Briefly, Phoenix Eco retroviral packaging cells (Orbigen), purchased within the last 2 years, were transfected with pMKO.1 puro PTEN shRNA or pMKO.1 puro EV retroviral expression vectors (Addgene Inc ., and supernatant containing recombinant retroviral vectors encoding PTEN shRNA or EV only was collected after 48 h.
By developing the assay in a clonal cell line in which stable lines were first generated from the receptor subtype followed by plasmid transfection, or transduction with a viral vector containing the recombinant DNA encoded sensor, one can robustly generate previously established receptor subtype clones containing the FRET based sensor.
After host cells containing recombinant expression vectors in LB-medium at 37°C reached an optical density (OD) of 0.5 0.8 at 600 nm, isopropyl-beta-D-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM to induce bacterial production of recombinant proteins.
Proteins were produced by Escherichia coli cells transformed with the prokaryotic expression vector pET30a, namely empty vector, or the recombinant vector containing an insert of the X. oryzae hpa1 gene or its truncated version hpa1 30–126 coding for the Hpa110 42 protein.
Also, the correct size of 1125 bp of the PCR product (dhaD gene), obtained from the recombinant vector containing the dhaD gene using forward and reverse primers, was confirmed by sequencing.
Recombinant vector containing inserts was transformed into competent Escherichia coli cells.
Lentiviruses were produced as previously described using the FCK(1.3 GW vector containing a 1.3-kb recombinant promoter of the mouse alpha-calcium/calmodulin-dependent protein kinase II (α-CaMKII) gene (Dittgen et al. 2004); this promoter drives expression specifically in pyramidal neurons (the vector backbone is based on a construct FUGW, described in Lois et al. 2002).
In vitro the single-strand recombinant phagemid vector containing the combination sequence exhibited 10 23 deoxyribozyme activity, and the linear transcript displayed hammerhead ribozyme activity.
To achieve this, mesenchymal stem cells (MSC) from the bone marrow of rabbit mandibles were isolated and genetically engineered using recombinant lentiviral vector containing hNGFβ.
