Use of Recombination-Mediated Genetic Engineering for Construction of Rescue Human Cytomegalovirus Bacterial Artificial Chromosome Clones

Kalpana Dulal,Hua Zhu,Benjamin Silver

doi:10.1155/2012/357147

Kalpana Dulal, Hua Zhu + Show 1 more

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https://doi.org/10.1155/2012/357147

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Abstract

Bacterial artificial chromosome (BAC) technology has contributed immensely to manipulation of larger genomes in many organisms including large DNA viruses like human cytomegalovirus (HCMV). The HCMV BAC clone propagated and maintained inside E. coli allows for accurate recombinant virus generation. Using this system, we have generated a panel of HCMV deletion mutants and their rescue clones. In this paper, we describe the construction of HCMV BAC mutants using a homologous recombination system. A gene capture method, or gap repair cloning, to seize large fragments of DNA from the virus BAC in order to generate rescue viruses, is described in detail. Construction of rescue clones using gap repair cloning is highly efficient and provides a novel use of the homologous recombination-based method in E. coli for molecular cloning, known colloquially as recombineering, when rescuing large BAC deletions. This method of excising large fragments of DNA provides important prospects for in vitro homologous recombination for genetic cloning.

Highlights

Human cytomegalovirus (HCMV), known as human herpesvirus-5, belongs to the betaherpesvirinae subfamily of the herpesviridae family
The plasmid was digested with AscI and FseI restriction enzymes to separate the vector sequence from the region IV region of interest (ROI). 500 ng of the purified digestion product was transformed into DY380 carrying the Toledo IV-Δ mutant Bacterial artificial chromosome (BAC) by electroporation and the transformants were cultured on LB agar with zeocin and hygromycin (50 μg/mL)
BAC cloning technique of viral genome has been a useful tool, especially for mutagenesis studies of large DNA viruses such as HCMV, in which the BAC DNA has been used to mutate or delete the individual genes to understand their functions in viral replications

Summary

Introduction

Human cytomegalovirus (HCMV), known as human herpesvirus-5, belongs to the betaherpesvirinae subfamily of the herpesviridae family. Delete large DNA fragments by inserting antibiotic resistance markers for proper and accurate selection of recombinant BAC clones and insert a luciferase reporter gene in order to measure expression of the recombinant virus. These new systems circumvented the problems associated with conventional genetic engineering as there was no longer a size restriction as seen when using restriction enzymes or other previous methods [12, 13]. Discussed below are the methods commonly used to make recombinant clones via this method

Uses of Recombination-Mediated Genetically Engineered BACs in Viral Research

Specific Recombineering Methods in Construction of Rescue BAC Clone

Discussion

Findings

Future Directions