| |
The Cignal Lenti Reporters are ready for transduction
right out of the box. There is no need to generate or propagate lentivirus
in your laboratory. These vectors are extremely useful for transient
transduction studies in difficult to transfect cells or for pathway sensor
cell line generation.
Transient Pathway Regulation Studies in Difficult to
Transfect Cells: Target cells are transduced with the Cignal Lenti
Pathway Reporter. The cells are typically cultured for 24 to 48 hours to
insure lentivirus integration. The cultures are then treated with the
biological agents of interest (siRNA, shRNA, chemical compound, viral
expression vector, protein, peptide). Reporter assays (firefly luciferase
or GFP) are carried out 18 to 36 hours post-treatment, depending upon the
treatment conditions.
Pathway Sensor Cell Line Generation: Target cells
are transduced with the Cignal Lenti Pathway Reporter. Following
transduction, the cells are cultured under puromycin selection to generate
a homogenous population of transduced cells. If necessary, single cell
cloning may be carried out in order to isolate a clonal pathway sensor
cell line. These pathway sensor cell lines serve as a valuable cell-based
assay platform, for subsequent screening and mechanism of action studies.
The Cignal Lenti Reporters are ready-to-transduce,
replication incompetent, HIV-based, VSV-G pseudotyped lentiviral particles.
The Cignal Lentiviral particles are safe to use. It is recommended that
they be treated as Risk Group Level 2 (RGL-2) organisms. Follow all
published RGL-2 guidelines for handling and waste decontamination. Details
on the requirements for creating a BSL-2 work environment are available in
the U.S. Department of Health and Human Services publication Biosafety
in Microbiological and Biomedical Laboratories, 4th edition.
The biosafety features engineered into these vectors include:
-
A deletion in the promoter/enhancer region of the U3 portion
of 3'LTR ensures self-inactivation of the lentiviral construct after
transduction and integration into the genomic DNA of target cells.
- The Cignal Lentiviral vector and plasmids expressing packaging proteins
contain no significant areas of homology, thereby minimizing any chance for
recombination.
-
None of the HIV-1 genes (gag, pol, rev) will be expressed in
transduced cells, as they are expressed from packaging plasmids lacking
packaging signal. Therefore, the lentiviral particles that are generated are
replication-incompetent
-
No virulence genes ( vpr, vif, vpu and nef) are present in
the Cignal Lentiviral vector.
| Feature |
Function |
| RSV-5' LTR; Hybrid Rous sarcoma Virus (RSV)
enhancer/promoter-U5 long terminal repeat |
Permits viral packaging and reverse
transcription of viral mRNA |
| Psi; Packaging signal |
Allow viral packaging |
| RRE; Rev response element |
Involved in packaging of viral transcript |
| cppt; Central polypurine tract |
Involved in nuclear translocation and
integration of transduced viral genome |
| Reporter gene (firefly luciferase or GFP) |
Allow quantification of transcription |
| hPGK; human phosphoglycerate kinase
eukaryotic promoter |
Permits high-level expression of the
mammalian selection marker (puromycin) |
| PuroR; puromycin resistance gene |
Can be used for mammalian selection |
| SIN/3'LTR; 3' self inactivating long terminal
repeat |
Modified 3'LTR that allows viral packaging
but self inactivates the 5'LTR for biosafety purpose. The element also
contains a polyadenylation signal for efficient transcription
termination |
| f1 ori; f1 origin of replication |
Allows episomal replication of plasmid in
eukaryotic cells
|
| AmpR; ampicillin resistance gene |
Allows selection of the plasmid in E.coli |
| TRE; Transcription response element |
Permits regulation of reporter gene
expression by a specific transcription factor |
| TATA box |
Act as an minimal promoter |
General performance
Average maximum response rate = 26.8
Average Z' factor at maximum response rate = 0.73
Average coefficient of variation (CV%) = 6.5%
Excellent signal to noise ratio
Cignal E2F reporter assay showed activation of E2F
cell cycle pathway: 293-H cells were transfected with E2F
reporter, negative control and positive control (for transfection protocol
refer our user manual). After 16 hours of transfection, medium was changed
to assay medium (Opti-MEM + 0.5% FBS + 0.1 mM NEAA + 1mM Sodium pyruvate +
100 U/ml penicillin + 100 ug/ml streptomycin). After 40 hours of
transfection, cells were treated with 10% serum, and 10% serum and 100 ng/ml
EGF for 6 hours. Dual Luciferase assay was performed, and promoter
activity values are expressed as arbitrary units using a Renilla reporter
for internal normalization. Experiments were done in triplicates, and the
standard deviation is indicated. Cignal E2F reporter assay measured 27
fold increase in E2F transcription activity in response to serum.
Back to Top
|
|
