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Lentiviral vectors offer a large packaging payload as well as possessing the ability to stably integrate into dividing and non-dividing cells, such as neurons. It
also enables us to study protein interactions that may aid in the modeling and treating of neurodegenerative disorders, such as Alzheimer's disease (AD).
AD is characterized by the hallmark lesions β-amyloid plaques and neurofibrillary tangles. Previous studies have indicated that the tau protein, which is
critically involved in the formation of neurofibrillary tangles in AD, can be overexpressed in the rat hippocampus via lentiviral vectors. Unfortunately, the level
of tau transgene expression in vivo is highly variable, even from the same batch of virus. To effectively use a lentiviral vector, it is essential to establish a
standardized titering protocol to consistently deliver the same concentration of transgene to transduced cells. I hypothesized that environmental factors –
specifically, the time between thawing and injection – affect expression levels, and that small variations in virus titer can have a strong influence on protein
expression. To test my hypotheses, I performed experiments to assess: 1) the effect temperature on viral infectivity, 2) the effects of multiple freeze-thaw
cycles on viral titer, and 3) that PCR is a viable technique to determine the infectious particle titer of lentiviral constructs. By testing the effects of common
laboratory factors such as time interval at which a viral prep is maintained at room temperature prior to infection, and the repeated use of a single prep of
virus following multiple freeze/thaw cycles, I have attempted to pinpoint the environmental source of the irregular expression observed in vivo. Based on
my experiments, I conclude that these environmental factors have almost no effect of the overall viral infectivity. The development of a standardized PCR-based
titering protocol will allow us to accurately deliver the same amount of virus from prep to prep as well as increasing our ability to titer viral constructs that do not
contain the enhance green fluoresce protein (eGFP) reporter gene.
A. Immunohistochemistry: To develop an improved method of quantifying EGFP expression in the brain, I performed preliminary experiments to compare
EGFP fluorescence to immunoreactivity of the protein using an antibody to EGFP. Tissues sections were examined from rats that were injected 3 months
previously with 1.5 μl of lenti-FUGW into the hippocampus. The contralateral hippocampus received a similar injection of lentivirus expressing a different protein.
The antibody to EGFP (SOURCE) was a goat polyclonal antibody. Tissue sections including the EGFP-injected hippocampus and the contralateral hippocampus
as control were placed in individual wells in a 24-well dish in cold PBS and washed three times with PBS for five minutes each. The tissue was then treated with
750 µl of 3% H2O2/methanol for 10 minutes at room temperature to reduce background due to endogenous peroxidase. After another three PBS washes for 5
min each, the tissue was put in 750 µl blocking solution consisting of 2% rabbit IgG and 0.2% Tween in PBS, for one hour at room temperature. The tissue then
was placed in EGFP antibody in PBS at concentrations of 1:10000 to 1:30000 and allowed to incubate on a shaker overnight at 4ºC. The following day the tissue
was washed with PBS three times for five minutes each. A secondary anti-goat antibody at 1:200 in PBS was applied for one hour at room temperature. After
another three PBS washes for five minutes each, the antigen-antibody complex was detected using the standard avidin-biotin (ABC) method (Vector Laboratories,
Burlingame, CA). The tissue was then washed with PBS three times for five minutes each, followed by incubation in diaminobenzidine (DAB) for two minutes.
Sections were then mounted onto slides, dehydrated, and cover-slipped for light-microscopic analysis.
B. Effects of Temperature and Freeze-Thaw on Infectivity: Low passage (<20) HEK293 cells (ATCC, Manassas, VA) were maintained in HEK media at 37ºC
and 5% CO2. Recombinant replication-deficient lentiviral vectors; FUGW (Lois et al., 2002) , SRFPUGW and SKRFPUGW were produced through Emory
University’s Viral Vector Core. Cells were plated at 50,000 cells/cm2 in 6-well culture dishes. Effects of storage at room temperature: Viral stocks were thawed
once then used immediately to infect one well of cells. The virus was maintained at room temperature and at every hour for the next 5 hours an additional well
was infected. A PBS vehicle-treated well was included as a negative-control. Effects of freeze-thaw cycles: 6-well plates were plated such that each well would
be confluent at the time of imaging, allowing infection to be performed serially at the same ratio of virus to cells. Every day for 4 days the virus was thawed and
one well was infected. Immediately after infection the virus was refrozen and kept at -80ºC until the next day. Replicate dishes were infected to ensure accuracy
of results. Direct EGFP fluorescence and phase-contrast images were captured using Metavue software on a Zeiss inverted microscope equipped with a
Hamamatsu monochromatic digital camera.
