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bioRxiv · 10.1101/2022.04.11.487972

Highly selective transgene expression through double-floxed inverted orientation system by using a unilateral spacer sequence

Matsushita, N.·Kato, S.·Nishizawa, K.·Sugawara, M.·Takeuchi, K.·Miyasaka, Y.·Mashimo, T.·Kobayashi, K.
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Abstract

The double-floxed inverted orientation (DIO) system with adeno-associated viral (AAV) vector provides a beneficial approach to express transgenes in specific cell populations having Cre recombinase. A significant issue with this system is the protection against non-specific expression of transgenes in tissues after vector injection. We here show that Cre-independent recombination in AAV genome carrying the DIO sequence occurs during the production of viral vectors in packaging cells, which results in transgene expression in off-target populations. Introduction of a relatively longer nucleotide sequence between two recognition sites at the unilateral side of the transgene cassette, termed a unilateral spacer sequence (USS), is useful to suppress recombination during the vector production, leading to the protection of non-specific transgene expression with enhanced gene expression selectivity. Our DIO/USS system offers a powerful strategy for highly specific Cre-dependent transgene expression, aiming at various applications for structural and functional analyses of target cell populations.

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Matsushita, N., Kato, S., Nishizawa, K., Sugawara, M., Takeuchi, K., Miyasaka, Y., Mashimo, T., Kobayashi, K.. 2022-04-12. Highly selective transgene expression through double-floxed inverted orientation system by using a unilateral spacer sequence. https://doi.org/10.1101/2022.04.11.487972

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