Lentiviral Vectors

Lentiviruses (lenti- is slow in Latin) are genus of slow viruses of the Retrovidae family, characterized by a long incubation period. Lentiviruses, including the HIV (Human), SIV (simian,) FIV (feline), are all pathogenic to humans. Lentiviruses can deliver significant amount of genetic information into the DNA of the host cell, and can target both dividing and non-dividing cells (unlike Retroviruses that infect only dividing cells). The native envelop of Lentivirus based vectors (the HIV-1) contains the Gp120 glycoprotein which is recognized by the CD4 receptor on the target cells (Lymphocytes). In order to broaden the range of target cells the native envelop was replaced (pseudotyped) by vesicular stomatitis virus G (VSVG).
All these unique features render Lentiviruses as the most efficient method for gene delivery. They are used to replace a damaged/missing gene in gene therapy, and in basic science are used to introduce or silence genes in a wide verity of models. It is an excellent method replacing non-efficient transfections, used for in vivo labeling, transgenic mice, high throughput screening and more.

The attractive features of Lentiviruses, especially with HIV-1 based Lentivirus vectors, raises Biosafety issues. Laboratory workers handling Lentivirus are the risk group. Lentivirus penetration may occur:

  • Through the skin - via puncture or absorption (scratches, cuts, dermatitis, or other lesions)
  • Through mucous membrane - eyes, nose, and mouth.

The goal of the safety and environment unit is to bring about a safe working environment. This goal can be achieved by your comprehensive implementation of our "Biosafety guidance and Safety protocol" provided herein.

Biosafety Guidance & Safety Protocol for Research

The basic principle is to avoid contamination with the recombinant virus, which can spread through droplets or aerosol. Therefore, all the work should be contained within a BSC (Bio Safety Cabinet, hood) in a Lentivirus approved room. According to the decision of the Institutional Biological Committee, which was approved by the institutional safety committee, Lentivirus work will be carried out within the facilities of each department or of a group and is under the responsibility of the principal investigator. This room can also serve as general tissue-culture room.
We recommend using the third generation plasmids which allow the production of replication-deficient virus, for example Invitrogen Virapower system (recommended by the NIH). This system incorporates several safety features as follows:

  • Only 3 viral genes are used in vector system (gag, pol on one plasmid, and rev on another). Tat is not expressed in the system.
  • HIV-1 Env is replaced by the VSV-G gene.
  • The genes encoding structural and other essential genes are separated onto 4 plasmids.
  • The vector is “self-inactivating” due to a deletion in the 3' LTR (U3).

Specific instructions:

