Lentivirus system transfection was performed using Lenti-Pac HIV Expression Packaging Kit (GeneCopoeia, Rockville, MD, USA) according to the manufacturer's instructions.
Packaging of the miR-29b constructs in pseudoviral particles was performed in 293Ta cells using the Lenti-Pac FIV Expression Packaging Kit (FPK-LvTR-20), according to the manufacturer's instructions (Genecopoeia, Rockville, MD, USA).
AAV-eGFP and AAV-Channelrhodopsin2(H134R -eYFP consisted of AAV-2-ITRs, tH134R -eYFPnapsin 1 gene promoter driving transgene expression, paconsisted AAV-6 capsids (AAV2/6 eGFP; AAV2/6 ChR2-eYFP).
Lenti-Pac HIV Expression Packaging mix (GeneCopoeia™), pLV-miR-137 or pLV-miR-NC were cotransfected into the 293Ta cells using EndoFectin Lenti transfection reagent and following the manufacturer's instructions.
Following the manufacturer's instructions, the CNE-2 and SUNE-1 cell lines were stably transfected using a Lenti-Pac™ HIV Expression Packaging Kit (GeneCopoeia, Rockville, MD, USA) and a plasmid encoding BRMS1 or a control vector plasmid.
Unfortunately, implicit in this model is the inability to make conclusions about changes in expression, packaging, and secretion of Paneth cell products in NEC (given the intentional damage to the Paneth cells).
Pseudo lentiviral particles for TK1 overexpression and the control were produced with Lv-TK1 or the control plasmid and Lenti-Pac HIV expression packaging kit (GeneCopoeia, USA) on 293T cells according to manufacturer's protocol.
The GDNF gene expression cassette vector and Lenti-Pac HIV Expression Packaging Kits (GeneCopoeia, Rockville, MD, USA), containing an HIV packaging mix of the three packaging plasmids, were cotransfected using EndoFectin Lenti transfection reagent into 293Ta cells to package capable lentiviruses, and were defined as pseudovirion siGDNF.
To express Brms1 in MB49 cells, Brms1/ORF (cat#: EX-Mm13102-Lv23), control vector with GFP (cat#: EX-EGFP-Lv23) and a lenti-pac FIV (cat#: FPK-LvTR-20) expression packaging kit were purchased from GeneCopeia to construct viral particles (Meyers-Needham et al, 2012; Salas et al, 2011).
In addition, we would hope to improve SuRFR's flexibility by i) linking it in with other R packages (for example, next-generation sequencing packages and methylation and expression analysis packages), and ii) provide additional utility for user customisation.
