Generation of retroviral particles for the spleen necrosis virus (SNV)-based vector system and their use in transduction of various cell types.
Genetically engineered retroviruses are widely used for gene delivery into human cells. A number of investigators have studied spleen necrosis virus (SNV) as a vehicle for gene delivery. Vectors developed from SNV and its closely associated avian reticuloendotheliosis virus strain A (REV-A) can be used for gene transfer into a variety of cells, including primary hematopoietic cells and human brain and post-mitotic neuronal cells that are difficult to transduce with other vector systems. SNV-based vector systems have the advantage of being quite safe, because wild-type SNV is unable to infect human cells and has less preference for integration into transcriptionally active sites or genes. However, the generation of retroviral vectors requires cotransfection of more than one plasmid into a packaging cell line, which is a tedious process. The development of stable packaging cell lines expressing envelope (Env) proteins and the structural proteins Gag-Pol will enhance mass production of retroviral vectors for future gene therapy experiments both in vitro and in vivo. This protocol describes the generation of retroviral particles for the SNV-based vector system. These particles can then be used for transduction of various cell types; as an example, a technique for transduction of post-mitotic neurons is also presented.
Zahida
Z
Parveen
Muhammad
M
Mukhtar
Roger J
RJ
Pomerantz
D002460:Cell Line; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D008938:Mitosis; D009474:Neurons / Q000166:cytology / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism; D053079:Trager duck spleen necrosis virus / Q000235:genetics*; D014161:Transduction, Genetic / Q000379:methods*; D014162:Transfection; D014771:Virion / Q000235:genetics*