PCR-based Lentivirus titering protocol
Protocol
Principle
The Lentivirus tittering protocol uses RRE (Rev Responsive Element) as the marker
to determine the copy number of integrated lentiviral constructs in
transduced cells. All lentiviruses encode a regulatory protein Rev that is essential for post-transcriptional
transport of the unspliced and incompletely spliced viral mRNAs from nuclei to cytoplasm. The Rev protein
acts via binding to to an RNA structural element know as the Rev Responsive Element. The RRE will be
integrated together with the lentiviral
expression construct (e.g. siRNA or cDNA) into the genomic DNA of
transduced cells. Therefore, the RRE copy number would
correspond to the copy number of the lentiviral expression construct
or MOI.
MOI (multiplicity of infection)—the average copy number of lentiviral
expression constructs per genome of target cell in the infected cell
population. MOI does not directly correspond to percentage of
infected cells, which can easily be determined as the % of GFPpositive
cells for lentiviral constructs with the GFP reporter. The
simple guideline below allows you to convert % of infected cells to
MOI and vice versa:
% transduced cells: 10 20 30 40 50 60 70 80 90 >90*
MOI: 0.1 0.23 0.36 0.51 0.7 0.93 1.22 1.64 2.3 >2.5*
* - Please note that MOI cannot be calculated if % of transduced
cells is more than 90%.
Pseudoviral titer: The relative titer (concentration) in ifu/ml
(infection units/ml) of infection-competent pseudoviral particles. We
measure the pseudoviral titer for lentiviral constructs by amplification of the lentivector-specific RRE
region from genomic DNA of HEK293 infected cells. The Pseudoviral
Titer is always specific to a given cell line (in which it is determined).
we use DNA from cells infected with a GFP reporter
construct at different multiplicity of infections (MOI) as control. By comparing the
amount of RRE amplified from sample and that from the
calibration standards, the virus tier will be determined. This protocol can be used to
determine the virus titer with any lentivector that
contains the RRE element, regardless of the type of selection
markers.
5x PCR buffer
50ul (from Promega, cat.# M300b)
dNTP (2mM each)
10ul
GoTaq polymerase 5ul/ul
1.5ul (from Promega)
RRE primer mis (50pmol/ul each) 10ul
Water
150ul
Total=
221.5ul
Aliquot
24ul each tube
Sample
3ul
each reaction tube=27ul
94O C 2 min
(94O C for 30sec; 68OC for 1min ) for 26 to 28 cycles
68O C for 5 min
15O C hold
For purification of genomic DNA
DNeasy Tissue Kit (QIAGEN, Cat.#69504)
Or Wizard Genomic DNA Purification Kit (Promega, Cat.#A1120)
RRE primers
RRE5’ 5’ -ATA GGA GCT TTG TTC CTT GGG TTC TTG GGA
RRE3’ 5’ -CCG TTC ACT AAT CGA ATG GAT CTG TCT CTG