Limits...
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-FIRE can transduce primary rat macrophages and EGFP expression is restricted to haemopoietic cells expressing Csf1r. (a) Rat bone marrow (BM) cells were differentiated into macrophages with rhCSF1 then transduced with Csf1r:EGFP-FIRE. Bar = 50 µm. (b) Rat BM cells were cultured in interleukin (IL)-6, IL-3, and SCF and transduced with Csf1r:EGFP-FIRE, then differentiated into macrophages with rhCSF1 and images taken via confocal microscopy on days (i) 0, (ii) 3, and (iii) 7 of differentiation. (iv) A phagocytosis assay was performed on transduced macrophages. Bar = 50 µm. (c) Rat BM cells were cultured in IL-6, FLT3-L, TPO, and SCF and transduced with Csf1r:EGFP-FIRE. After 3 weeks in selection with blasticidin, cells were FACS sorted for EGFP− and EGFP+ populations. (d) mRNA was prepared from sorted EGFP− and EGFP+ populations and used in RT-PCR to analyze Csf1r expression. Each image is representative of two experiments. (e) Murine RAW264.7 cells were transduced with 1 × 105 viral titer units per 3 × 105 cells and FACS analysis performed for EGFP expression following blasticidin selection. Histogram is representative of three independent experiments. RT-PCR, reverse transcriptase PCR; SCF, stem cell factor; TPO, thrombopoietin.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4362345&req=5

fig3: Csf1r:EGFP-FIRE can transduce primary rat macrophages and EGFP expression is restricted to haemopoietic cells expressing Csf1r. (a) Rat bone marrow (BM) cells were differentiated into macrophages with rhCSF1 then transduced with Csf1r:EGFP-FIRE. Bar = 50 µm. (b) Rat BM cells were cultured in interleukin (IL)-6, IL-3, and SCF and transduced with Csf1r:EGFP-FIRE, then differentiated into macrophages with rhCSF1 and images taken via confocal microscopy on days (i) 0, (ii) 3, and (iii) 7 of differentiation. (iv) A phagocytosis assay was performed on transduced macrophages. Bar = 50 µm. (c) Rat BM cells were cultured in IL-6, FLT3-L, TPO, and SCF and transduced with Csf1r:EGFP-FIRE. After 3 weeks in selection with blasticidin, cells were FACS sorted for EGFP− and EGFP+ populations. (d) mRNA was prepared from sorted EGFP− and EGFP+ populations and used in RT-PCR to analyze Csf1r expression. Each image is representative of two experiments. (e) Murine RAW264.7 cells were transduced with 1 × 105 viral titer units per 3 × 105 cells and FACS analysis performed for EGFP expression following blasticidin selection. Histogram is representative of three independent experiments. RT-PCR, reverse transcriptase PCR; SCF, stem cell factor; TPO, thrombopoietin.

Mentions: We currently have Csf1r-EGFP-positive transgenic mice, which have been widely used.12 A similar reporter in rat would have great utility, but rat transgenesis is less straightforward and lentiviral delivery would provide an alternative route. The Csf1r:EGFP-FIRE lentivirus was also active in primary cells and in the rat. Rat BM-derived macrophages were transduced with the lentivirus and the majority of transduced cells were EGFP+ as determined by fluorescent microscopy (Figure 3a). We therefore wished to determine whether the virus, once integrated into progenitors, would maintain expression during differentiation. Rat BM cells were cultured for 2 days in IL-3, IL-6, and stem cell factor (SCF) to support myeloid progenitors and then transduced with Csf1r:EGFP-FIRE. Two days following transduction, growth factors were replaced with rhCSF1 to allow macrophage development. Prior to addition of rhCSF1, few cells were EGFP+ (Figure 3b, i), but as the cells differentiated into macrophages, the number of EGFP+ increased. After 7 days, the large majority of cells were EGFP+ (Figure 3b, iii) and were confirmed as functional macrophages by their ability to phagocytose fluorescently labeled zymosan particles (Figure 3b, iv).


