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

## TL;DR

Researchers created a modified mouse cytomegalovirus vector that can carry large DNA inserts and deliver genes efficiently to various mammalian and even some non-mammalian cells.

## Contribution

A high-capacity MCMV vector with a 46 kbp cloning capacity was developed, enabling stable transduction across diverse cell types.

## Key findings

- Deletions up to 26% of the MCMV genome delayed virus reconstitution, while smaller deletions retained wild-type kinetics.
- Insertion of large DNA segments (up to 35 kbp) into the vector Q4 was achieved without compromising stability.
- The vector successfully transduced cells from multiple species, including human, monkey, bovine, bat, and chicken origins.

## Abstract

We investigated the basic characteristics of a new murine cytomegalovirus (MCMV) vector platform. Using BAC technology, we engineered replication-competent recombinant MCMVs with deletions of up to 26% of the wild-type genome. To this end, we targeted five gene blocks (m01-m17, m106-m109, m129-m141, m144-m158, and m159-m170). BACs featuring deletions from 18% to 26% of the wild-type genome exhibited delayed virus reconstitution, while smaller deletions (up to 16%) demonstrated reconstitution kinetics similar to those of the wild type. Utilizing an innovative methodology, we introduced large genomic DNA segments, up to 35 kbp, along with reporter genes into a newly designed vector with a potential cloning capacity of 46 kbp (Q4). Surprisingly, the insertion of diverse foreign DNAs alleviated the delayed plaque formation phenotype of Q4, and these large inserts remained stable through serial in vitro passages. With reporter-gene-expressing recombinant MCMVs, we successfully transduced not only mouse cell lines but also non-rodent mammalian cells, including those of human, monkey, bovine, and bat origin. Remarkably, even non-mammalian cell lines derived from chickens exhibited successful transduction.