61] Procedures for isolation of murine alpha interferon gene and expression of a murine leukocyte interferon in E. coli

Abstract

This chapter discusses the procedures for the isolation of murine alpha interferon genes (Mu-IFN-αA) and expression of a murine leukocyte interferon in E.coli . Mu-IFN-αA is isolated from a genomic DNA library in phage lambda with a human alpha interferon DNA as a probe. The probe consisted of the 642 bp Ava II fragment from the plasmid pLeIFA25, which represented 90% of the coding region. The conditions for lambda phage screening and hybridization are described in which this fragment is successfully used to isolate a genomic mouse alpha interferon gene. The method by which its expression is achieved in E. coli is also described. Hybridization of the murine genomic library to the 32 P-labeled human alpha interferon gene resulted in strong positive signals with virtually no background. The probe chosen represented over 90% of the coding region and contained no poly(A) sequences and no introns. Because there is cross-hybridizing between species, and because the extent of the homology between human and mouse interferon genes is unknown, the stringency of the hybridization solution is lowered from a standard concentration of 50% formamide to a lower stringency of 40% formamide. This method proves successful in the isolation of a genomic Mu-IFN-αA.