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A Strategy for the Generation of an Infectious Transmissible Gastroenteritis Coronavirus from Cloned cDNA

  • Fernando Almazan
  • Jose M. Gonzalez
  • Zoltan Penzes
  • Ander Izeta
  • Enrique Calvo
  • Luis Enjuanes
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Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 494)

Abstract

To date reverse genetics of Coronavirus has been possible by targeted recombination following the procedure initially developed by Masters’ group (Koetzner et al., 1992). However, the construction of a full-length cDNA clone, from which infectious RNA may be transcribed, will considerably improve the genetic manipulation of coronaviruses. Unfortunately, the size of the Coronavirus genome and the instability in bacteria of plasmids carrying Coronavirus replicase sequences have hampered the construction of a full-length cDNA clone (Masters, 1999). To overcome these problems we have combined three strategies: (i) a two-step amplification system that couples transcription in the nucleus from the cytomegalovirus (CMV) promoter with a second amplification step in the cytoplasm by the viral Polymerase; (ii) the construction of the full-length cDNA from a defective minigenome (DI) that was stably and efficiently replicated by the helper virus (Izeta et al., 1999); and (iii) the full-length cDNA was cloned as a bacterial artificial chromosome (BAC), a low-copy number plasmid, which is present in one or two copies per cell.

Keywords

Bacterial Artificial Chromosome Bacterial Artificial Chromosome Cloning Parental Virus Hepatitis Delta Virus Transmissible Gastroenteritis Virus 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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Copyright information

© Springer Science+Business Media New York 2001

Authors and Affiliations

  • Fernando Almazan
    • 1
  • Jose M. Gonzalez
    • 1
  • Zoltan Penzes
    • 1
  • Ander Izeta
    • 1
  • Enrique Calvo
    • 1
  • Luis Enjuanes
    • 1
  1. 1.Centro Nacional de Biotecnología, CSIC, Department of Molecular and Cell BiologyCampus Universidad AutónomaCantoblancoMadridSpain

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