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-FIRE can transduce primary rat macrophages and EGFP expression is restricted to haemopoietic cells expressing Csf1r. (a) Rat bone marrow (BM) cells were differentiated into macrophages with rhCSF1 then transduced with Csf1r:EGFP-FIRE. Bar = 50 µm. (b) Rat BM cells were cultured in interleukin (IL)-6, IL-3, and SCF and transduced with Csf1r:EGFP-FIRE, then differentiated into macrophages with rhCSF1 and images taken via confocal microscopy on days (i) 0, (ii) 3, and (iii) 7 of differentiation. (iv) A phagocytosis assay was performed on transduced macrophages. Bar = 50 µm. (c) Rat BM cells were cultured in IL-6, FLT3-L, TPO, and SCF and transduced with Csf1r:EGFP-FIRE. After 3 weeks in selection with blasticidin, cells were FACS sorted for EGFP− and EGFP+ populations. (d) mRNA was prepared from sorted EGFP− and EGFP+ populations and used in RT-PCR to analyze Csf1r expression. Each image is representative of two experiments. (e) Murine RAW264.7 cells were transduced with 1 × 105 viral titer units per 3 × 105 cells and FACS analysis performed for EGFP expression following blasticidin selection. Histogram is representative of three independent experiments. RT-PCR, reverse transcriptase PCR; SCF, stem cell factor; TPO, thrombopoietin.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig3: Csf1r:EGFP-FIRE can transduce primary rat macrophages and EGFP expression is restricted to haemopoietic cells expressing Csf1r. (a) Rat bone marrow (BM) cells were differentiated into macrophages with rhCSF1 then transduced with Csf1r:EGFP-FIRE. Bar = 50 µm. (b) Rat BM cells were cultured in interleukin (IL)-6, IL-3, and SCF and transduced with Csf1r:EGFP-FIRE, then differentiated into macrophages with rhCSF1 and images taken via confocal microscopy on days (i) 0, (ii) 3, and (iii) 7 of differentiation. (iv) A phagocytosis assay was performed on transduced macrophages. Bar = 50 µm. (c) Rat BM cells were cultured in IL-6, FLT3-L, TPO, and SCF and transduced with Csf1r:EGFP-FIRE. After 3 weeks in selection with blasticidin, cells were FACS sorted for EGFP− and EGFP+ populations. (d) mRNA was prepared from sorted EGFP− and EGFP+ populations and used in RT-PCR to analyze Csf1r expression. Each image is representative of two experiments. (e) Murine RAW264.7 cells were transduced with 1 × 105 viral titer units per 3 × 105 cells and FACS analysis performed for EGFP expression following blasticidin selection. Histogram is representative of three independent experiments. RT-PCR, reverse transcriptase PCR; SCF, stem cell factor; TPO, thrombopoietin.
Mentions: We currently have Csf1r-EGFP-positive transgenic mice, which have been widely used.12 A similar reporter in rat would have great utility, but rat transgenesis is less straightforward and lentiviral delivery would provide an alternative route. The Csf1r:EGFP-FIRE lentivirus was also active in primary cells and in the rat. Rat BM-derived macrophages were transduced with the lentivirus and the majority of transduced cells were EGFP+ as determined by fluorescent microscopy (Figure 3a). We therefore wished to determine whether the virus, once integrated into progenitors, would maintain expression during differentiation. Rat BM cells were cultured for 2 days in IL-3, IL-6, and stem cell factor (SCF) to support myeloid progenitors and then transduced with Csf1r:EGFP-FIRE. Two days following transduction, growth factors were replaced with rhCSF1 to allow macrophage development. Prior to addition of rhCSF1, few cells were EGFP+ (Figure 3b, i), but as the cells differentiated into macrophages, the number of EGFP+ increased. After 7 days, the large majority of cells were EGFP+ (Figure 3b, iii) and were confirmed as functional macrophages by their ability to phagocytose fluorescently labeled zymosan particles (Figure 3b, iv).

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