和元李记

Lentiviral vector

原文链接：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).

微信截图_20240410110410.png

　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
Expressing cre	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.