A novel and simple method for construction of recombinant adenoviruses

Abstract

Recombinant adenoviruses have been widely used for various applications, including protein expression and gene therapy. We herein report a new and simple cloning approach to an efficient and robust construction of recombinant adenoviral genomes based on the mating-assisted genetically integrated cloning (MAGIC) strategy. The production of recombinant adenovirus serotype 5-based vectors was greatly facilitated by the use of the MAGIC procedure and the development of the Adeasy™ adenoviral vector system. The recombinant adenoviral plasmid can be generated by a direct and seamless substitution, which replaces the stuff fragment in a full-length adenoviral genome with the gene of interest in a small plasmid in Escherichia coli. Recombinant adenoviral plasmids can be rapidly constructed in vivo by using the new method, without manipulations of the large adenoviral genome. In contrast to other traditional systems, it reduces the need for multiple in vitro manipulations, such as endonuclease cleavage, ligation and transformation, thus achieving a higher efficiency with negligible background. This strategy has been proven to be suitable for constructing an adenoviral cDNA expression library. In summary, the new method is highly efficient, technically less demanding and less labor-intensive for constructing recombinant adenoviruses, which will be beneficial for functional genomic and proteomic researches in mammalian cells.

Constructing recombinant herpesvirus BAC vectors with mating-assisted genetically integrated clone method

“…Plasmid pBAD-TOPO and pEGFP-1 were from Invitrogen or Clontech, respectively. Plasmid pRTRA was previously constructed in our lab (Tan et al 2006). Plasmid pML300 and E. coli DH10b were provided by Prof. S. J. Elledge (Li and Elledge 2005).…”

“…The kanamycin (Kan) resistant gene cassette (H1-I-kan r -I-H2) was amplified from pShuttleK (Tan et al 2006) with primers F1 and R1 (Supplementary Table 1). Each of the primers bears 50 bp sequences homologous to pBecker2 TK gene.…”

“…Lines: M, k/EcoT14 DNA marker; 1, an expected 1.4 kb fragment was amplified with primers F7 and R7 from pBecker2-KH; 2-9, a fragment about 1.7 kb was amplified from the colonies containing expected recombinant pBecker2-gfp, excepted 7 pRTRA1. The gfp fragment was amplified from pEGFP-1 with the primers F4 and R4 (Supplementary Table 1) and inserted into Bsu36I-digested pRTRA1 to obtain plasmid pRThGA (Tan et al 2006). …”

“…In this study, a simple method of generating PRV expression vectors was developed based on MAGIC (Li and Elledge 2005;Tan et al 2006). Any target gene cloned into a small donor vector can be transferred into a recipient PRV vector via homologues recombination in E. coli.…”

Tumor-targeted gene therapy using Adv-AFP-HRPC/IAA prodrug system suppresses growth of hepatoma xenografted in mice

“…The integrity of the HRPC gene constructs was confirmed by DNA sequencing analysis. The recombinant plasmid (pAdv-AFP-HRPC) was constructed using mating-assisted genetically integrated cloning methods, as described before, 22,23 and then transfected into 293T cells to produce infectious recombinant adenovirus (Adv-AFP-HRPC). Viral particle number was calculated by detecting the absorbance at 260 nm (A 260 ).…”