Strategies to insulate lentiviral vector-expressed transgenes.
Lentiviruses are capable of infecting many cells irrespective of their cycling status, stably inserting DNA copies of the viral RNA genomes into host chromosomes. This property has led to the development of lentiviral vectors for high-efficiency gene transfer to a wide variety of cell types, from slowly proliferating hematopoietic stem cells to terminally differentiated neurons. Regardless of their advantage over gammaretroviral vectors, which can only introduce transgenes into target cells that are actively dividing, lentiviral vectors are still susceptible to chromosomal position effects that result in transgene silencing or variegated expression. In this chapter, various genetic regulatory elements are described that can be incorporated within lentiviral vector backbones to minimize the influences of neighboring chromatin on single-copy transgene expression. The modifications include utilization of strong internal enhancer-promoter sequences, addition of scaffold/matrix attachment regions, and flanking the transcriptional unit with chromatin domain insulators. Protocols are provided to evaluate the performance as well as the relative biosafety of lentiviral vectors containing these elements.
Department of Anatomy and Regenerative Biology, The George Washington University Medical Center, Washington, DC, USA.
Ali
A
Ramezani
Robert G
RG
Hawley
D004742:Enhancer Elements, Genetic; D020868:Gene Silencing; D005822:Genetic Vectors; D006801:Humans; D020014:K562 Cells; D016086:Lentivirus / Q000235:genetics*; D011401:Promoter Regions, Genetic; D019076:Transgenes