Using the Sleeping Beauty Transposon System for Doxycycline-inducible Gene Expression in RAW264.7 Macrophage Cells to Study Phagocytosis
Macrophages are known for engulfing and digesting pathogens and dead cells through a specialized form of endocytosis called phagocytosis. Unfortunately, many macrophage cell lines are refractory to most reagents used for transient transfections. Alternative transient approaches, such as electroporat...
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Bio-protocol LLC
2025-02-01
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| author | Parsa Kamali Gregory Fairn |
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| description | Macrophages are known for engulfing and digesting pathogens and dead cells through a specialized form of endocytosis called phagocytosis. Unfortunately, many macrophage cell lines are refractory to most reagents used for transient transfections. Alternative transient approaches, such as electroporation or transduction with lentiviral vectors, typically cause cell death (electroporation) or can be time-consuming to generate numerous lentivirus when using different genes of interest. Therefore, we use the Sleeping Beauty system to generate stably transfected cells. The system uses a “resurrected” transposase gene named Sleeping Beauty found in salmonid fish. Experimentally, the system introduces two plasmids: one carrying the Sleeping Beauty transposase and the other with an integration cassette carrying the gene of interest, a reverse-doxycycline controlled repressor gene, and an antibiotic resistance gene. The construct used in this protocol provides puromycin resistance. Stable integrations are selected by culturing the cells in the presence of puromycin, and further enrichment can be obtained using fluorescence-activated cell sorting (FACS). In this protocol, we use the Sleeping Beauty transposon system to generate RAW264.7 cells with doxycycline-inducible inositol polyphosphate 4-phosphatase B containing a C-terminal CaaX motif (INPP4B-CaaX). INPP4B-CaaX dephosphorylates the D-4 position of phosphatidylinositol 3,4-bisphosphate and inhibits phagocytosis. One benefit is that generating stable cell lines is substantially faster than selecting for random integrations. Without FACS, the method typically gives ~50% of the cells that are transfected; with sorting, this approaches 100%. This makes phagocytosis experiments easier since more cells can be analyzed per experiment, allowing for population-based measurements where a ~10% transient transfection rate is insufficient. Finally, using the doxycycline-promoter allows for low near endogenous expression of proteins or robust overexpression. |
| format | Article |
| id | doaj-art-549e36f2a2f842e78b58a71cd6f1404e |
| institution | Kabale University |
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| language | English |
| publishDate | 2025-02-01 |
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| spelling | doaj-art-549e36f2a2f842e78b58a71cd6f1404e2025-02-07T08:16:46ZengBio-protocol LLCBio-Protocol2331-83252025-02-0115310.21769/BioProtoc.5178Using the Sleeping Beauty Transposon System for Doxycycline-inducible Gene Expression in RAW264.7 Macrophage Cells to Study PhagocytosisParsa Kamali0Gregory Fairn1Dept of Biochemistry and Molecular Biology, Dalhousie University, Halifax, NS, CanadaDept of Biochemistry and Molecular Biology, Dalhousie University, Halifax, NS, CanadaDept of Pathology, Dalhousie University, Halifax, NS, CanadaMacrophages are known for engulfing and digesting pathogens and dead cells through a specialized form of endocytosis called phagocytosis. Unfortunately, many macrophage cell lines are refractory to most reagents used for transient transfections. Alternative transient approaches, such as electroporation or transduction with lentiviral vectors, typically cause cell death (electroporation) or can be time-consuming to generate numerous lentivirus when using different genes of interest. Therefore, we use the Sleeping Beauty system to generate stably transfected cells. The system uses a “resurrected” transposase gene named Sleeping Beauty found in salmonid fish. Experimentally, the system introduces two plasmids: one carrying the Sleeping Beauty transposase and the other with an integration cassette carrying the gene of interest, a reverse-doxycycline controlled repressor gene, and an antibiotic resistance gene. The construct used in this protocol provides puromycin resistance. Stable integrations are selected by culturing the cells in the presence of puromycin, and further enrichment can be obtained using fluorescence-activated cell sorting (FACS). In this protocol, we use the Sleeping Beauty transposon system to generate RAW264.7 cells with doxycycline-inducible inositol polyphosphate 4-phosphatase B containing a C-terminal CaaX motif (INPP4B-CaaX). INPP4B-CaaX dephosphorylates the D-4 position of phosphatidylinositol 3,4-bisphosphate and inhibits phagocytosis. One benefit is that generating stable cell lines is substantially faster than selecting for random integrations. Without FACS, the method typically gives ~50% of the cells that are transfected; with sorting, this approaches 100%. This makes phagocytosis experiments easier since more cells can be analyzed per experiment, allowing for population-based measurements where a ~10% transient transfection rate is insufficient. Finally, using the doxycycline-promoter allows for low near endogenous expression of proteins or robust overexpression.https://bio-protocol.org/en/bpdetail?id=5178&type=0 |
| spellingShingle | Parsa Kamali Gregory Fairn Using the Sleeping Beauty Transposon System for Doxycycline-inducible Gene Expression in RAW264.7 Macrophage Cells to Study Phagocytosis Bio-Protocol |
| title | Using the Sleeping Beauty Transposon System for Doxycycline-inducible Gene Expression in RAW264.7 Macrophage Cells to Study Phagocytosis |
| title_full | Using the Sleeping Beauty Transposon System for Doxycycline-inducible Gene Expression in RAW264.7 Macrophage Cells to Study Phagocytosis |
| title_fullStr | Using the Sleeping Beauty Transposon System for Doxycycline-inducible Gene Expression in RAW264.7 Macrophage Cells to Study Phagocytosis |
| title_full_unstemmed | Using the Sleeping Beauty Transposon System for Doxycycline-inducible Gene Expression in RAW264.7 Macrophage Cells to Study Phagocytosis |
| title_short | Using the Sleeping Beauty Transposon System for Doxycycline-inducible Gene Expression in RAW264.7 Macrophage Cells to Study Phagocytosis |
| title_sort | using the sleeping beauty transposon system for doxycycline inducible gene expression in raw264 7 macrophage cells to study phagocytosis |
| url | https://bio-protocol.org/en/bpdetail?id=5178&type=0 |
| work_keys_str_mv | AT parsakamali usingthesleepingbeautytransposonsystemfordoxycyclineinduciblegeneexpressioninraw2647macrophagecellstostudyphagocytosis AT gregoryfairn usingthesleepingbeautytransposonsystemfordoxycyclineinduciblegeneexpressioninraw2647macrophagecellstostudyphagocytosis |