Abstract
Since cytomegalovirus (CMV) infection is highly species-specific, it is necessary to study animal cytomegaloviruses to assess viral factors which contribute to pathogenesis. The generation of recombinant viruses carrying reporter genes would provide useful tools for studying the genetics of CMV pathogenicity in vivo. We evaluated whether the guinea pig cytomegalovirus (GPCM-V) was amenable to such manipulation. Metabolic selection using the guanosylphosphoribosityl transferase (gpt) gene facilitated recovery of a recombinant virus, vAM403, containing a gpt/green fluorescent protein (eGFP) cassette introduced into the HindIII "N" region of the viral genome. This virus had replication kinetics identical to wild-type virus. We next attempted to clone the GPCMV genome as a bacterial artificial chromosome (BAC). A BAC plasmid containing a gpt/eGFP cassette and the chloramphenicol resistance marker was introduced into HindIII "N" to generate another GPCMV recombinant, vAMBGPCM-V. Circular viral DNA isolated from vAMBGPCMV-infected cells was used to transform Escherichia coli. Restriction profiles revealed that the GPCMV genome had been cloned as a BAC plasmid, and transfection of BAC plasmid DNA confirmed that the BAC clone was infectious. A novel strategy based on a unique PmeI site was devised to quickly modify the BAC GPCMV plasmid. Recombinants retained the capability to replicate and express reporter genes in guinea pigs, suggesting that these viruses will be useful for in vivo pathogenesis studies.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 15-26 |
| Number of pages | 12 |
| Journal | Molecular Genetics and Metabolism |
| Volume | 72 |
| Issue number | 1 |
| DOIs | |
| State | Published - 2001 |
Bibliographical note
Funding Information:We acknowledge the gift of plasmid pCHN to the Children’s Hospital Division of Infectious Diseases from Harriet Isom. We thank J. Vieira for providing us with plasmid pQ106 and also M. Messerle and U. Koszinowski for supplying the BAC plasmid, pKSO. We thank Dr. Nancy Sawtell with assistance on microscopy and photography. We are also grateful for the advice and assistance of Fernando Bravo in animal studies. These studies were supported by March of Dimes Basic Research Grant 6-FY98-0416, and National Institutes of Health HD38416-01 and AI65289.
Copyright:
Copyright 2017 Elsevier B.V., All rights reserved.
Keywords
- Bacterial artificial chromosome (BAC)
- Cytomegalovirus (CMV)
- Green fluorescent protein
- Guinea pig