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Original link：Metaorganisms - lentiviral vectors
Lentivirus is a type of viral vector modified from human immunodeficiency virus (HIV), which is a type of retrovirus. The genome is RNA, and its toxic genes have been removed and replaced by exogenous target genes. Lentivirus belongs to the pseudotype virus, which can integrate foreign genes into the genome to achieve stable expression and has the characteristics of infecting dividing and non dividing cells.
After the lentivirus genome enters the cell, it is reverse transcribed into DNA in the cytoplasm, forming a DNA pre integration complex. After entering the nucleus, DNA is integrated into the cell genome. The integrated DNA is transcribed into mRNA and returned to the cytoplasm to express the target protein or produce small RNA. The gene expression or small RNA interference mediated by lentivirus is sustained and stable, and divides with the division of the cell genome (Figure 1).
Figure 1 Process of Slow Virus Packaging and Infection of Cells
Slow virus vectors have the characteristics of wide host range, low immunogenicity, large gene capacity, and long-term expression. Capable of effectively infecting various types of cells in culture, including tumor cells, liver cells, myocardial cells, neurons, endothelial cells, stem cells, etc.
The infection of lentivirus has the characteristic of integration, which can effectively integrate foreign genes into the host chromosome, achieve persistent expression, and thus construct stable cell lines for the study of gene cellular function. The use of lentiviral vectors to modify T cells can be used for CAR-T cell therapy.
Slow virus packaging and purification
Slow virus packaging uses a three plasmid or four plasmid system for packaging, collects cell culture supernatant, and concentrates, purifies, and collects viruses through high-speed centrifugation.
Slow virus titer detection
Detection method: Quantitative PCR is used to detect the copy number of exogenous DNA in the cell genome after infection
Experimental principle: lentivirus mediates the integration of exogenous genes into the target cell genome through reverse transcription
Advantages of lentiviral vectors
① Long expression time: Slow viruses can integrate exogenous genes into the host cell genome, achieving stable gene expression for a long time without loss during cell division and passage. It is the preferred choice for cell experiments and is commonly used to construct stable strains.
② High safety: No pathogenicity was found, and lentiviral vectors were used to modify T cells for CAR-T cell therapy.
③ Low immunogenicity: Direct injection into live tissue does not easily cause an immune response and is suitable for animal experiments.
Comparison of Biological Characteristics of Different Viruses
