What Is a Slow Virus?

Lentivirus vector is a gene therapy vector developed based on HIV-1 (Human Immunodeficiency Virus Type I virus). Different from general retroviral vectors, it has the ability to infect both dividing and non-dividing cells .

Lentivirus

Lentivirus vector is a gene therapy vector developed based on HIV-1 (Human Immunodeficiency Virus Type I virus). Difference in general

• Lentivirus

  Lentiviruses are also widely used in the study of RNAi expression. Because some types of cells

  Commonly used viral vectors are adenoviruses, retroviruses and lentiviruses. Retroviral vectors can only infect

  For some cells that are difficult to transfect, such as primary cells, stem cells, and undifferentiated cells, the transduction efficiency of the target gene can be greatly improved, and the probability of integration of the target gene into the host cell genome is greatly increased. This is RNAi, The study of cDNA cloning and reporter genes provides an advantageous approach.

  Screening of stable cell lines;

  Provide high-quality virus liquid containing the target gene for the experiment of living animal models;

  1 operation of lentivirus

  2 specifications for safe use of lentivirus

  3 Suspended cell infection method summary

  4 related technical terms (for details, please refer to the company website FAQ)

  5 Related parameters of cell culture vessels

  1. Lentivirus operating manual

  1.1 Storage and dilution of lentivirus:

  1.1.1 Virus storage: After receiving the virus solution, users can use the lentivirus to perform experiments in a short period of time.

  It should be stored at 4 for a long time; if it needs to be stored for a long time, it should be stored at -80 (the virus is placed in a cryopreservation tube and sealed with a parafilm)

  A. The virus can be stored at -80 ° C for more than 6 months; however, if the virus is stored for more than 6 months, we recommend that

  Virus titer needs to be re-titrated before use

  B. Repeated freezing and thawing will reduce virus titer: each freeze and thaw will reduce virus titer by 10%; therefore, during virus use

  Repeated freezing and thawing should be avoided only as far as possible. To avoid repeated freezing and thawing, we strongly recommend that customers receive the virus according to the amount of each use.

  Perform packing.

  1.1.2 Virus dilution: When the user needs to dilute the virus, please take out the virus and put it in an ice bath to melt it.

  Cells are mixed in PBS or serum-free medium (containing serum or double antibodies does not affect viral infection) and stored at 4 ° C (please try as far as possible)

  Use within three days) Use after dispensing.

  1.2 Lentivirus for In Vitro Experiments: Infection and Culture of Primary Cells and Lines

  cell

  1.2.1 Lentiviruses have different affinity for various cells and tissues. Users can pass the lentivirus provided by Invabio before using

  Consult relevant literature to learn about the affinity of lentivirus for your target cells, the multiplicity of infection (MOI value) and in vivo (In

  Vivo) The amount of virus needed for the injection. If there is no relevant literature support, you can obtain the appropriate multiplicity of infection (MOI value) through pre-infection experiments (using a 24-well plate to detect the virus' affinity for the target cells)

  1.2.2 Pre-experiment of lentivirus-infected target cells

  1.2.2.1 Pre-experimental precautions for target cells infected with lentivirus

  A. To determine the affinity of lentivirus for target cells, it is necessary to set cells with high affinity for lentivirus (HEK293T, Hela) as control cells for parallel experiments.

  B. When performing lentiviral infection experiments, it can be diluted with complete medium (for culture of target cells); theoretically, it contains

  Complete media of serum, monoclonal antibodies, or other nutritional factors do not affect the efficiency of lentivirus infection.

  C. The unit of virus provided by Invabio is TU / ml, which means the number of biologically active virus particles per ml. Such as:

  The virus titer is> 1X10 ⁸ TU / ml, which means that there are at least 1X10 ⁸ biologically active lentiviral particles per ml of virus solution.

  1.2.2.2 Using a 24-well culture plate as an example, pre-infection experiments with target cells and HEK293T cells

  Set different infection wells according to different MOIs before the experiment, and calculate the amount of virus required according to the MOI and the number of cells, such as

  It is necessary to dilute the virus stock solution with PBS solution or serum-free medium

  On the first day, prepare the cells: inoculate several wells in a 24-well culture plate, and inoculate 3 ~ 5X10 ⁴ target cells in each well.

