Autoregulating Transgene Silencing via Feedback Control

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Solution Overview

Problem

Transgenes in plants are often susceptible to small RNA (sRNA) silencing, which limits their expression and confers undesirable traits such as reduced herbicide and pest tolerance.

Innovation Solution

Concomitant transcription of RNA from a transgene and an sRNA silencing pathway gene is used to autoregulate expression, suppressing the sRNA silencing pathway and reducing susceptibility to silencing, thereby maintaining transgene expression.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Productivity

If transgenes are highly expressed to provide novel traits, then the desired trait expression is improved, but susceptibility to sRNA silencing increases

Engineering Contradiction:

Improvetransgene expression levelVSAvoidtransgene expression stability

Core Design Contradiction:

ProductivityVSReliability

Solution Approach 1:

The invention uses feedback control by introducing an sRNA silencing regulator gene that is co-transcribed with the transgene. When sRNA silencing is activated against the transgene, it simultaneously suppresses the silencing regulator gene, creating a negative feedback loop that reduces silencing susceptibility and maintains transgene expression stability.

Inventive Principle:

Principle #23Feedback

Solution Approach 2:

The sRNA silencing regulator gene acts as an intermediary element that mediates between the transgene and the sRNA silencing pathway. By co-transcribing the regulator gene with the transgene, the system creates an intermediate control mechanism that modulates silencing susceptibility without directly altering the transgene sequence.

Inventive Principle:

Principle #24Intermediary (Mediator)

2Reliability

If sRNA silencing is activated against transgene RNA, then transgene expression is suppressed, but concomitant transcription of silencing regulator gene suppresses the silencing pathway

Engineering Contradiction:

Improvetransgene expression stabilityVSAvoidDNA construct structure

Core Design Contradiction:

ReliabilityVSDevice complexity

Solution Approach 1:

The invention merges the transgene and sRNA silencing regulator gene into a single DNA construct that is co-transcribed by a common promoter. This combining approach allows both genes to be expressed simultaneously from the same transcriptional unit, simplifying the overall system architecture while achieving autoregulation.

Inventive Principle:

Principle #5Merging (Combining)

Solution Approach 2:

The sRNA silencing regulator gene serves multiple functions: it is co-transcribed with the transgene, acts as a target for sRNA silencing, and simultaneously suppresses the sRNA silencing pathway when activated. This multi-functionality reduces the need for separate regulatory components.

Inventive Principle:

Principle #6Universality (Multi-functionality)

Data Source

PatentUS9222100B2Methods and DNA constructs for autoregulating transgene silencing

Publication Date: 2015.12.29 MONSANTO TECHNOLOGY LLC

AI summary

This invention provides a method to autoregulate expression of a transgene susceptible to sRNA silencing by concomitantly transcribing RNA from DNA of a transgene and RNA from DNA from at least one sRNA silencing pathway gene. An aspect of the invention provides use of a recombinant DNA construct that includes DNA of a transgene and DNA of an sRNA silencing regulator. Also disclosed are transgenic cells and organisms having in their genome a recombinant DNA construct that includes DNA of a transgene and DNA of an sRNA silencing regulator.