Carrier selection
HarmonyBio has a rich range of lentiviral products for manipulating both coding and non coding genes, such as lncRNA microRNA、circRNA， The following slow virus vectors are available for you to choose from (not limited to this list):
Regulatory methods | Carrier number | Carrier name (order of carrier components) | Eukaryotic resistance | fluorescence | Promoter | Default label | Carrier capacity |
Overexpression | GL127 | pSLenti-CMV-EGFP-3xFLAG-WPRE | N/A | EGFP | CMV | 3FLAG | 4.8kb |
GL121 | pSLenti-EF1-EGFP-CMV-MCS-WPRE | N/A | EGFP | CMV | N/A | 4.4kb | |
GL129 | pSLenti-CMV-mCherry-3xFLAG-WPRE | N/A | mCherry | CMV | 3FLAG | 4.8kb | |
GL119 | pSLenti-CMV-MCS-3xFLAG-PGK-Puro-WPRE | Puro | N/A | CMV | 3FLAG | 4.4kb | |
GL120 | pSLenti-SFH-EGFP-P2A-Puro-CMV-MCS-3xFLAG-WPRE | Puro | EGFP | CMV | 3FLAG | 3.5kb | |
GL107 | pSLenti-EF1-EGFP-P2A-Puro-CMV-MCS-3xFLAG-WPRE | Puro | EGFP | CMV | 3FLAG | 3.7kb | |
GL109 | pSLenti-EF1-EGFP-F2A-Puro-CMV-MCS-WPRE | Puro | EGFP | CMV | N/A | 3.8kb | |
GL122 | pSLenti-EF1-EGFP-F2A-BSR-CMV-MCS-WPRE | blasticidin | EGFP | CMV | N/A | 3.9kb | |
GL130 | pSLenti-CMV-EGFP-3xFLAG-PGK-Puro-WPRE | Puro | EGFP | CMV | 3FLAG | 3.7kb | |
GL132 | pSLenti-EF1-EGFP-F2A-Puro-WPRE2-CMV-MCS | Puro | EGFP | CMV | N/A | 3.7kb | |
GL123 | pSLenti-EF1-mCherry-P2A-Puro-CMV-MCS-3xFLAG-WPRE | Puro | mCherry | CMV | 3FLAG | 3.7kb | |
GL125 | pSLenti-CMV-mCherry-3xFLAG-PGK-Puro-WPRE | Puro | mCherry | CMV | 3FLAG | 3.7kb | |
GL133 | pSLenti-EF1-mCherry-F2A-Puro-WPRE2-CMV-MCS | Puro | mCherry | CMV | N/A | 3.7kb | |
H114 | pLenti-CMV-Luc2-IRES-Puro-WPRE | Puro | N/A | CMV | N/A | 2.2kb | |
GL124 | pSLenti-EF1-Luc2-F2A-Puro-CMV-MCS-WPRE | Puro | N/A | CMV | N/A | 2.8kb | |
Expressing cre | H126 | pLenti-CMV-NLS-Cre-3xFLAG-WPRE | N/A | N/A | CMV | 3FLAG | 3.9kb |
H15108 | pLenti-CMV-DIO-MCS-WPRE | N/A | N/A | CMV | 3FLAG | 5.3kb | |
Three standards | H7656 | pLenti-CBh-3xFLAG-Luc2-tCMV-mNeonGreen-F2A-Puro-WPRE | Puro | mNeonGreen | CBh | 3FLAG | 1.3kb |
H7657 | pLenti-CBh-3xFLAG-Luc2-tCMV-tdTomato-F2A-Puro-WPRE | Puro | tdTomato | CBh | 3FLAG | 0.6kb | |
H9911 | pLenti-CBh-3xFLAG-Luc2-tCMV-mNeonGreen-F2A-BSR-WPRE | blasticidin | mNeonGreen | CBh | 3FLAG | 1.5kb | |
H9912 | pLenti-CBh-3xFLAG-Luc2-tCMV-tdTomato-F2A-BSR-WPRE | blasticidin | tdTomato | CBh | 3FLAG | 0.8kb | |
CircRNA overexpression | H8384 | pLenti-EF1-EGFP-F2A-Puro-CMV-S-circRNA-WPRE | N/A | EGFP | CMV | N/A | 3.0kb |
H8807 | pLenti-CMV-S-circRNA-WPRE | N/A | N/A | CMV | N/A | 4.8kb | |
H8399 | pLenti-EF1-EGFP-F2A-Puro-CMV-L-circRNA-WPRE | Puro | EGFP | CMV | N/A | 1.0kb | |
H8810 | pLenti-CMV-L-circRNA-WPRE | N/A | N/A | CMV | N/A | 2.8kb | |
MiRNA overexpression | GL109 | pSLenti-EF1-EGFP-F2A-Puro-CMV-MCS-WPRE | Puro | EGFP | CMV | N/A | N/A |
H3919 | pCLenti-U6-miR30(miRNA)-CMV-EGFP-F2A-Puro-WPRE | Puro | EGFP | U6 | N/A | N/A | |