  The fusion rate of the cells is about 50%, and the volume of the culture medium per well is 100 l; the degree of fusion of the cells during virus infection is about

  About 70%.

  The next day, prepare the virus: Remove the virus stored at 4 ° C and centrifuge it for 20 seconds using a desktop centrifuge (so that the virus is completely suspended in the

  The bottom of the centrifuge tube is sufficient); if it is frozen at -80 , the virus needs to be thawed on ice before use. Also based on experiments

  The actual situation of the chamber will be diluted into the culture medium according to the MOI accurately calculated, and as much as possible to ensure that the obtained

  The total volume of the lentivirus culture medium is the minimum volume in order to obtain the best infection efficiency.

  The next day, infect the target cells: After the virus is ready, remove the cells from the incubator, and first observe the cell growth status.

  If the cells are in good condition, start the experiment.

  a Pipette the exact volume of virus solution into the prepared medium

  b Aspirate the medium from the culture vessel (if the cells are growing well and the density is appropriate, do not change the solution)

  cAdd the calculated virus solution to the target cells and control cells, respectively.

  d After mixing, put in a carbon dioxide incubator (37 ° C, 5% CO2) and incubate overnight

  Note: The state of the cells before infection has a great impact on the final infection effect, so be sure to ensure that the cells before adding the virus

  In good growth

  You can also add a mixture of the prepared culture medium and lentivirus directly to the culture vessel

  Lentivirus has low infection efficiency on target cells. It can increase virus infection efficiency by increasing the MOI value, but when

  When the MOI is higher than 20, we recommend that customers add ploybrene (about 8 g / ml) to the medium to increase the virus infection.

  effectiveness.

  A. On the third day, replace the culture medium: Generally, the culture medium containing lentivirus is replaced with normal culture medium after 24 hours;

  Observe the cell status after infection. If lentivirus has obvious toxic effects on cells and affects cell growth status,

  Shortly after the virus is added for 4 hours, replace the fresh culture medium and continue the culture (recommended to replace it in 8-12 hours). First change

  After the culture, if the lentivirus does not have a significant toxic effect on the cells, the culture is changed according to the normal culture conditions.

  B. On the sixth day, the detection of infection efficiency: observe the fluorescence in an inverted fluorescence microscope to estimate the efficiency of the target cells infected by lentivirus;

  How to choose a vector that cannot carry the Marker Gene because the target gene is large, you can use Real-time

  The expression of the target gene was detected by RT-PCR to evaluate the infection efficiency.

  note:

  Invabio provides a viral vector with GFP green fluorescent protein, which can be inverted by the user after 96 hours of virus infection

  Observe the green fluorescence of GFP with a fluorescence microscope to observe the infection of the target cells by the virus.

  If the lentiviral vector carries other Marker Genes such as RFP \ BFP, you can use a fluorescence microscope at the corresponding excitation light wave.

  Observe the fluorescence expression

  The expression time of lentivirus is slow and the time required for fluorescent expression is long. It is recommended to observe the fluorescent expression 96 hours after infection. infection

  After the cells can be continuously cultured for a week, the target cells can be determined by observing the expression time and intensity of fluorescence.

  Infection. During the infection, please change the cells in time according to the growth of the cells to ensure good cell growth.

  status.

  2. Safe use of lentivirus

  The lentivirus provided by Invabio is a "suicide" virus, that is, the virus will not infect other cells after infecting the cells of interest, nor will it

  Host cells are used to generate new virus particles. The virulence genes in the lentivirus have been knocked out and removed by the foreign gene of interest

  Instead, it is a pseudotyped virus and therefore has no toxic effect. But the virus still has a potential biological danger,

  Invabio recommends against using pseudotyped viruses that encode genes that are known or may cause cancer. Unless a basis is fully recognized

  Because it is certainly not carcinogenic, otherwise it is not recommended to use pseudotyped viruses for biological experiments. If in doubt, please contact us.

  Please refer to the following for experimentation:

  2.1. It is best to use biological safety cabinets for virus operations. If you use a normal ultra-clean workbench to operate the virus,

  Please do not turn on the exhaust fan.

