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 ofdUTP 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.The method of in situ hybridization has been a powerful tool for the localization of specific sequences linearly arranged on a chromosome (1). This approach has been modified for the detection of RNA transcripts within individual cells. In some studies of this type total polyadenylylated RNA has been detected by using poly(U) probes (2-4). Other approaches have used complementary RNA or DNA reverse transcripts as probes either for viral sequences (5-7) or for specific messages within individual cells (8-10). More recently, a purified recombinant DNA clone has been used to detect specific cellular sequences (11). All ofthese approaches have used radiolabeled probes and the in situ hybridization has been detected by autoradiography. The inherent lack of resolution in tritium-labeled probes due to the track of the decay particle and the thickness of the emulsion makes the approach undesirable for precise morphological work. Recently, a number of workers have attempted the use of nucleic acid probes conjugated with fluorescent molecules for the detection of in situ hybridizations (12)(13)(14)(15) The investigation of gene expression by using recombinant DNA methods focuses on the molecular aspects of cell differentiation without consideration of the diversity of morphology inherent in the cell population containing these molecules. Because differentiation is often defined on the basis of cell morphology, we have attempted to develop a method relating cellular structure to the molecular aspects ofgene expression. This method requires high-resolution in situ hybridization and preservation of morphological detail. This work reports the initial results we have obtained by using the muscle tissue culture system for the detection of actin gene expression in situ and a recombinant actin plasmid labeled with biotin. This culture system is well suited to the investigation because both the morphology of cell differentiation (fusion of myoblasts into a syncytial myotube) and gene expression during this fusion process (e.g., the actin gene) have been well defined (18-22). MATERIALS AND METHODSPreparat...

Intravenous somatic gene transfer with antisense tissue factor restores blood flow by reducing tumor necrosis factor-induced tissue factor expression and fibrin deposition in mouse meth-A sarcoma.

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“…For immunocytochemistry staining, tumors were harvested after the animal had been perfused with 30 ml of PBS to remove unclotted material and instantaneously frozen in isopentan, cooled in liquid N 2 (32,33). After cutting, sections were fixed in 4% paraformaldehyde in 1 ϫ PBS for 20 min and washed three times for 10 min with 1 ϫ PBS.…”

Demonstration of pseudorabies virus DNA in the mouse inner ear by an in situ nucleic acid hybridization technique in plastic embedded bony material

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An Improved Method for Genomic In situ Hybridization in Oryza Species

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