Peipro

The plasmid pLKO.1 containing short hairpin RNA (shRNA) sequences targeting cadherin-11 was obtained from Sigma-Aldrich together with a scrambled negative control. These plasmids were co-transfected with third generation lentiviral packaging and envelope vectors; pMD2.G, pRSV-Rev, and pMDLg/pRRE (Addgene plasmid #12259, #12253, and #12251, respectively, which were gifts from Didier Trono^{15 (link)}), into HEK-293T cells using PEIpro (VWR) transfection reagent. Lentiviral particles were harvested 48 and 72 h after transfection. The harvested viral supernatant was filtered and stored at −80 °C until use. Five milliliters of viral supernatant were used to transduce hMSCs seeded at 5000 cells/cm² in a T225 flask and incubated for 48 h. After transduction, positive cells were selected with 2 µg/mL puromycin dihydrochloride (Sigma-Aldrich) in growth media for 7 days and were then used for subsequent experiments.

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.

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.