  2.2. Wear laboratory clothes, masks, and gloves when handling viruses.

  2.3. Take special care when handling viruses to avoid aerosols or splashes. If there is a virus on the clean bench during operation

  Contamination, please wipe off immediately with 70% ethanol and 1% SDS solution. Touched the tip of the virus,

  Centrifuge tubes, culture plates, and soak the culture solution in 84 disinfectant solution or 1% SDS overnight and discard.

  2.4. Follow these steps when observing cell infection with a microscope: screw the culture bottle or cover the culture

  board. After cleaning the outer wall of the flask with 70% ethanol, observe and take a picture at the microscope. Leave microscope experiment

  Before the stage, clean the microscope experimental stage with 70% ethanol.

  2.5. If centrifugation is needed, use a well-sealed centrifuge tube, or centrifuge after sealing with a parafilm.

  Also try to use a centrifuge in a tissue culture room.

  2.6. After removing gloves, wash your hands with soap and water.

  3. Summary of suspension cell infection methods

  1.1 Collect the cells by centrifugation in a 1.5ml tube according to the amount of cells and then dilute the cells with 100-200ul of serum-free medium

  Precipitation, subject to complete immersion of cells in culture medium

  1.2 Calculate the number of virus particles according to MOI, pipette the virus solution into the cells, and place the 1.5ml tube in a 37 ° C incubator.

  Incubate for 30 minutes

  1.3 Aspirate the mixed solution from the tube and add it to the petri dish or well

  1.4 Add sufficient amount of fresh culture medium

  Change fluid after 1.512 hours

  Observe the cell positive rate after 1.696 hours

  4. Related technical terms

  (For details, please refer to the company website FAQ)

  Common terms:

  MOI: Multiple Virus Infections The traditional concept of MOI originated from the study of phage-infected bacteria. Phage

  The ratio to the number of bacteria, which is the average number of phages infected by each bacterium. The number of phage units is pfu.

  It is generally considered that MOI is a ratio and has no unit. In fact, the implicit unit is pfu number / cell. MOI was later

  Commonly used in the study of virus-infected cells, meaning the ratio of virus to cell number at the time of infection

  MOI uses this concept.

  However, the MOI has different meanings due to the different ways of representing the number of viruses. Can produce fine

  Cytolytic effect viruses such as herpes simplex virus and so on still use pfu to express the number of viruses, so the meaning of their MOI

  Same as traditional concept.

  MOIstands for "Multiplicity of Infection." It is the ratio of infectiousvirus particles to the number of cells being infected. The recommended MOI isin the range of 0.1 (meaning that you inoculate 1 virus particle for every 10cells) to 0.01. The general theory behind MOI is to introduce one infectiousvirus particle to every host cell that is present in the culture. However,

  more than one virus may infect the same cell which leaves a percentage of cells uninfected. This

  occurrence can be reduced by using a higher MOI to ensure that every cell is infected.

  5. Related parameters of cell culture vessels

  Flask / Dish	Surface (mm)	Cell number	Media Volume
  96 well plate	50	1.5-5.0 × 10	100; l
  48 well plate	100	3.0 × 104-1.0 × 10	200; l
  24 well plate	200	8.0 × 104-2.0 × 10	500; l
  12 well plate	401	1.6-4.0 × 10	1.0 ml
  6 well plate	962	3.0-8.0 × 10	2.0 ml
  35mm	962	3.0-8.0 × 10	2.0 ml
  60mm	2827	1.0-2.5 × 10	6.0 ml
  100mm	7854	2.5-6.4 × 10	10.0 ml

  Packaging and purification of lentiviral vectors

  1 experimental process

  The lentiviral shuttle plasmid and its auxiliary packaging original carrier plasmid were prepared. The four plasmid vectors were extracted with high-purity endotoxin-free and co-transfected with 293T cells. The complete medium was replaced 8 hours after transfection. The cell supernatant containing lentiviral particles was concentrated to obtain a high-titer lentivirus concentrate, and the viral titer was detected by quantitative PCR after infection with Hela cells. In vivo and in vitro experiments have different requirements for lentivirus titers, and lentiviral particles of different titers can be obtained by concentration during production.

