Use of simian immunodeficiency virus vectors for simian embryonic stem cells.
The ability to stably introduce genetic material into primate embryonic stem (ES) cells could allow broader application. In this chapter, we describe a method of gene transfer into simian (cynomolgus macaque) ES cells using a simian immunodeficiency virus-based lentivirus vector. When cynomolgus ES cells are transduced with a simian immunodeficiency virus vector encoding the green fluorescent protein (GFP) gene, a large fraction of cells (greater than 50%) fluoresce, and high levels of GFP expression persist for months as assessed by flow cytometry and real-time polymerase chain reaction. Thus, the use of GFP as a reporter gene allows direct and simple detection of successfully transduced ES cells and facilitates monitoring of ES cell proliferation and differentiation both in vitro and in vivo. In addition, this highly efficient gene transfer method allows faithful gene delivery to primate ES cells with potential for both research and therapeutic applications.
Asano
Takayuki
T
Division of Regenerative Medicine, Center for Molecular Medicine, Jichi Medical School, Tochigi, Japan.
Shibata
Hiroaki
H
Hanazono
Yutaka
Y
D000818:Animals; D001483:Base Sequence; D018929:Cell Culture Techniques / Q000379:methods; D002478:Cells, Cultured; D017931:DNA Primers / Q000235:genetics; D004622:Embryo, Mammalian / Q000166:cytology*; D005434:Flow Cytometry; D017930:Genes, Reporter; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D008252:Macaca fascicularis / Q000196:embryology*; D051379:Mice; D039904:Pluripotent Stem Cells / Q000166:cytology* / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D015302:Simian Immunodeficiency Virus / Q000235:genetics*; D014161:Transduction, Genetic
D017931:DNA Primers; D049452:Green Fluorescent Proteins