The goal of this proposal is to develop a simple, efficient and sensitive methodology for high resolution visualization of the expression of individual genes within single cells. We have developed a method of in situ hybridization wherein nucleic acids of known sequence are exposed to preserved cells and allowed to hybridize (base-pair) with the complementary strand of messenger RNA within the cell. The morphology of the cell is maintained during this procedure so that the messenger RNA is detected at its precise cellular location. We have used an actin recombinant DNA clone into which a nucleotide analog containing biotin has been enzymatically inserted. This biotin moiety is then detected by antibodies to biotin, or by avidin, coupled to a fluorescent group. This procedure is rapid and yields results of high resolution. We have been able to visualize the expression of actin messenger RNA during the differntiation of chicken muscle cells in tissue culture. Accompanying this process is the expression of actin messenger RNA in large amounts. By in situ hybridization we can see the increase in the actin messages as fluorescent signal and observe the distribution of these messages with indidual cells. This fluorescent signal can be detected and quantitated by use of a digital image processing computer and a low light level SIT camera. The theoretical limit of detection is 200-500 fluorochromes using this system, possibly one messenger RNA molecule. We wish to use this approach to investigate the """"""""turning on"""""""" of a specific gene, the alpha actin expressed in differentiated muscle. We intend to exploit the high resolution capabilities of the biotin analog to study the intracellular distribution of mRNAs using the electron microscope. This approach would lead to a more precise understanding of the control of gene expression and could eventually be applied to more complex events such as morphogenesis. A method for investigating specific genes within individual cells in embryological systems would have important consequences for the detection and understanding of causes and effects operating in normal and abnormal development.
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