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Mast cell-specific Cre/loxP-mediated recombination in vivo.

Scholten J, Hartmann K, Gerbaulet A, Krieg T, Müller W, Testa G, Roers A - Transgenic Res. (2007)

Bottom Line: Mast cells are important effectors of type I allergy but also essential regulators of innate and adaptive immune responses.Efficient Cre-mediated recombination was observed in mast cells from the peritoneal cavity and the skin while only minimal reporter gene expression was detected outside the mast cell compartment.Our results show that the Mcpt5 promoter can drive Cre expression in a mast cell-specific fashion.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, University of Cologne, Kerpener Str. 62, 50937, Cologne, Germany.

ABSTRACT
Mast cells are important effectors of type I allergy but also essential regulators of innate and adaptive immune responses. The aim of this study was to develop a Cre recombinase-expressing mouse line that allows mast cell-specific inactivation of genes in vivo. Following a BAC transgenic approach, Cre was expressed under the control of the mast cell protease (Mcpt) 5 promoter. Mcpt5-Cre transgenic mice were crossed to the ROSA26-EYFP Cre excision reporter strain. Efficient Cre-mediated recombination was observed in mast cells from the peritoneal cavity and the skin while only minimal reporter gene expression was detected outside the mast cell compartment. Our results show that the Mcpt5 promoter can drive Cre expression in a mast cell-specific fashion. We expect that our Mcpt5-Cre mice will be a useful tool for the investigation of mast cell biology.

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Efficiency of Cre-mediated reporter gene activation in mast cells. EYFP expression is demonstrated in cells from peritoneal lavage fluid and single cell suspensions of skin of Mcpt5-Cre ROSA26-EYFP double transgenic mice. Peritoneal mast cells were stained for CD117 and FcεRIα (n = 7) and skin mast cells for CD117 and CD45 (n = 4). Mast cell populations were gated and their EYFP fluorescence displayed in histogram plots. The black graph represents Mcpt5-Cre ROSA26-EYFP double transgenic mice, the shaded graph represents the Cre-negative but ROSA26-EYFP-positive control (littermates in most instances)
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Fig2: Efficiency of Cre-mediated reporter gene activation in mast cells. EYFP expression is demonstrated in cells from peritoneal lavage fluid and single cell suspensions of skin of Mcpt5-Cre ROSA26-EYFP double transgenic mice. Peritoneal mast cells were stained for CD117 and FcεRIα (n = 7) and skin mast cells for CD117 and CD45 (n = 4). Mast cell populations were gated and their EYFP fluorescence displayed in histogram plots. The black graph represents Mcpt5-Cre ROSA26-EYFP double transgenic mice, the shaded graph represents the Cre-negative but ROSA26-EYFP-positive control (littermates in most instances)

Mentions: In order to drive Cre expression in mast cells, we used a BAC clone containing the entire Mcpt5 gene along with abundant upstream and downstream flanking DNA (Fig. 1). Neighboring genes were excluded by shortening the BAC on both ends to avoid possible effects of altered gene dosage in the transgenic mice. In a next step, the coding part of the first exon of Mcpt5 was replaced by the Cre cassette (see Methods and Fig. 1). The construct was released from the BAC backbone by NotI digest (Fig. 1) and injected into the pronuclei of C57BL/6 oocytes. Six Mcpt5-Cre transgenic founder animals were obtained and mated to a ROSA26 reporter line (Srinivas et al. 2001) which expresses enhanced yellow fluorescent protein (EYFP) under the control of the ubiquitous ROSA26 promoter following Cre-mediated deletion of a loxP-flanked stop element. Mcpt5-CreROSA26-EYFP double transgenic animals were analyzed for EYFP expression in peritoneal and skin mast cells by FACS as described below (Fig. 2). Reporter gene expression was detected in mast cells from three of the six founder lines. One of these lines showed EYFP expression in only 80% of peritoneal mast cells and was not investigated further. Close to 100% of peritoneal mast cells were EYFP positive in two lines, which, because of autosomal inheritance of the transgene in one line and X-linked inheritance in the other, were called “A-Mcpt5-Cre” and “X-Mcpt5-Cre”, respectively.Fig. 2


Mast cell-specific Cre/loxP-mediated recombination in vivo.

