Inducible methods for repressing gene function

Abstract

Methods for the rapid repression of gene function in eucaryotic cells are disclosed including inducible means for both shutting down a targeted gene's transcription and rapidly removing a targeted gene's polypeptide product.

Description

1. BACKGROUND OF THE INVENTION[0001] Genetics is essentially an approach to understanding biological processes through the systematic elimination of gene function. Historically, the genetic approach has involved the development of "screens" for mutations in genes which affect a specific phenotypic trait of an organism. The great advantage of this approach has been that no prior knowledge of the molecular nature of the genes involved is required because the "screen" identifies the affected genes by marking them with mutations. The mutation involved is frequently a change in the gene's sequence which results in a loss-of-function of the encoded gene product. Unfortunately the genetic approach has many limitations. Indeed the study of essential genes, required for cell viability, is exceedingly difficult using a purely genetic approach. As the famous molecular biologist David Botstein once put it, "death is not a phenotype;" an expression which encapsulates the frustration of attemptin...

AI Technical Summary

Benefits of technology

[0003] In general, the present invention provides a rapid and effective means for inactivating target genes, including target genes involved in important biological pathways. The invention also provides a system for the rapid and reliable repression of gene function regardless of whether the gene of interest is known or suspected of being an essential gene.

Problems solved by technology

The mutation involved is frequently a change in the gene's sequence which results in a loss-of-function of the encoded gene product.

Unfortunately the genetic approach has many limitations.

Indeed the study of essential genes, required for cell viability, is exceedingly difficult using a purely genetic approach.

Nevertheless, the exact biological processes in which the essential gene is involved are difficult to determine due to the inability to isolate and / or study the doomed knockout yeast spore or the inviable homozygous mouse zygote.

Thus the downstream effects on specific aspects of cell function following removal of the essential gene product cannot be readily determined using these traditional "knockout" studies.

Furthermore, while traditional gene "knockout" experiments may be useful in demonstrating that a given gene is essential for the life of the organism, they provide no data on precisely how important the gene is or to what extent so-called "second-site suppressing" mutations can arise which restore cell viability following the removal of the essential gene.

However, the generality with which the thermosensitive degron can be successfully applied to specific gene targets has yet to be determined and the necessity of relying upon thermal induction for the resulting system is a major drawback.

Furthermore, the requirement for induction by heat shock precludes useful application to mammalian transgenic animal systems.

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