koerber  (Addgene inc)

Journal: Scientific Reports

Article Title: Development and characterization of in vitro inducible immortalization of a murine microglia cell line for high throughput studies

doi: 10.1038/s41598-025-87543-1

Figure Lengend Snippet: Primary mouse microglia can be immortalized through HRAS and CMYC transcriptional factors. ( a ) Timeline depicting the transduction of primary CD1 E17.5 microglial cells at DIV4, followed by doxycycline induction of CMYC T58A and HRAS G12V at DIV5. ( b ) Immunocytochemistry results showing IBA1 + (Green) non-transduced control microglia, indicating a high-purity microglial culture. ( c ) Live imaging of cell confluence using IncuCyte for microglial cells transduced with TET-O-CMYC T58A-HRAS G12V with doxycycline (DOX), compared to non-transduced primary microglia at DIV16-23. ( d ) FACS gating strategy for isolating CD11B + LY6C – cells for microglial single-cell colony expansion. ( e ) Relative expression levels of microglial genes C1qa and P2ry12 , as well as human oncogenes CMYC and HRAS, normalized to Gadph. This was assessed using qRT-PCR on CD11b + LY6C − microglial cells, comparing control and transduced TET-O-CMYC T58A-HRAS G12V with DOX microglial cells. Statistical significance was evaluated using an unpaired t-test ( N = 3 replicates), with bars representing mean ± SEM. ( f ) Normalized counts per million (norm CPM) of murine proliferation genes Myc , Hras , Mki67 , Mcm2 and Pcna in primary MG and 2E11 clonal line without doxycycline (2E11 CTRL) and with doxycycline (2E11 DOX) from RNA-sequencing of total mRNA.

Article Snippet: A mutant form of HRAS G12V, containing a P2A sequence, was synthesized by GenScript based on the published HRAS G12V sequence . pUC57 plasmid containing HRAS G12V was subsequently cloned into a lentiviral vector with a tetracycline response element with CMYC T58A (19775, Addgene) using BsaBI and Xba1 restriction enzymes.

Techniques: Transduction, Immunocytochemistry, Control, Imaging, Expressing, Quantitative RT-PCR, RNA Sequencing

Journal: Scientific Reports

Article Title: Development and characterization of in vitro inducible immortalization of a murine microglia cell line for high throughput studies

doi: 10.1038/s41598-025-87543-1

Figure Lengend Snippet: Comparison of commonly used microglia cell lines examining microglial markers and function in vitro. Table modified from Stansley, Post, and Hensley (2012).

Article Snippet: A mutant form of HRAS G12V, containing a P2A sequence, was synthesized by GenScript based on the published HRAS G12V sequence . pUC57 plasmid containing HRAS G12V was subsequently cloned into a lentiviral vector with a tetracycline response element with CMYC T58A (19775, Addgene) using BsaBI and Xba1 restriction enzymes.

Techniques: Comparison, In Vitro, Modification, Transformation Assay

Journal: Scientific Reports

Article Title: Development and characterization of in vitro inducible immortalization of a murine microglia cell line for high throughput studies

doi: 10.1038/s41598-025-87543-1

Figure Lengend Snippet: Experimental conditions tested for immortalization of microglia of human and mouse.

Article Snippet: A mutant form of HRAS G12V, containing a P2A sequence, was synthesized by GenScript based on the published HRAS G12V sequence . pUC57 plasmid containing HRAS G12V was subsequently cloned into a lentiviral vector with a tetracycline response element with CMYC T58A (19775, Addgene) using BsaBI and Xba1 restriction enzymes.

Techniques: Concentration Assay

Journal: Scientific Reports

Article Title: Development and characterization of in vitro inducible immortalization of a murine microglia cell line for high throughput studies

doi: 10.1038/s41598-025-87543-1

Figure Lengend Snippet: Primary mouse microglia can be immortalized through HRAS and CMYC transcriptional factors. ( a ) Timeline depicting the transduction of primary CD1 E17.5 microglial cells at DIV4, followed by doxycycline induction of CMYC T58A and HRAS G12V at DIV5. ( b ) Immunocytochemistry results showing IBA1 + (Green) non-transduced control microglia, indicating a high-purity microglial culture. ( c ) Live imaging of cell confluence using IncuCyte for microglial cells transduced with TET-O-CMYC T58A-HRAS G12V with doxycycline (DOX), compared to non-transduced primary microglia at DIV16-23. ( d ) FACS gating strategy for isolating CD11B + LY6C – cells for microglial single-cell colony expansion. ( e ) Relative expression levels of microglial genes C1qa and P2ry12 , as well as human oncogenes CMYC and HRAS, normalized to Gadph. This was assessed using qRT-PCR on CD11b + LY6C − microglial cells, comparing control and transduced TET-O-CMYC T58A-HRAS G12V with DOX microglial cells. Statistical significance was evaluated using an unpaired t-test ( N = 3 replicates), with bars representing mean ± SEM. ( f ) Normalized counts per million (norm CPM) of murine proliferation genes Myc , Hras , Mki67 , Mcm2 and Pcna in primary MG and 2E11 clonal line without doxycycline (2E11 CTRL) and with doxycycline (2E11 DOX) from RNA-sequencing of total mRNA.

Article Snippet: The lentiviral TET-O-CMYC T58A-P2A-HRAS G12V and rTTA plasmids (19780, Addgene) were amplified using SURE2 Super competent cells (200152, Agilent) to produce stable plasmids.

Techniques: Transduction, Immunocytochemistry, Control, Imaging, Expressing, Quantitative RT-PCR, RNA Sequencing

Journal: Scientific Reports

Article Title: Development and characterization of in vitro inducible immortalization of a murine microglia cell line for high throughput studies

doi: 10.1038/s41598-025-87543-1

Figure Lengend Snippet: Experimental conditions tested for immortalization of microglia of human and mouse.

Article Snippet: The lentiviral TET-O-CMYC T58A-P2A-HRAS G12V and rTTA plasmids (19780, Addgene) were amplified using SURE2 Super competent cells (200152, Agilent) to produce stable plasmids.

Techniques: Concentration Assay