H119 | pLenti-CMV-TurboGFP-IRES-Puro-miR30(miRNA)-WPRE | Puro | TurboGFP | CMV | N/A | N/A | |
H3928 | pCLenti-U6-miR30(miRNA)-CMV-mCherry-F2A-Puro-WPRE | Puro | mCherry | U6 | N/A | N/A | |
H146 | pCLenti-EF1-Puro-CMV-EGFP-3xFLAG-Sponge(miRNA)-WPRE | Puro | EGFP | CMV | 3FLAG | N/A | |
H7505 | pCLenti-U6-TuD(miRNA)-CMV-EGFP-F2A-BSR-WPRE | blasticidin | EGFP | CMV | N/A | N/A | |
H7506 | pCLenti-U6-TuD(miRNA)-CMV-EGFP-F2A-Puro-WPRE | Puro | EGFP | CMV | N/A | N/A | |
Overexpression (Tet on) | H125 | pLenti-TRE-EGFP-EF1-rtTA3-IRES-Puro-WPRE | Puro | EGFP | TRE | N/A | 2.0kb |
H121 | pLenti-TRE-EGFP-3xFLAG-PGK-Puro-WPRE | Puro | EGFP | TRE | 3FLAG | 3.3kb | |
H2057 | pLenti-EF1-rtTA3-IRES-Puro-WPRE | Puro | N/A | EF1 | N/A | N/A | |
ShRNA interference | GL401 | pCLenti-U6-shRNA-CMV-Puro-WPRE | Puro | N/A | U6 | N/A | N/A |
GL404 | pCLenti-U6-shRNA-CMV-EGFP-WPRE | N/A | EGFP | U6 | N/A | N/A | |
GL427 | pSLenti-U6-shRNA-CMV-EGFP-F2A-Puro-WPRE | Puro | EGFP | U6 | N/A | N/A | |
GL428 | pSLenti-U6-shRNA-CMV-mCherry-F2A-Puro-WPRE | Puro | mCherry | U6 | N/A | N/A | |
H7615 | pCLenti-U6-shRNA-CMV-mCherry-F2A-BSR-WPRE | blasticidin | mCherry | U7 | N/A | N/A | |
CRISPR Knockout | H5070 | pLenti-U6-spgRNA v2.0-CMV-Puro-P2A-3xFLAG-spCas9-WPRE | Puro | N/A | U6 | N/A | N/A |
H7072 | pLenti-U6-spgRNA v2.0-CMV-BSR-P2A-3xFLAG-spCas9-WPRE | blasticidin | N/A | U6 | N/A | N/A | |
H6825 | pLenti-U6-spgRNA v2.0-CMV-sfGFP-P2A-3xFLAG-spCas9-WPRE | N/A | sfGFP | U6 | N/A | N/A | |
H5068 | pLenti-U6-spgRNA v2.0-CMV-EGFP-WPRE | N/A | EGFP | U7 | N/A | N/A | |
H5450 | pLenti-CMV-Puro-P2A-3xFLAG-espCas9_1.1-WPRE | Puro | N/A | CMV | 3FLAG | N/A | |
CRISPRa transcriptional activation | H9517 | pCLenti-U6-gRNA-MS2-EFS-dCas9-VP64-T2A-BSD-WPRE | blasticidin | N/A | U6 | N/A | N/A |
E2577 | pCLenti-EF1a-MCP-P65-HSF1-T2A-Hygro-WPRE | hygromycin | N/A | EF1a | N/A | N/A | |
H7281 | pLenti-CMV-dCas9-VP64-T2A-Puro-WPRE | Puro | N/A | CMV | N/A | N/A |
Conventional applications
In vitro infected cells
Slow virus vectors can effectively infect various types of primary cells and most cell lines, including tumor cells, liver cells, myocardial cells, neurons, endothelial cells, stem cells, etc.
The infection of lentivirus has integration characteristics, which can effectively integrate foreign genes into the host chromosome, thus achieving persistent expression and constructing stable transgenic cell lines. In vitro cellular functional experiments, including cell proliferation, invasion, migration, apoptosis, etc., are conducted. Stable expression of target genes in tumor cell lines can further construct tumor animal models for in vivo gene function validation, pharmacokinetic analysis, in vivo imaging, and other detection to further study the occurrence, development, metastasis, and drug treatment effects of tumors in vivo.
