Profection mammalian transfection system

Plasmids encoding the human and mouse CAR constructs in a SFG γ-retroviral vector (27 (link)) were used to transfect gpg29 fibroblasts (H29) with the ProFection Mammalian Transfection System (Promega) according to manufacturer’s instructions. The retroviral supernatants were used for transduction of Galv9 293 or Phoenix ECO cell lines to generate stable retroviral particle-producing cell lines. All vectors were generated by restriction enzyme digest and Gibson Assembly (New England BioLabs). For the human CARs, we used human EGFR cDNA without the intracellular domain, a T2A self-cleaving peptide, cDNA of the scFvs, the Flag-tag sequence (DYKDDDDK), the human CD28 transmembrane and intracellular domain, the human 4-1BB intracellular domain, the human CD3ζ intracellular domain, a P2A self-cleaving peptide, and the mature, processed form of human IL-18. For the murine CARs, we used mCherry, a T2A self-cleaving peptide, cDNA of the SC16.8 scFv with Flag-tag, the murine CD28 transmembrane and intracellular domain, the murine CD3ζ intracellular domain, an internal ribosome entry site, a fusion gene encoding the complete murine IL-12 with a serine-glycine repeat between the p35 and p40 chain-coding domains (provided by Alan Houghton and Jedd Wolchok, MSKCC), a P2A self-cleaving peptide, and the mature form of murine IL-18.

Viral stocks containing infectious pseudotyped viral particles were generated by transfection of 5 × 10⁶ HEK293T cells with: (1) 10 μg of lentiviral plasmid encoding LINC00973 (LV2) that was inserted into LEGO-iG2 vector or with an empty plasmid (LV1, used as a control). The transfection mix also included 10 μg of pMDL gag-pol plasmid, 5 μg of REV-encoding plasmid and 2 μg of pVSV-G expression vector. Cells were transfected using ProFection Mammalian Transfection System (Promega, Madison, WI, USA) and plated in a 10-cm Petri dish. Six hours after transfection, the medium was changed to DMEM containing 20 mM HEPES. After 16 h, supernatants containing viral particles were aspirated and filtered using Millex-GP 0.22 μM filter (Merk KGaA, Darmstadt, Germany). Titers were measured as described [34 (link)] and were found to be consistently higher than 5 × 10⁶ units/mL. Supernatants containing VSV-G pseudotyped viral particles and 8 μg/mL hexadimethrin bromide (Sigma-Aldrich, Saint Louis, MO, USA) were used to infect HT-29 cells at MOI = 0.15. Transduced HT-29 with ectopic over-expression of LINC00973 (LV2) and control cells expressing only GFP marker gene (LV1) were obtained by selection using BioRad S3 cell sorter (BIO-RAD, Hercules, CA, USA).

Medium-density hippocampal neurons were prepared from E18 rat embryos and maintained in Neurobasal medium (Invitrogen) containing B-27 supplement (Invitrogen). For Co-IP or biotinylation, dissociated hippocampal neurons were plated on 10 cm dishes coated with poly-l-lysine (PLL); and experiments were done on 14 days in vitro (DIV). For electrophysiology recordings, dissociated hippocampal neuron cultures were plated on glass coverslips coated with PLL and transfected with the plasmids using ProFection® Mammalian Transfection System (Promega) on 11 DIV; and electrophysiological recordings were performed on 13–14 DIV.
For immunostaining surface GluA1 or GluA2, cultured hippocampal neurons were prepared from embryonic day 18. The hippocampal cells were plated on glass coverslips coated with PLL at a density of 300,000 per 60 mm dish. The coverslips were inverted over a feeder layer of astrocytes in Neurobasal medium (Invitrogen) supplemented with B27 (Invitrogen). Cytosine arabinoside (5 μM) was added to neuron culture dishes on 3 DIV to prevent overgrowth of glial cells. Neurons were transfected with YFP-p2a-p97 or YFP as control before plating using nucleofection (AMAXA Biosystems), and immunostaining was performed on 14 DIV.