Construction and Characterization of a High-Capacity Replication-Competent Murine Cytomegalovirus Vector for Gene...

Area of Science:

• Virology
• Molecular Biology
• Genetic Engineering

Background:

• Murine cytomegalovirus (MCMV) is a widely used viral vector system.
• Existing MCMV vectors have limitations in terms of genome capacity and transduction efficiency across diverse cell types.

Purpose of the Study:

• To engineer and characterize a novel, replication-competent MCMV vector with enhanced genomic capacity.
• To assess the transduction capabilities of the new MCMV vector in various mammalian and non-mammalian cell lines.

Main Methods:

• Engineered replication-competent recombinant MCMVs using Bacterial Artificial Chromosome (BAC) technology with targeted gene block deletions.
• Introduced large genomic DNA segments (up to 35 kbp) and reporter genes into a novel MCMV vector (Q4) with a 46 kbp cloning capacity.
• Assessed virus reconstitution kinetics, plaque formation, insert stability through in vitro passages, and transduction efficiency in diverse cell lines.

Main Results:

• MCMV BACs with 18-26% genome deletions showed delayed reconstitution, while smaller deletions (up to 16%) were similar to wild-type.
• The Q4 vector accepted large DNA inserts (up to 35 kbp), which alleviated its delayed plaque formation phenotype and remained stable.
• Recombinant MCMVs successfully transduced mouse, human, monkey, bovine, bat, and chicken cell lines.

Conclusions:

• The novel MCMV vector platform (Q4) demonstrates a significantly expanded cloning capacity and stable maintenance of large foreign DNA inserts.
• This MCMV vector exhibits broad tropism, enabling efficient transduction of diverse mammalian and non-mammalian cells.
• The engineered MCMV vector holds promise for various applications in gene delivery and biomedical research.