  2 experimental materials

  2.1 Lentiviral vectors, packaging cells and strains

  Lentiviral vector system (Tronolab) The virus packaging system is a four-plasmid system consisting of pRsv-REV, pMDlg-pRRE, and pMD2G, and the target interference plasmid. The target interference plasmid can express green fluorescent protein (GFP).

  Cell line 293T, a lentiviral packaging cell, is an adherent-dependent epithelial-like cell, and the growth medium is DMEM (containing 10% FBS). Adherent cells grow and proliferate to form monolayer cells after culture.

  Strains; E. coli strain DH5. Used to amplify lentiviral vectors and auxiliary packaging vector plasmids.

  2.2 Main reagents

  Reagent name

  Manufacturer

  Article number

  Packaging cell 293T cell line

  Shanghai Institute of Cells, Chinese Academy of Sciences

  E. coli strain DH5

  Invitrogen

  Fetal bovine serum

  Invitrogen

  Trypsin

  Invitrogen

  Restriction enzyme

  Fermentas

  T4DNA ligase

  NEB company

  M0202V

  Plasmid DNA extraction kit

  Axygen

  Preparation of Competent Kits

  BIOSCIENCES

  Gel recovery kit

  Qiagen

  1kb / 100bpladder

  Fermentas

  2.3; main instrument

  equipment name

  Manufacturer

  Article number

  PCR instrument

  Applied Biosystems

  Electrophoresis

  Bio-rad

  Fluorescence inverted microscope

  Olympus

  Refrigerated ultracentrifuge

  Hitachi

  Cell incubator

  healforce

  Gel imager stabilized DNA electrophoresis instrument

  Tianneng

  Constant temperature shaker

  Shanghai Boxun Instrument

  Heated Incubators

  Shanghai Boxun Instrument

  Pipette

  eppendorf

  Electric thermostatic water bath

  Shanghai Yiheng Experimental Equipment Co., Ltd.

  Digital gel processing system

  Tianneng Scientific Instruments

  Disposable dishes

  Cell-star

  Flask

  3 Basic information and map of lentiviral vector system plasmid

  The virus packaging system is a four-plasmid system consisting of pRsv-REV, pMDlg-pRRE, and pMD2G, and a destination shuttle plasmid. The shuttle plasmid (target shuttle plasmid) contains green fluorescent protein (GFP); pRsv-REV, pMDlg-pRRE, and pMD2G contain the necessary components for virus packaging.

  3.1 Shuttle plasmid: the target shuttle plasmid

  3.2pRsv-REV

  3.3pMDlg-pRRE

  3.4pMD2G

  4 Lentivirus production experimental steps

  4.1 Lentiviral packaging cells were transfected with trypsinized 293 T cells in logarithmic growth phase at a cell density of 0.5 × 10; re-seeded in a 25 mL 15cm cell culture dish, 37 ° C, 50 mL / L CO2 incubator to cultivate;

  4.2 Preparation of four plasmid DNA solutions in the lentivirus packaging system;

  pRsv-REV 10 g,

  pMDlg-pRRE 15 g,

  pMD2G 7.5 g,

  Interference plasmid 20 ug

  Add sterile water to make up to 1800 L, and then add 200 L of CaCl2 (2.5 mol / L) solution, mix well, add 2000 × L of 2 × BBS buffered saline solution, and let stand at room temperature for 20-30 minutes;

  4.3; Transfection when the cell density reaches 60% to 70%. Transfer the mixed solution of DNA and calcium phosphate to the culture solution containing monolayer cells, mix well, discard the culture solution containing the transfection mixture after 12 h Add 15 mL of PBS, discard after shaking, and repeat this step 3 times;

  4.4 Add 15 mL of cell culture solution containing 100 mL / L calf serum to each bottle of cells, and continue to culture for 48 h.

  4.5 Collect the supernatant of 293T cells transfected for 72 h;

  4.6 Centrifuge the collected supernatant at 4 ° C, 4000 g for 10 min, and collect the supernatant;

  4.7 Filter the supernatants of various RNAi plasmids through 0.45 m filters;

  4.8 Centrifuge in a 40 mL ultracentrifuge tube at 4 ° C and 25000 r / min for 20 min.

  4.9 Then dissolve in 500 ul Dmem with ice PBS solution, and resuspend the virus pellet in 500 ul PBS overnight at 4 ° C.