Allele-specific oligonucleotide hybridization detects transgene expression in RAW 264.7 macrophages
RNA prepared from macrophages, brain or spleen tissues from B10.L-Lshr congenic mice or RAW 264.7 transfectants following expansion were treated with DNAase, reverse transcribed and a specific Nramp fragment covering the susceptibility-associated mutation prepared by PCR. Aliquots of the PCRs were alkali-denatured, spotted in duplicate (1 and 2) onto membrane and probed with oligonucleotides corresponding to either the resistant (R) or susceptible (S) sequences. Posthybridization washing conditions were selected to achieve specificity for allelic forms. In Panel (a), a series of antibiotic transfectants clones were analyzed and a subset selected (b) to confirm that the resistant signal was derived from expressed RNA (+RT) rather than contaminating expression vector-derived DNA (−RT). As a positive resistant control, cDNA from B10.L-Lshr macrophages was used, resulting in positive hybridization in the RT negative control. Subclones from the strongest expressing clones from Panel (a) were analysed in Panel (c), including several clones prepared with a susceptible allele Nramp expression construct.