Split Cre Recombinase Reporter System for Stable HBV Infection Assessment
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Solution Overview
Problem
Current recombinant reporter systems for hepatitis B virus (HBV) have limitations such as restricted cargo size, inhibition of HBV replication, inefficient replication, and transient expression of reporter genes due to instability of cccDNA in cell culture models, making it challenging to assess the capacity of substances to treat or prevent HBV infection effectively.
Innovation Solution
A split recombinase reporter system is used, where a first fragment of the Cre recombinase is integrated into the HBV genome and a second fragment is expressed in mammalian hepatocytes, allowing the two fragments to associate and excise a stop cassette flanked by recombination sites, enabling permanent expression of a reporter gene like DsRed, facilitating the assessment of HBV infection and treatment capacity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If a reporter gene is integrated into the HBV genome, then the reporter gene can be expressed upon infection, but the HBV replication is inhibited and cargo size is restricted
Solution Approach 1:
The Cre recombinase is divided into two non-functional fragments: the first fragment is integrated into the HBV genome while the second fragment is expressed by the host cell. Only when both fragments are present do they reassemble into functional Cre recombinase, which then excises the stop cassette to enable reporter gene expression. This segmentation allows HBV replication to proceed without inhibition while still enabling reporter function upon co-expression of both fragments.
Solution Approach 2:
The stop cassette flanked by loxP sites acts as an intermediary element that prevents reporter gene expression until the split Cre fragments reassemble and excise it. This intermediary mechanism ensures that reporter expression only occurs when both viral and cellular components are present, without interfering with HBV replication.
2Measurement precision
If the reporter gene expression is based on HBV cccDNA stability, then the reporter can be expressed, but the expression is transient because cccDNA is lost over time in cell culture
Solution Approach 1:
The host cell is pre-engineered to constitutively express the second fragment of Cre recombinase and to carry the stop-cassette-reporter construct. Upon infection with HBV containing the first Cre fragment, the fragments reassemble and immediately excise the stop cassette, enabling permanent reporter expression that persists independently of cccDNA stability.
3Adaptability or versatility
If foreign sequences are introduced into HBV vector, then reporter genes can be carried, but HBV replication is inhibited
Solution Approach 1:
The reporter system is segmented between two hosts: the HBV carries only the essential first Cre fragment (minimal foreign sequence), while the host cell carries the second Cre fragment, the stop cassette, and the reporter gene. This distribution minimizes the foreign genetic load on HBV, allowing efficient replication while maintaining reporter functionality.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This approach allows for stable and efficient assessment of HBV infection and treatment capacity by enabling long-term expression of reporter genes, overcoming the limitations of transient expression and replication inefficiencies in existing systems.
Implementation Method 1
The reporter cell comprises a stop cassette flanked by two recombination sites fused to a reporter gene, wherein the presence of the stop cassette suppresses expression of the reporter gene. The stop cassette is then excised by the recombinase to permanently enable the expression of the reporter gene
Data Source
AI summary
The present invention discloses a method for assessing the capacity of a substance to treat or prevent hepadnavirus infection. A reporter virus carrying genetic information for a first fragment of a recombinase and a reporter cell expressing a second fragment of the recombinase are used. When the reporter virus infects the reporter cell, the two fragments of the recombinase associate and excise a stop cassette that is flanked by two recombination sites and blocks the expression of a reporter gene. Accordingly, the present invention relates to a method of assessing the capacity of a substance to treat or prevent hepadnavirus infection, a hepadnavirus comprising a nucleic acid encoding a first fragment of a recombinase and a mammalian hepatocyte or hepatoma cell comprising a nucleic acid encoding a second fragment of a recombinase and a nucleic acid comprising a stop cassette flanked by two recombination sites fused to a reporter gene.