C. Development of PCR-based Titer Protocol: Viral stocks were serial diluted 10-fold in PBS until a final dilution of 10-10 was achieved (Figure 1). Starting
with 1 µl undiluted virus, a separate well was infected with 10 µl of each dilution. A PBS vehicle-treated well was included as a negative-control (Figure 2).
Replicate dishes were infected to ensure accuracy of results. At 72 hrs post-infection, direct EGFP fluorescence and phase-contrast images were captured
using Metavue software on a Zeiss inverted microscope equipped with a Hamamatsu monochromatic digital camera. Once images were captured, cells were
harvested in PBS, pelleted at 16,000 x g for 5min after which point genomic DNA was extracted using a DNeasy kit (Qiagen, Valencia, CA). PCR using a
primer set to specifically amplify a portion of the ubiquitin-C promoter was run on the extracted DNA according to the following profile: 98C for 5 min followed by
40 cycles: 1) 95C for 30 sec., 2) 60C for 30 sec., 3) 72C for 90 sec.; followed by 5min at 72C. PCR products were resolved by agarose gel electrophoresis and
imaged by an Innotech gel documentation system.
The Problem: Lentivirus-mediated expression of focally administered tau protein in the rat hippocampus is highly variable
Hypotheses:
• Variability is due to environmental factors such as temperature and freeze-thaw cycles of the virus
• The specific titer of the virus must be precisely established to reduce variability of transgene expression
• Dependent variable: Expression of green fluorescent protein as a specific readout:
The hippocampus was bilaterally injected with FUGW. Only the right hippocampus in both animals shows EGFP expression.
Room Temperature and Freeze-Thaw have little effect on EGFP protein Expression
Both the Time trials and the Freeze-Thaw trials showed no decrease in EGFP Expression under these common laboratory situations
SRFPUGW Bigenic Serial Dilution
This Bigenic Lentivirus shows a dramatic decrease in the number of cells producing EGFP after each tenfold dilution. The top pictures are of EGFP
producing cells and the bottom pictures are Differential Interference Contrast (DIC) images of these cells in the plane.
FUGW Serial Dilution
The EGFP positive cells were identified in the well infected with a 10-4 dilution yielding a viral titer of 107 Infectious units per mL.
Kozak Bigenic Virus Serial Dilution
Both of the bigenic lentiviruses showed expression out to a 10-6 dilution. The Kozak lentivirus contains a special sequence specific to eukaryotes.
Standard PCR-based Titer Protocol
CMV LTR, FLAP, and SIN LTR are structural elements required for packaging
recombinant lentivirus. hUbi-C and hSYN1 are the human ubiquitin-c and synapsin-1 promoters, respectively. RFP and eEGFP are the red fluorescent and
enhanced green fluorescent proteins, respectively. WPRE is the woodchuck hepatitis posttranscriptional regulatory element, which enhances transgene
expression. The location of the PCR primers are indicated by the arrowheads.
PCR-based titer = 1 x 109 IU/ml and EGFP-based titer = 1 x 109 IU/ml
PCR is a highly sensitive and adaptable approach for titering recombinant lentiviral vectors.
• The bigenic virus used in these experiments was unaffected by both varying time intervals at room temperature prior to infection and exposure to multiple
freeze-thaw cycles.
• A drastic change in expression is seen from one tenfold dilution to the next using an EGFP reporter lentivirus.
• Οur standardized PCR-based infection assay is adaptable to use with multiple viral constructs and can be utilized to calculate virus titer accurately and efficiently
• Τhe titer of both Bigenic viruses used is 109 IU per mL
Future Directions
Our standardized titering protocol will allow lentiviral vectors to be used to accurately infect at a known concentration with little variability. Future experiments
can now be performed using non-reporter gene constructs with startling accuracy.
• Carlos Lois, Elizabeth J. Hong, Shirley Pease, Eric J. Brown, David Baltimore, “Germline Transmission and Tissue-Specific Expression of Transgenes
Delivered by Lentiviral Vectors,” Science 295, 868-872 (2002)
• L. Sastry, T. Johnson, M.J. Hobson, B. Smucker, K. Cornetta, “Titering lentiviral vectors: comparison of DNA, RNA and marker expression methods,” Gene
Therapy 9, 1155-1162 (2002)
• Gregory Liz, Joeri L. Aerts, Monica I. Gonzales, Nachimuthu Chinnasamy, Richard A. Morgan, and Suzanne L. Topalian, “Real-Time Quantitative Reverse
Transcriptase-Polymerase Chain Reaction as a Method for Determining Lentiviral Vector Titers and Measuring Transgene Expression,” Human Gene Therapy
14, 497-507 (2003)
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