A second rBCG strain, designated AFRO-1, was generated by incorporating an expression plasmid encoding three mycobacterial antigens, Ag85A, Ag85B and TB10.4, into AERAS-401.
The AFRO-1 strain was generated by incorporating an expression plasmid encoding for three mycobacterial antigens, Ag85A (GenBank accession number P0A4V2), Ag85B (GenBank accession number P12942) and TB10.4 (GenBank accession number AF2122/97) into the Pfo-expressing BCG strain AERAS-401.
We extended the functional analyses by creating an expression plasmid encoding for the CTF only.
The specificity of our system was checked by transfecting an expression plasmid for fsNOD2, a mutant unable to activate NF-κB in response to MDP.
Functional validations were performed either by siRNAs or by transfection of an expression plasmid.
The response to TNF-α was restored by transfection with an expression plasmid containing a p47phox cDNA inserted in the sense direction.
The miR-433/127 loci cloned from human, rat, or dog was overexpressed in mouse Hepa-1 cells by transfection of an expression plasmid containing the loci of each species (Figure S2, see below description), respectively.
Transfection efficiency was followed by cotransfection with an expression plasmid coding for green fluorescent protein (GFP, pEGFP-N1) at a pcDNA3.1+with insert:pEGFP-N1ratio of 1 10.
BFTC-173-12 cells were derived from BFTC-905 cells by transfection with V173L, an expression plasmid of a dominant-negative mutant p53 with a substitution of leucine for valine at codon 173 (Chen et al, 1993), followed by selection of stable transfectants with G418 (Chang and Lai, 2001).
Once recombinant monoclonal antibodies with the desired specificity have been obtained by screening, the corresponding V genes can be inserted into an expression plasmid by TS-HR, which can in turn be used for large-scale antibody production.
