Lentiviral Vectors for Gene Delivery to the Nervous System - Gene Delivery and Therapy for Neurological Disorders - page 49

Fluorescent Lypophilic Carbocyanines

16 hpt

2hrs

Transfect 293T cells Replace medium with

DiO/Dil/DiD containing
medium

Replace medium with low
serum NaBut containing
medium

gp120

DiO Dil DiD DiR

lipid
membrane

RNA

gp41

capsid

matrix

Reverse
Transcriptase

500 600 700 800 900

Wavelength (nm)

Fig. 3 Lipophilic labeling of lentiviral vectors. Sixteen hours post transfection medium is replaced with Opti-
MEM containing Vybrant dye and incubated at 37 °C for 2 h before replacing with low serum media containing
sodium butyrate. Labeling of the envelope with lipophilic dyes allows highly effi cient labeling of the viral vec-
tors without affecting their biological titer

size of the transgene, (2) the cell line chosen for titration, (3) the
age and storage conditions of the viral stock (virus can usually be
kept at −80 °C in cryovials for up to a year), and (4) the number of
freeze-thaw cycles as each freeze-thaw cycle results in 2-4-fold
decrease in titer.
LV titers can be determined with several methods. Some of
these are based on the determination of the number of vector par-
ticles present in a stock, whereas other on the number of proviral
copies in transduced target cells. In general it can roughly be divided
into functional and nonfunctional titration methods. Vector parti-
cle number can be determined by real-time PCR based on the pres-
ence of strong-stop cDNA virions [ 116 ]. Relative particle titers can
be obtained by measuring the amount of viral proteins, such as viral
capsid protein p24, present in vectors stocks by ELISA [ 117 ]. More
accurate functional (biological) titration assays are based on
reporter-gene expression encoded by the vector. LVs expressing
fl uorescent proteins such as GFP or red fl uorescent protein (RFP)
can easily be titrated by fl uorescence-activated cell sorting (FACS)
analysis, which detects the encoded protein in transduced cells [ 29 ,
118 ]. It is not necessary to immunostain GFP and RFP before
FACS analysis. Transduced cells expressing the reporter protein can
easily be detected by FACS using the appropriate excitation laser.