow-derived mast cells (BMMCs) were differentiated from bone marrow of C57BL/6 or CD25KO mice (14) as described (15) and cultured in the presence of IL-3 as survival/proliferation factor. Cells were fully differentiated after 3-4 wk of culture and were routinely used for experiments between weeks 4 and 9.…”

“…Lentiviral vectors were generated using standard cloning techniques, and BMMCs were transduced at the second week of differentiation exactly as described (15). Murine CD25 was cloned by PCR from T cells activated with anti-CD3 and anti-CD28 Abs.…”

Dnmt3a restrains mast cell inflammatory responses

“…Heterozygous intercrosses were used to generate age-and sex-matched KO and WT littermates for use in all experiments shown. Mast cells were differentiated in the presence of IL-3 as the essential survival and proliferation factor, as described (58). All animal studies were performed in accordance with Swiss Federal Veterinary Office guidelines and approved by the Cantonal animal experimentation committee, Dipartimento della Sanità e della Socialità Cantone Ticino (authorization nos.…”

Histone Deacetylase-3 Mediates Positive Feedback Relationship between Anaphylaxis and Tumor Metastasis

“…miR-221 influences effector functions and actin cytoskeleton in mast cells (46), and it acts as a positive transcriptional regulator of c-kit (47). The loss of miR-21 significantly enhances the Th1-associated delayed-type hypersensitivity cutaneous responses (18).…”