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Lentiviral vectors containing mouse Csf1r control elements direct macrophage-restricted expression in multiple species of birds and mammals.

Pridans C, Lillico S, Whitelaw B, Hume DA - Mol Ther Methods Clin Dev (2014)

Bottom Line: We have created a lentiviral construct containing mouse FIRE and promoter.The lentivirus is capable of directing macrophage-restricted reporter gene expression in mouse, rat, human, pig, cow, sheep, and even chicken.Rat bone marrow cells transduced with the lentivirus were capable of differentiating into macrophages expressing the reporter gene in vitro.

View Article: PubMed Central - PubMed

Affiliation: The Roslin Institute, Royal (Dick) School of Veterinary Studies, University of Edinburgh, Scotland , UK.

ABSTRACT
The development of macrophages requires signaling through the lineage-restricted receptor Csf1r. Macrophage-restricted expression of transgenic reporters based upon Csf1r requires the highly conserved Fms-intronic regulatory element (FIRE). We have created a lentiviral construct containing mouse FIRE and promoter. The lentivirus is capable of directing macrophage-restricted reporter gene expression in mouse, rat, human, pig, cow, sheep, and even chicken. Rat bone marrow cells transduced with the lentivirus were capable of differentiating into macrophages expressing the reporter gene in vitro. Macrophage-restricted expression may be desirable for immunization or immune response modulation, and for gene therapy for lysosomal storage diseases and some immunodeficiencies. The small size of the Csf1r transcription control elements will allow the insertion of large "cargo" for applications in gene therapy and vaccine delivery.

No MeSH data available.


Related in: MedlinePlus

Csf1r:EGFP lentiviral constructs. Schematic of murine Csf1r:EGFP constructs. All constructs contained a blasticidin-resistance gene (BSD) driven by the EM7/SV40 promoter. (a) Csf1r:EGFP-FIRE (b) Csf1r:EGFP-sFIRE (c) Csf1r:myr:EGFP-FIRE. cPPT, central polypurine tract; FIRE, Fms-Intronic Regulatory Element; ψ, HIV-1 packaging signal; LTR, long terminal repeat; RRE, rev response elements; sFIRE, short FIRE; myr, myristoylated motif.
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fig1: Csf1r:EGFP lentiviral constructs. Schematic of murine Csf1r:EGFP constructs. All constructs contained a blasticidin-resistance gene (BSD) driven by the EM7/SV40 promoter. (a) Csf1r:EGFP-FIRE (b) Csf1r:EGFP-sFIRE (c) Csf1r:myr:EGFP-FIRE. cPPT, central polypurine tract; FIRE, Fms-Intronic Regulatory Element; ψ, HIV-1 packaging signal; LTR, long terminal repeat; RRE, rev response elements; sFIRE, short FIRE; myr, myristoylated motif.

Mentions: Our recent study suggested that the function of FIRE, in situ, is position and orientation dependent,13 but it has previously been reported to have conventional enhancer activity.14 In order to drive expression of enhanced green fluorescent protein (EGFP) specifically in monocytes and macrophages, the Csf1r promoter (including start codon) was fused to the coding sequence of EGFP and cloned upstream of FIRE in a lentiviral vector (Figure 1). mCherry constructs (data not shown) were also created as well as one in which expression of EGFP or mCherry was localized to membranes using a myristoylated motif (Figure 1c). The specificity of the lentivirus was initially tested by transduction of the murine cell lines EL4 (T lymphocyte) and RAW264.7 (monocyte/macrophage) with Csf1r:EGFP-FIRE. After 10 days of selection in blasticidin, only RAW264.7 cells expressed EGFP when viewed by confocal microscopy (Figure 2a). Cells transduced with Csf1r:myr:EGFP-FIRE displayed EGFP expression at slightly lower levels via flow cytometry (Figure 2b). However, in these cells, as anticipated, the EGFP was only localized to the cell membrane and also to membranes of intracellular vesicles (Figure 2c). Lentiviral transduction of RAW264.7 cells with the alternative vector containing mCherry also resulted in reporter expression as detected by confocal microscopy (Figure 2c). In summary, with all reporters, the expression was not only restricted to macrophages, but was sufficient to enable ready visualization.


