[Expression of green fluorescent protein gene in mouse T lymphocytes mediated by lentiviral vector].
This study was purposed to constructe the three-plasmid system of the lentiviral vector carrying the green fluorescent protein (GFP) gene and to investigate the expression of GFP in T lymphocytes of the mouse. The polypurine tract (PPT) element, ubiquinone promoter (PUB) and GFP were ligated to plasmid pLO134 using subcloning technology to construct plasmid pTK153. Human kidney 293T cells were co-transfected with the three-plasmid system containing packaging plasmid DeltaNRF, plasmid pTK153 and envelope plasmid VSV-G by using calcium phosphate DNA precipation and the expression of GFP was observed under fluorescence microscope after 12 hours. The viral particles were collected after transfection 72 hours, were frozen at -80 degrees C and were used to infect mouse T lymphocytes at multiplicity of infection (m.o.i.) of 3. The expression of GFP in mouse T lymphocytes was observed by fluorescence microscopy and fluorescence-activated cell sorting (FACS). The results showed that the transfection efficacy was 63.04 +/- 7.24% in 293T cells analysed by FACS and the viral titer was (3.09 +/- 0.61) x 10(6) U/ml. The expression of GFP was also evident in mouse T lymphocytes and the transduction efficacy was (37.98 +/- 6.26)%. It is concluded that the three-plasmid system of lentiviral vector containing GFP gene is successfully constructed and the transduction efficacy is high in mouse T lymphocytes.
Li
Zhen-Yu
ZY
Department of Hematology, The Affiliated Hospital of Xuzhou Medical College, Xuzhou 221002, China. lizhenyumd@163.com
Xu
Kai-Lin
KL
Pan
Xiu-Ying
XY
Sun
Hai-Ying
HY
Gao
Fei
F
Lu
Qun-Xian
QX
Li
De-Peng
DP
He
Xu-Peng
XP
D000818:Animals; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000096:biosynthesis* / Q000235:genetics; D016086:Lentivirus / Q000235:genetics*; D051379:Mice; D008807:Mice, Inbred BALB C; D012367:RNA, Viral / Q000032:analysis; D013601:T-Lymphocytes / Q000378:metabolism*; D014161:Transduction, Genetic
D012367:RNA, Viral; D049452:Green Fluorescent Proteins