Scholten J, Hartmann K, Gerbaulet A, Krieg T, Müller W, Testa G, Roers A - Transgenic Res. (2007)

Efficiency of Cre-mediated reporter gene activation in mast cells. EYFP expression is demonstrated in cells from peritoneal lavage fluid and single cell suspensions of skin of Mcpt5-Cre ROSA26-EYFP double transgenic mice. Peritoneal mast cells were stained for CD117 and FcεRIα (n = 7) and skin mast cells for CD117 and CD45 (n = 4). Mast cell populations were gated and their EYFP fluorescence displayed in histogram plots. The black graph represents Mcpt5-Cre ROSA26-EYFP double transgenic mice, the shaded graph represents the Cre-negative but ROSA26-EYFP-positive control (littermates in most instances)
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2268725&req=5

Fig2: Efficiency of Cre-mediated reporter gene activation in mast cells. EYFP expression is demonstrated in cells from peritoneal lavage fluid and single cell suspensions of skin of Mcpt5-Cre ROSA26-EYFP double transgenic mice. Peritoneal mast cells were stained for CD117 and FcεRIα (n = 7) and skin mast cells for CD117 and CD45 (n = 4). Mast cell populations were gated and their EYFP fluorescence displayed in histogram plots. The black graph represents Mcpt5-Cre ROSA26-EYFP double transgenic mice, the shaded graph represents the Cre-negative but ROSA26-EYFP-positive control (littermates in most instances)
Mentions: In order to drive Cre expression in mast cells, we used a BAC clone containing the entire Mcpt5 gene along with abundant upstream and downstream flanking DNA (Fig. 1). Neighboring genes were excluded by shortening the BAC on both ends to avoid possible effects of altered gene dosage in the transgenic mice. In a next step, the coding part of the first exon of Mcpt5 was replaced by the Cre cassette (see Methods and Fig. 1). The construct was released from the BAC backbone by NotI digest (Fig. 1) and injected into the pronuclei of C57BL/6 oocytes. Six Mcpt5-Cre transgenic founder animals were obtained and mated to a ROSA26 reporter line (Srinivas et al. 2001) which expresses enhanced yellow fluorescent protein (EYFP) under the control of the ubiquitous ROSA26 promoter following Cre-mediated deletion of a loxP-flanked stop element. Mcpt5-CreROSA26-EYFP double transgenic animals were analyzed for EYFP expression in peritoneal and skin mast cells by FACS as described below (Fig. 2). Reporter gene expression was detected in mast cells from three of the six founder lines. One of these lines showed EYFP expression in only 80% of peritoneal mast cells and was not investigated further. Close to 100% of peritoneal mast cells were EYFP positive in two lines, which, because of autosomal inheritance of the transgene in one line and X-linked inheritance in the other, were called “A-Mcpt5-Cre” and “X-Mcpt5-Cre”, respectively.Fig. 2

Bottom Line: Mast cells are important effectors of type I allergy but also essential regulators of innate and adaptive immune responses.Efficient Cre-mediated recombination was observed in mast cells from the peritoneal cavity and the skin while only minimal reporter gene expression was detected outside the mast cell compartment.Our results show that the Mcpt5 promoter can drive Cre expression in a mast cell-specific fashion.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, University of Cologne, Kerpener Str. 62, 50937, Cologne, Germany.

ABSTRACT
Mast cells are important effectors of type I allergy but also essential regulators of innate and adaptive immune responses. The aim of this study was to develop a Cre recombinase-expressing mouse line that allows mast cell-specific inactivation of genes in vivo. Following a BAC transgenic approach, Cre was expressed under the control of the mast cell protease (Mcpt) 5 promoter. Mcpt5-Cre transgenic mice were crossed to the ROSA26-EYFP Cre excision reporter strain. Efficient Cre-mediated recombination was observed in mast cells from the peritoneal cavity and the skin while only minimal reporter gene expression was detected outside the mast cell compartment. Our results show that the Mcpt5 promoter can drive Cre expression in a mast cell-specific fashion. We expect that our Mcpt5-Cre mice will be a useful tool for the investigation of mast cell biology.

Show MeSH
Related in: MedlinePlus