lentiGuide-Puro (Addgene, no. 52963) was digested with Mlu1 and BsiWI. dTomato was amplified from AAV-hSyn1-GCaMP6f-P2A-NLS-dTomato (Addgene, no.51085). HygroR sequence was amplified from lenti MS2-P65-HSF1_Hygro (Addgene, no. 61426). mTagBFP2 was amplified form pBAD-mTagBFP2 (Addgene, no. 3463). The P2A self-cleaving peptide sequence was amplified using a reverse primer of HygroR and forward primer of mTagBFP2. All gRNA sequences are provided in Table S2 .
Pbad mtagbfp2
Manufactured by Addgene
The PBAD-mTagBFP2 is a plasmid that expresses the mTagBFP2 fluorescent protein under the control of the arabinose-inducible PBAD promoter. mTagBFP2 is a bright, photostable blue fluorescent protein that can be used as a reporter or fusion tag.
Automatically generated - may contain errors
Lab products found in correlation
Puromycin dihydrochloride, by Merck Group (2 mentions)
Plenti6.2 mtagbfp2, by Addgene (2 mentions)
Plenti6.2 mneongreen2, by Addgene (2 mentions)
Peipro, by Avantor (2 mentions)
Pmdlg prre, by Addgene (2 mentions)
Prsv rev, by Addgene (2 mentions)
Lentiguide puro, by Addgene (1 mentions)
Lenti ms2 p65 hsf1 hygro, by Addgene (1 mentions)
Hek293ft cells, by Thermo Fisher Scientific (1 mentions)
Pmd2 g, by Addgene (1 mentions)
4 protocols using pbad mtagbfp2
1
Lentiviral Construct Engineering for Fluorescent Imaging
2
AAV-mediated Cerebellar GC Labeling
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AAV vectors with estimated titers of approximately 1013 vector genome copies per mL were produced as described previously [66 (link)]. AAV constructs were made by cloning in plasmids for AAV-GABRα6 that were used in our previous study [66 (link)]. The cDNA fragment for dT was obtained from AAV-CaMKIIa-GCaMP6f-P2A-nls-dTomato (Addgene, #51087) and the fragment of mTB was obtained from pBAD-mTagBFP2 (Addgene, #34632). To trigger expression of fluorescent protein in GCs with PFs located at deep (D-GCs), middle (M-GCs), or superficial (S-GCs) sublayer of the ML, AAV-GABRα6 (about 1.5 μl total) were stereotaxically injected into cerebellar lobe IV/V of the cerebellar vermis at P7, P9, or P13, respectively. When two groups of GCs were labeled, for the second injection, the hole made in the cranium for the first injection was reused. After the surgery, mice were kept on a heating pad until they recovered from the anesthesia and then were returned to their home cages.
3
Lentiviral transduction of α and β cells
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The open reading frames of mTagBFP2 and mNeongreen2 were amplified by PCR from donor vectors pBAD-mTagBFP2 (Addgene, #34632) and pSFFV_mNG2 (11)1–10 (Addgene, #82610) respectively, and ligated in a pLenti6.2 destination vector. Both vectors were verified by Sanger sequencing and deposited to Addgene as pLenti6.2_mTagBFP2 (#113725) and pLenti6.2_mNeonGreen2 (#113727).
Stable cell lines were generated using the lentiviral transduction of α cells and β cells using pLenti6.2_mTagBFP2 and pLenti6.2_mNeonGreen2, respectively. Both plasmids were co-transfected separately with third-generation lentiviral packaging and envelope vectors [pMD2. G (Addgene, #12259), pRSV-Rev (Addgene, #12253), and pMDLg/pRRE (Addgene, #12251)] into HEK-293T cells using the PEIpro (VWR) transfection reagent. After 24 h, the viral supernatant was collected and used to transduce α and β cells. Positive cells were selected 48 h after transduction using 1 μg/ml puromycin dihydrochloride (Sigma-Aldrich) added to growth media for at least 7 days. Transduction efficiency was assessed by fluorescence microscopy after a minimal culture period of 7 days.
Stable cell lines were generated using the lentiviral transduction of α cells and β cells using pLenti6.2_mTagBFP2 and pLenti6.2_mNeonGreen2, respectively. Both plasmids were co-transfected separately with third-generation lentiviral packaging and envelope vectors [pMD2. G (Addgene, #12259), pRSV-Rev (Addgene, #12253), and pMDLg/pRRE (Addgene, #12251)] into HEK-293T cells using the PEIpro (VWR) transfection reagent. After 24 h, the viral supernatant was collected and used to transduce α and β cells. Positive cells were selected 48 h after transduction using 1 μg/ml puromycin dihydrochloride (Sigma-Aldrich) added to growth media for at least 7 days. Transduction efficiency was assessed by fluorescence microscopy after a minimal culture period of 7 days.
4
Distinct Fluorescent Labeling of Pancreatic Alpha and Beta Cells
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Mouse alpha cells were labelled with BFP2 and rat beta cells were labelled with mNeonGreen to distinguish the different cell types. To generate the vectors, the open reading frames of mTagBFP2 and mNeongreen2 were amplified by PCR from donor vectors pBAD-mTagBFP2 (Addgene #34632) and pSFFV_mNG2(11)1-10 (Addgene #82610), respectively, and ligated in a pLenti6.2 destination vector. After verification by Sanger sequencing, they were deposited as pLenti6.2_mTagBFP2 (Addgene #113725) and pLenti6.2_mNeonGreen2 (Addgene #113727). The plasmids were then co-transfected separately with third-generation lentiviral packaging and envelope vectors: pMD2.G (Addgene #12259), pRSV-Rev (Addgene #12253), and pMDLg/pRRE (Addgene #12251) into HEK293ft cells (Thermo Fisher R70007) using the PEIpro (VWR) transfection reagent. After 24 h, the viral supernatant was collected in either alphaTC1 or INS1E medium and filtered. Stable cell lines were generated by diluting the lentivirus 1:10 and 1:20 in cell type–specific medium and transducing the alphaTC1 and INS1E using pLenti6.2_mTagBFP2 and pLenti6.2_mNeonGreen2, respectively. Selection with 1 μg/ml puromycin dihydrochloride (Sigma-Aldrich) began 48 h after transduction and continued for at least 7–14 days. The transduction efficiency was assessed by flow cytometry and was determined to be 66.9% for INS1E cells and 80.4% for alphaTC1 cells.
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