Retrovirus and lentivirus vector design and methods of cell conditioning

Methods Enzymol. 2012;507:29-57. doi: 10.1016/B978-0-12-386509-0.00003-X.


Retroviruses are useful tools for the efficient delivery of genes to mammalian cells, owing to their ability to stably integrate into the host cell genome. Over the past few decades, retroviral vectors have been used in gene therapy clinical trials for the treatment of a number of inherited diseases and cancers. The earliest retrovirus vectors were based on simple oncogenic gammaretroviruses such as Moloney murine leukemia virus (MMLV) which, when pseudotyped with envelope proteins from other viruses such as the gibbon ape leukemia virus envelope protein (GALV) or vesicular stomatitis virus G protein (VSV-G), can efficiently introduce genes to a wide range of host cells. However, gammaretroviral vectors have the disadvantage that they are unable to efficiently transduce nondividing or slowly dividing cells. As a result, specific protocols have been developed to activate cells through the use of growth factors and cytokines. In the case of hematopoietic stem cells, activation has to be carefully controlled so that pluripotency is maintained. For many applications, gammaretroviral vectors are being superseded by lentiviral vectors based on human immunodeficiency virus type-1 (HIV-1) which has additional accessory proteins that enable integration in the absence of cell division. In addition, retroviral and lentiviral vector design has evolved to address a number of safety concerns. These include separate expression of the viral genes in trans to prevent recombination events leading to the generation of replication-competent viruses. Further, the development of self-inactivating (SIN) vectors reduces the potential for transactivation of neighboring genes and allows the incorporation of regulatory elements that may target gene expression more physiologically to particular cell types.

MeSH terms

  • Cell Culture Techniques
  • Cloning, Molecular
  • Gammaretrovirus / genetics*
  • Gammaretrovirus / isolation & purification
  • Gammaretrovirus / physiology
  • Gene Transfer Techniques
  • Genes, Viral
  • Genetic Engineering
  • Genetic Markers
  • Genetic Therapy / methods
  • Genetic Vectors
  • HEK293 Cells
  • Hematopoietic Stem Cells / metabolism
  • Hematopoietic Stem Cells / physiology
  • Hematopoietic Stem Cells / virology
  • Humans
  • Lentivirus / genetics*
  • Lentivirus / isolation & purification
  • Lentivirus / physiology
  • Promoter Regions, Genetic
  • Viral Load
  • Viral Tropism


  • Genetic Markers