Lentiviral vectors containing mouse Csf1r control elements direct macrophage-restricted expression in multiple species of birds and mammals.

Pridans C, Lillico S, Whitelaw B, Hume DA - Mol Ther Methods Clin Dev (2014)

Csf1r:EGFP lentiviral constructs. Schematic of murine Csf1r:EGFP constructs. All constructs contained a blasticidin-resistance gene (BSD) driven by the EM7/SV40 promoter. (a) Csf1r:EGFP-FIRE (b) Csf1r:EGFP-sFIRE (c) Csf1r:myr:EGFP-FIRE. cPPT, central polypurine tract; FIRE, Fms-Intronic Regulatory Element; ψ, HIV-1 packaging signal; LTR, long terminal repeat; RRE, rev response elements; sFIRE, short FIRE; myr, myristoylated motif.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4362345&req=5

fig1: Csf1r:EGFP lentiviral constructs. Schematic of murine Csf1r:EGFP constructs. All constructs contained a blasticidin-resistance gene (BSD) driven by the EM7/SV40 promoter. (a) Csf1r:EGFP-FIRE (b) Csf1r:EGFP-sFIRE (c) Csf1r:myr:EGFP-FIRE. cPPT, central polypurine tract; FIRE, Fms-Intronic Regulatory Element; ψ, HIV-1 packaging signal; LTR, long terminal repeat; RRE, rev response elements; sFIRE, short FIRE; myr, myristoylated motif.
Mentions: Our recent study suggested that the function of FIRE, in situ, is position and orientation dependent,13 but it has previously been reported to have conventional enhancer activity.14 In order to drive expression of enhanced green fluorescent protein (EGFP) specifically in monocytes and macrophages, the Csf1r promoter (including start codon) was fused to the coding sequence of EGFP and cloned upstream of FIRE in a lentiviral vector (Figure 1). mCherry constructs (data not shown) were also created as well as one in which expression of EGFP or mCherry was localized to membranes using a myristoylated motif (Figure 1c). The specificity of the lentivirus was initially tested by transduction of the murine cell lines EL4 (T lymphocyte) and RAW264.7 (monocyte/macrophage) with Csf1r:EGFP-FIRE. After 10 days of selection in blasticidin, only RAW264.7 cells expressed EGFP when viewed by confocal microscopy (Figure 2a). Cells transduced with Csf1r:myr:EGFP-FIRE displayed EGFP expression at slightly lower levels via flow cytometry (Figure 2b). However, in these cells, as anticipated, the EGFP was only localized to the cell membrane and also to membranes of intracellular vesicles (Figure 2c). Lentiviral transduction of RAW264.7 cells with the alternative vector containing mCherry also resulted in reporter expression as detected by confocal microscopy (Figure 2c). In summary, with all reporters, the expression was not only restricted to macrophages, but was sufficient to enable ready visualization.

Bottom Line: We have created a lentiviral construct containing mouse FIRE and promoter.The lentivirus is capable of directing macrophage-restricted reporter gene expression in mouse, rat, human, pig, cow, sheep, and even chicken.Rat bone marrow cells transduced with the lentivirus were capable of differentiating into macrophages expressing the reporter gene in vitro.

View Article: PubMed Central - PubMed

Affiliation: The Roslin Institute, Royal (Dick) School of Veterinary Studies, University of Edinburgh, Scotland , UK.

ABSTRACT
The development of macrophages requires signaling through the lineage-restricted receptor Csf1r. Macrophage-restricted expression of transgenic reporters based upon Csf1r requires the highly conserved Fms-intronic regulatory element (FIRE). We have created a lentiviral construct containing mouse FIRE and promoter. The lentivirus is capable of directing macrophage-restricted reporter gene expression in mouse, rat, human, pig, cow, sheep, and even chicken. Rat bone marrow cells transduced with the lentivirus were capable of differentiating into macrophages expressing the reporter gene in vitro. Macrophage-restricted expression may be desirable for immunization or immune response modulation, and for gene therapy for lysosomal storage diseases and some immunodeficiencies. The small size of the Csf1r transcription control elements will allow the insertion of large "cargo" for applications in gene therapy and vaccine delivery.

No MeSH data available.


Related in: MedlinePlus