  1. The allocated tissue-culture room should be equipped for work with lentiviral vectors (see item 2). The room must be approved by the Biosafety officer.
  2. Obligatory equipment in the allocated tissue-culture room:
    • BSC Class II
    • Centrifuge and sealed safety centrifuge tubes
    • Microscope
    • Incubator
    • Biosafety waste disposal equipment.
    • A set of micro pipettes.
  3. Signs should be posted on the entrance stating:
    • The use of Lentivirus
    • Restricted entrance for authorized personnel only.
  4. Before starting your work:
    • Wear a laboratory-coat (preferably disposable) and 2 pairs of gloves
    • Prepare decontamination solutions as specified under decontamination
    • Prepare biohazard bag for solid infectious waste inside the BSC and another biohazard bag in the biohazard bucket.
    • Use of sharps is not recommended, if you can not avoid using sharps prepare a separate bucket for sharp disposal.
  5. Disposable equipment (pipettes, flasks/plates) must be decontaminated before discarding it to the biohazard bag (details under decontamination).
  6. Use tips and pipettes containing filter. Collect the used tips in a screw-cup plastic bottle (cleaned used plastic medium bottle), dispose into the biohazard bag after you have closed the bottle.
  7. Shut the T.C door while working with Lentivirus (collection and infection).
  8. No other vectors are allowed in BSC during the work with Lentivirus production.
  9. Do not leave virus-containing solutions unattended in the hood or in the centrifuge.
  10. All disposable personal protective equipment (gloves and disposable lab-coat) should be discarded in a biohazard bag located inside the approved room.
  11. You must not leave the Lentivirus approved room to any other room wearing the disposable lab-coat. You may leave the room wearing a clean fabric lab-coat and gloves.
  12. Cells for virus packaging and for infection should preferably be grown in flasks (not in plates).
  13. Plates/flasks should be placed in the incubator on a tray such as a sterile plate cover, bigger then your lentivirus containing plate.
  14. Pay special attention to avoid aerosols and splashes.
  15. Do not touch anything outside the BSC with contaminated gloves. Replace the gloves upon leaving the BSC and throw the gloves to the biohazard bag (inside the BSC). Wear new gloves for opening the incubator or using other facility in the room.
  16. To visualize the cells under microscope take the following steps:
    • Take the flask/plate to the microscope on the designated tray
    • Before leaving the microscope clean the stage with 70% ethanol
  17. Biohazard bags used in the hood must be sealed (not tightly) and transferred to the biohazard bag placed in the biohazard bucket. When full (do not over fill) seal and carry it inside the bucket to the red collection bin.
  18. Small volume liquid waste (up to 500 ml) may be decontaminated in the BSC using 500 ml cleaned used plastic medium bottle containing 1:10 of the liquid waste volume of 6% Sodium Hypochlorite, wait 30min and pour the decontaminated liquid to the sink.
  19. Large volumes of liquid waste may be decontaminated using a vacuum pump/line protected by double trap system. Never use glass pipettes. We recommend to use a 1000 μl disposable sterile tip attached to a 10 cm cut 5 ml pipette (pipette edge intact, facing the tip) permanently inserted in the rubber hose. The collection bottle should contain 6% Sodium Hypochlorite in an amount of 1:10 of the bottle volume (decontaminate for 1hr as above).
  20. At the end wash the rubber hose with 0.6% Sodium Hypochlorite.
  21. At the end of each session, wipe the hood, the incubator handle and the equipment used (microscope, centrifuge, etc) with 70% Ethanol.
  22. After removing the gloves, wash hands with soap and water.
  23. Centrifugation must be carried out in aerosol sealed tubes in a centrifuge located in the approved room.
  24. If you have to use an ultracentrifuge (located in another room) you should follow these instructions:
    • Post a clear sign notifying the use of Lentivirus, as well as information specifying the length of your run, your name and group, your mobile phone number
    • Balance the plastic tubes by adding an exact volume of medium containing virus inside the BSC of the approved room
    • When filling, do not exceed 75% of the tubes volume
    • Gently insert the plastic tube into the metal bucket, paying attention to avoid splashes
    • Cover the metal bucket using the provided metal screw and O-ring
    • Spray the exterior of the metal bucket and covers with 70% Ethanol, change your glove and the rest of your personal disposable protection, leaving them in the Lentivirus approved room. You may leave the room wearing a clean fabric lab-coat to access departmental ultracentrifuge room
    • Take the sealed tubes to the centrifuge using the provided stand and place them inside a carrier such as a cooler
    • After centrifugation open the lentivirus containing tubes only inside the BSC in the Lentivirus approved room. Clean the metal centrifuge buckets and covers (inside and outside) with 70% Ethanol in the BSC. The ultracentrifuge and the rotor should be decontaminated using 70% Ethanol even if you did not notice a spill.

 

Decontamination

In general, decontamination is done using Sodium Hypochlorite. WIS warehouse offers a 6% Sodium Hypochlorite solution item number 020015987.

  • Keep in the hood a bottle of freshly prepared 0.6 % Sodium Hypochlorite (it is good for one week only)
  • Prepare 70% Ethanol sprayer
  • Keep a facial shield in the approved laboratory

Liquid waste decontamination

Decontamination of liquid waste (conditioned medium and virus containing samples) should be performed in final 0.6% Sodium Hypochlorite for 1 hr. Decontaminating a small volume spill
Cover the spill with paper towel and gently pour on top 0.6% Sodium Hypochlorite.
Collect the paper towel to the biohazard bag.

 

 

Decontaminating a large volume spill:

Wear a face protection shield (prevents splashes to your face while decontaminating), cover the spill with paper towel and gently pour on top 6% Sodium Hypochlorite (pour1/10 of the spill volume).
Collect the paper towel to the biohazard bag.

 

 

Decontaminating a splash:

Cover the splash with paper towel, spray on top with either 0.06% Sodium Hypochlorite or 70% Ethanol sprayer. Collect the paper towel to the biohazard bag.
Solid waste decontamination
Used pipettes has to be dipped for 30min in an oval bucket (item number 050003076) containing 0.06% Sodium Hypochlorite before discarding it to the biohazard bag. The volume of the Sodium Hypochlorite in the bucket should be enough to cover the lower edge of the pipette. .
Used plates/flasks have to be decontaminated with 0.6% Sodium Hypochlorite for 30min.
Discard all decontaminated solid waste in the biohazard bag.