Common MOI List
Cell name | Chinese name | Chronic viral infection (MOI) (Reference value) |
MGC80-3 | Human gastric cancer cells | 10 |
HT-29 | human colon cancer cell | 20 |
RKO | Human colon adenocarcinoma cells | 10 |
Caki-1 | Human renal clear cell carcinoma skin metastatic cells | 10 |
5637 | Human bladder cancer cells | 10 |
U-118 MG | Human astroblastoma | 10 |
RWPE-1 | Normal human prostate epithelial cells | 20 |
HeLa | Human cervical cancer cells | 10~20 |
Ca Ski | Human cervical cancer intestinal metastatic cells | 10 |
MCF7 [MCF-7] | Human breast cancer cells | 10 |
A549 | Human non-small cell lung cancer cells | 10 |
NCI-H1299 | Human non-small cell lung cancer cells | 20 |
U-87 MG | Human astroblastoma | 10 |
U251 | Human glioma cells | 10 |
A172 | Human glioblastoma cells | 10 |
K-562 | Human chronic myeloid leukemia cells | 10 |
HL-60 | Human myeloid leukemia cells | 10 |
GES-1 | Human gastric epithelial cells | 20 |
U266 | Human myeloma cells | 20 |
CAL 27 | Human tongue squamous cell carcinoma cells | 20 |
PC9 | Human lung cancer cells | 5 |
PC14 | Human lung cancer cells | 10 |
MADB-106 | Rat breast cancer cells | 20 |
F98 | Rat glioma cells | 20 |
MHCC-97H | Human liver cancer cells (high metastasis) | 10~20 |
MOI stands for multiplicity of infection, also known as the plural number of infections, which refers to the ratio of the number of viruses to cells during infection. Generally, in an experiment, when a certain cell is infected and reaches 80%, it is defined as the MOI value of that cell, that is, the ratio of the number of viruses to the number of cells; The amount of virus added (μ l)=number of cells x MOI/titer (.../ml) x 1000
Application cases of lentivirus in tumor research
1.Nature Communications. (IF=12.353). Shi Y,et.al. (2017). Tumour-associated macrophages secrete pleiotrophin to promote PTPRZ1 signalling in glioblastoma stem cells for tumour growth.[lentivirus, glioma]
Slow virus interference： Lenti-shNT/shPTN/shPTPRZ1
2. Hepatology. (IF=14.079). Ma JZ,et.al. (2016). METTL14 suppresses the metastatic potential of HCC by modulating m6 A-dependent primary miRNA processing. [lentivirus, liver cancer]Slow virus overexpression/interference:pLV-CMV-PGK-EGFP-T2A-puro/ pLV-U6-shRNA-CMV-EGFP-T2A-puro
The application of lentivirus in the nervous system (in vivo injection)
1．Nature Neuroscience. (IF=19.912). Ding XL,et.al. (2017). Activity-induced histone modifications govern Neurexin-1 mRNA splicing and memory preservation.[Slow virus, learning and memory]
Virus type: lentivirus
Carrier name：LV-Suv39h1-RNAi（Uncertain details）
Injection site: DG area of hippocampus
Virus injection volume:2 μL，9.98 × 108 TU/mL
Testing time: 2 weeks
2．Nature Communications. (IF=12.353). Yao XH,et.al. (2016). Electrical coupling regulates layer 1 interneuron microcircuit formation in the neocortex.[lentivirus, neural circuit]
Virus type: lentivirus
Carrier name：LV- CX36-shRNA-EGFP（Uncertain details）
Injection site: P1 mouse neocortex L1
Virus injection volume：1μL
Testing time: 2 weeks
3．Molecular Psychiatry. (IF=11.64). Guo DJ,et.al. (2019). Autism-like social deficit generated by Dock4 deficiency is rescued by restoration of Rac1 activity and NMDA receptor function. [Chronic virus, autism]
Virus type: lentivirus
Carrier name:Lenti-CMV-EGFP-P2A-3FLAG-Rac1
Injection site: CA1 area of mouse hippocampus
Virus injection volume: 1 μ L
Testing time: 4 weeks
Slow virus extension service
The production and quality control of metabiotic lentiviruses have adopted internationally recognized standard processes, using a three plasmid or four plasmid system for packaging in 293T cells. The obtained virus particles were purified by ultracentrifugation and the viral gene copies were titrated by qPCR. In general, the titer of our lentivirus is between 108~109TU/ml, which can fully meet the requirements of various experiments.
