Distinction of Mouse Interferon-α Subtypes by Polymerase Chain Reaction Utilizing Consensus Primers and Type-Specific Oligonucleotide Probes

Abstract

We have developed a novel method to study the subtype-specific expression of interferon-α (IFN-α) in the mouse system: we synthesized and used consensus oligonucleotide primers to allow simultaneous polymerase chain reaction (PCR) amplification of multiple murine IFN-α gene sequences. In addition, a set of subtype-specific oligomer probes were designed and used to distinguish between IFN-α genes that differ by only a few bases. The consensus primers, corresponding to two regions highly conserved among murine IFN-α subtypes, were used in reverse transcription and PCR amplification of total cellular RNA, isolated from IFN-γ-treated murine L-929 cells, to yield a fragment of the anticipated ∼520-bp size. Southern analysis of the amplified product using an internal consensus oligomer probe confirmed specific amplification of murine IFN-α gene(s). Subtype-specific oligonucleotide probes indicate that IFNs-α1, -α2, and -α5 are present following IFN-γ treatment, whereas IFN-α4 remains virtually absent. Our results indicate that the expression of specific IFN-α subtypes may be subject to complex regulation, dependent upon inducing agents and cell types involved, and countless other factors. The procedure described here represents a novel method for studying the subtleties of the murine IFN-α mRNA expression.