Actin gene expression visualized in chicken muscle tissue culture by using in situ hybridization with a biotinated nucleotide analog

Abstract

The chicken muscle tissue culture system has been used for visualizing actin gene expression after in situ hybridization. Cell differentiation is morphologically distinguishable in this system as the myoblasts fuse into myotubes. This differentiation involves the production of large amounts of actin required for myofibrils. The presence of actin mRNA has been observed in cells preserved with ethanol and paraformaldehyde by hybridizing a recombinant plasmid into which a biotinated analog of dUTP was incorporated by nick-translation. The biotin was then detected by using an anti-biotin antibody and a rhodamine-conjugated second antibody. Alternatively, avidin conjugated to rhodamine or avidin complexed to biotinated peroxidase has been used for mRNA detection. The procedure described preserved morphological detail yet is compatible with hybridization conditions and reveals the disposition of actin mRNA during gene expression.