Analysis of human class A scavenger receptor (hSR-A) III transgene RNA expression. (a) Northern blot of isolated macrophage (Mφ) populations. Total RNA was prepared from peritoneal cells isolated from transgenic line III.11 (Tg + ) mice and non-transgenic (Tg – ) littermates, either 1 or 4 days after injection of 1 ml of thioglycollate broth. Bone marrow-derived Mφs (BMDMs) were generated by the culture of bone marrow precursors in medium containing macrophage colony-stimulating factor (M-CSF) and total RNA isolated from adherent cells cultured for 6 days. Total RNA (5 µg) was denatured and separated on a 1% formaldehyde–agarose gel prior to capillary transfer to a nylon membrane. Blots were hybridized with [α- 32 P]dATP-labelled DNA probes for β-actin (a 2-kb fragment of human β-actin; Clontech) or hSR-A III transgene (a 1·2-kb fragment of type III hSR-A coding sequence generated by digestion of expression plasmid pcDNA 3 with Hin dIII and Xba I) and exposed to film. (b) Northern blot of adult tissues. Total tissue RNA (10 µg) prepared using organs collected from transgenic line III.4 (Tg + ) mice and non-transgenic (Tg – ) littermates, and from human monocyte-derived Mφ total RNA (10 µg), was denatured and separated on a 1% formaldehyde–agarose gel prior to transfer and hybridization as described in (a). The different sizes of SR-A hybridizing bands in the human and transgenic RNA samples are caused by the absence of 3′ untranslated sequences in the CD68 SR-A type III transgene. (c) Reverse transcription–polymerase chain reaction (RT–PCR) of adult tissues. Total tissue RNA from organs collected from transgenic line III.4 (Tg + ) mice was reverse transcribed in the presence (+) or absence (–) of Moloney murine leukemia virus reverse transcriptase. cDNA synthesized from 100 ng of total RNA served as a template for subsequent PCR using primers specific for hypoxanthine phosphoribosyl transferase (HPRT), macrosialin or the hSR-A III transgene. To enable a comparison of the relative levels of RNA for macrosialin and the hSR-A III transgene, PCR was performed with both oligonucleotide pairs in the same reaction tube. Primers for HPRT and macrosialin do not generate a detectable reaction product from contaminating genomic DNA (gDNA) under the conditions used, while the product corresponding to the amplification of the hSR-A III transgene genomic DNA is 83 bp larger owing to the presence of the first intron (IVS-1) sequence, which is spliced out during the processing of the primary RNA transcript. Reaction products were separated on a 1·2% agarose gel and visualized by ethidium bromide staining.