One is tagged with a C-terminal MYC/DDK tag (Cat# PS100064) for easy antibody detection and purification. OriGene recommends 4C5-AntiDDK high-affinity mAb (Cat #TA50011) for this work. The other form is tagged with either GFP or RFP which can be visualized under a fluorescence microscope and also be detected by Western blotting with anti-GFP or anti-RFP specific antibodies.
The fusion protein is under a CMV promoter for strong constitutive expression or EF1 promoter. The ORF insert can also be easily shuttled into over 70 destination vectors to create different tagging options via a simple “cut-and-ligate” mechanism/ OriGene offers the RapidShuttling Kits specifically for this use (origene.com/rapid-shuttling-kit). SV40 or allows for replication in mammalian cells, the chloramphenicol resistant gene confers the selection of the plasmid in E. coli. Both 5’ and 3’ truncated LTR can be used for viral RNA transcription and packaging of viral particles.
Day 1, plate target cells in three 10 cm plates at a density that will produce approximately 60% confluency in 24 hours. Note: other size formats can also be used depending on the nature of your experiment. Adjust the reagent amount accordingly.
2. Day 2, Remove the growth media from the plates prepared the day before. To plate 1, add 4.5 mL of fresh growth medium and 0.5 mL of Lentiviral particles; To plate 2, add 4.0 mL of growth medium and 1 mL of Lentiviral particles; To plate 3,
add 2.5 mL of growth medium and 2.5 mL of Lentiviral particles (for a low titer viral preparation, the amount of virus added can be increased to 5 mL). Mix the solution with gentle swirling.
3. Incubate the cells at 37 oC with 5% CO2 for 4 hours. Remove the transduction medium and add 10 mL of fresh growth medium. Incubate the cells for three more days.
Frequently Asked Questions
Is there any safety issue with this pLenti vector?
Answer: The pLenti vector is a third-generation lentiviral vector and it is the safest lentiviral vector because both LTRs are truncated. Please contact the biosafety office at your institution prior to the use of the pLenti vector for permission and for further institution-specific instructions.
BL2/(+) conditions should be used at all times when handling lentivirus. All decontamination steps should be performed using 70% ethanol/1% SDS. Gloves should be worn at all times when handling lentiviral preparations, transfected cells, or the combined transfection reagent and lentiviral DNA.
What is unique about the 3rd generation of Lentiviral vectors?
Answer: The 3rd generation lentiviral vectors are safer than the 2nd generation vectors. The 3rd generation packaging systems express gag and pol from one packaging vector. and rev from another. The 3rd generation packaging systems DO NOT express tat (Trans-Activator of Transcription).
What cell line should be used in order to produce lentivirus?
Answer: HEK293T cells are commonly used to produce lentivirus. The HEK293T cell line for producing lentiviral particles can be obtained from ATCC (www.atcc.org).
How do I propagate the pLenti vector in E. coli?
Answer: The lentiviral vector can be amplified using high-efficiency, competent E. coli cells (≥ 1×108 CFU/μg DNA) following the manufacturer’s transformation protocol. Plate the transformants on LB-agar plates supplemented with 34 μg/ml chloramphenicol.
Can I use the pLenti vector for stable selection in mammalian cells?
Answer: Only a subset of the pLenti vectors have mammalian selectable markers and those without a mammalian selection marker can not be used for mammalian selection. You can make stable cell lines using the pLenti-C-Myc-DDK-IRES-Puro vector. You might also be able to get stable cells by GFP sorting using the pLenti-C-Myc-DDKIRES-GFP vector.
How do I clone an insert into the pLenti vector?
Answer: The multiple-cloning site of the pLenti vector is compatible with OriGene’s PrecisionShuttling system, a simple cut-and-ligation process. Please refer to the corresponding protocols in the TrueORF application guide.
What is the size limit for the ORF that is to be cloned into the pLenti vector?
Answer: In general, lentiviral vectors have the capacity to accommodate an insert of 9 kb. However, ORFs larger than 4kb will dramatically decrease the packaging efficiency.
Can pLenti vectors be used in direct transfections as opposed to making viruses?
Answer: OriGene’s pLenti vectors can also be used in transient transfections to achieve expression of the transgene. This usually involves lower levels of protein production due to diminished transfection efficiency.
What is the difference between a lentivirus and a retrovirus?
Answer: Lentiviruses are a subtype of retrovirus. The main difference between lentiviruses and standard retroviruses from an experimental standpoint is lentiviruses are capable of infecting both non-dividing and actively dividing cell types whereas standard retroviruses can only infect mitotically active cell types.
Both lentiviruses and standard retroviruses use the gag, pol, and env genes for packaging. However, the isoforms of these proteins used by retroviruses and lentiviruses are different and lentiviral vectors may not be efficiently packaged by each other’s packaging systems.
Can I use a second-generation packaging system with the pLenti vectors?
Answer: Yes, a second-generation packaging system should work with OriGene’s third-generation Plenti vectors although we have not explicitly tested this. You can use OriGene’s third-generation packaging kit, cat# TR30002 for pLenti vectors.