General Guideline about Viral Vectors in Cell Immortalization

The pressure and need to develop stable cell lines has led to numerous approaches to produce stable, well-characterized cells as biomedical research tools. Big pharmaceutical companies in particular rely on stable cell lines for drug screening and toxicology studies. Due to the large numbers of cells that can be generated, embryonic stem cells and induced pluripotent stem cells invoke some powerful arguments for their utility, while there are also some limitations with respect to consistent and reliable differentiation into target cells of interest. To this end, production of cell lines using primary cells, coupled with inducible transgenic technologies, can impart regulatory control over cell division and offer an alternative strategy. Cell immortalization technology based on gene transfer has been successfully used to generate cell lines from a wide variety of cell types.

Primary cells are known to be resistant to transfection, but can be transduced by recombinant viral vector, particularly adenoviral and lentiviral vectors. To facilitate cell immortalization, scientists have developed comprehensive, ready-to-use viral vectors for cell immortalization. The following sections outline the basic characteristics of different viral vectors.

Recombinant Adenoviral Vector

Recombinant adenoviral vector has proven to be the most efficient viral vector to date. All types of human cells (except for blood cells lacking the adenovirus receptor) can be transduced with adenoviral vectors at 100% efficiency. However, adenoviral vectors cannot integrate into the target cell genome, leading to transient transgene expression. The vector DNA will be degraded or diluted with subsequent cell division in host cells. Thus, primary cells transduced by Adeno-SV40 or Adeno-hTERT can only express SV40 T antigen or hTERT for 1-2 weeks, depending on the rate of cell division.

Recombinant Retroviral Vector

Since retroviral vectors cannot transport across the nuclear membrane, retroviral vectors are capable of transducing actively dividing cells. During cell division, the nuclear membrane is disintegrated and thus the viral DNA can access the genome. Once the nucleus is bypassed, retroviruses can be integrated into the host genome efficiently, resulting in permanent and stable gene expression. However, the transduction efficiency of target cells using retroviral vectors is low, especially when cell division is slow.

Recombinant Lentiviral Vector

The newly developed lentiviral vectors can be used to transduce both dividing and non-dividing cells, as lentiviral vectors can actively pass though the nuclei membrane. Furthermore, like a retroviral vector, lentiviral vector will integrates into the host cell genome it enters the nucleus. Therefore, lentiviral vectors are becoming increasingly popular in both in vitro and in vivo applications of gene transduction. One disadvantage of lentiviral vectors is the insertion size. For most lentiviral vectors, the maximum insertion size is 5.0 kb. Insertion size longer than 3.0 kb will significantly decrease virus titers. As the SV40 genome is over 5.0 kb, the expected Lenti-SV40 titer is relatively low.

Assay Procedure

The following procedure outlines how to infect target cells with cell immortalizing genes by using retroviral and lentiviral vectors.

• Thaw the recombinant retrovirus supernatant in a 37°C water bath and remove it once thawed.
• Prepare polybrene storage solution with a concentration of 0.8 mg/mL.
• Infect the target cells in a 6-well plate with 2 mL/well supernatant in the presence of 2-10 μg/mL polybrene. Place the remainder viral supernatant in the fridge for the second infection in the afternoon.

  Note: Polybrene is a polycation that neutralizes charge interactions and increases binding between the pseudo-viral capsid and the cellular membrane. The optimal concentration of polybrene depends on the cell type and may need to be determined empirically (usually 2-10 μg/mL). Excessive exposure to polybrene (>12 hours) can be toxic to certain cells.

• After 6-8 hours, remove the viral supernatant (from the first infection) and re-infect the cells with 2 mL of fresh supernatant (with polybrene).

  Note: For lentiviral vector, one infection (incubate overnight) works well for most target cells. Dilute lentiviral vector with fresh complete medium (1:1) if cytotoxicity is a problem.

• The next day, remove viral supernatant and add the appropriate complete growth medium to the cells and incubate at 37°C.
• After 72 hours incubation, subculture the cells into 100 mm dishes and add appropriate selection drug for stable cell-line generation.
• For the EGFP control retrovirus, the selection marker is puromycin. For most cell lines, the selection concentration is between 1-10 μg/mL.
• 10-15 days after selection, pick clones for expansion and screen for positive ones.

  Note: After thawing, it is recommend that the supernatant not be frozen again for future use, since the virus-titer will decrease significantly. Infection of MDA-MB-468 cells would be a good control for the EGFP virus.

The following procedure outlines how to utilize adenoviral vectors to infect target cells with cell immortalizing genes.

Note: Target cells can be transduced by recombinant adenoviral vectors either by viral supernatant or purified adenovirus. For most in vitroapplication, target cells can be transduced at 100% efficiency with viral supernatant. However, purified high titer of adenoviral preparation is necessary for in vivoapplications, which requires adenoviral preparations free of FBS or other contaminants.

• Prepare target cells in a 6-well plate or a 10 cm dish at 70% confluency at the time of transduction.
• Aspirate the culture medium and overlay with viral culture supernatant (1 mL for 6 well plate, 4-5 mL for 10 cm dishes) to cover the cells for 1 hour in an incubator.
• Remove the medium containing the virus and replace it with fresh complete medium.
• Gene transduction can be evaluated 48-72 hours after transduction by different assays, such as western blot, qPCR analysis, or microscope observation if there is a color generating reporter gene.

Table 1. The selection markers commonly used in viral vectors.

Note: Antibiotic selection should not be necessary. Non-immortalized cells will die after several passage rounds as they will reach senescence. In these cases, passaging can be considered as the selection process. If antibiotics must be used, the optimal concentrations will need to be evaluated by the end user for their cells.

References

1. Goldring M. B. Immortalization of human articular chondrocytes for generation of stable, differentiated cell lines. Cartilage and Osteoarthritis, 2004, 100: 23-35.
2. Wall B. I. et al.; Recent advances in conditional cell immortalization technology. Cell & Gene Therapy, 2016, 339-355.

For research use only. Not for any other purpose.