Linear expression elements: a rapid, in vivo, method to screen for gene functions

Abstract

The increasing accumulation of genomic sequence information has accentuated the need for new methods to efficiently assess gene function and to prepare reagents to study these functions. Toward solving this general problem in functional genomics, we report a method by which any PCR-amplified open-reading frame (ORF) can be noncovalently linked to a eukaryotic promoter and terminator, and directly injected into animals to produce local gene expression. We also demonstrate that ORFs can be delivered into mice to produce antibodies specific for the encoded foreign protein by simply attaching mammalian promoter and terminator sequences. This technology makes it possible to screen large numbers of genes rapidly for their functions in vivo or to produce immune responses without the necessity of cloning, bacterial propagation, or protein purification.

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Figure 1: Delivery and expression of a PCR-amplified luciferase gene in mice.
Figure 2: Noncovalently linked PCR products can be delivered and expressed in vivo.
Figure 3: LEEs can be delivered into animals biolistically or by needle injection.
Figure 4: Introduction of a bimolecular LEE encoding AAT generates specific antibodies in mice at titers comparable to those produced from a plasmid encoding this test antigen.
Figure 5: Tri-molecular LEEs expressing M. tuberculosis ORFs can be delivered into mice to generate polypeptide-specific antibodies.

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Acknowledgements

We thank Mike Barry for initial observation of PCR-product encoded gene expression and colleagues at the Center for Biomedical Inventions for encouragement and discussion. This work was supported by a grant from DARPA to S.A.J.

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Correspondence to Stephen Albert Johnston.

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Sykes, K., Johnston, S. Linear expression elements: a rapid, in vivo, method to screen for gene functions. Nat Biotechnol 17, 355–359 (1999). https://doi.org/10.1038/7908

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