The most common viral vectors used in biomedical research are lentivirus, adenovirus, adeno-associated virus (AAV) and retroviruses, and each vector type has distinct advantages and disadvantages.

Considerations

Many different factors affect the ideal vector type for each experiment. Some of the key things to consider include: What is the target cell type? Are they dividing? Do you want transient transduction or stable integration into the host genome? Will an immune response to the virus affect your experiment? What transduction efficiency is needed?

Lentivirus

This is the most common viral system for gene delivery. Lentivirus is a highly efficient vehicle for introducing genes permanently into mammalian cells. This system has broad tropism (i.e. can infect a wide range of cell types) for both dividing and non-cycling cells, with relatively low cellular immune response. Live lentivirus can be produced at high titer (>10⁸ TU/ml), and transduction efficiency for cultured cells can approach 100% under optimal conditions.

Retrovirus

Similar to lentivirus are retrovirus such as MMLV (Moloney Murine Leukemia Virus). These viruses also have broad tropism and stably integrate into the host cell genome, allowing long-term, stable gene expression. However, MMLV does not efficiently infect non-dividing cells, and can produce a more significant cellular immune response than lentivirus. Additionally, the viral titer of MMLV and similar retrovirus is usually only about one tenth that of lentivirus.

Adenovirus

These viral vectors are non-integrating, remaining in an episomal state within infected target cells, with no disruption of the host genome. Expression of transduced genes is usually transient, particularly in rapidly dividing cells which will lose adenovirus over time. Many cell types (both diving and non-dividing) can be transduced with adenovirus, but certain cell types lack the appropriate cell surface receptor, and cannot be efficiently transduced. Cellular and in vivo immune responses due to adenoviral infection can be significant, and may interfere with certain experiments. Adenovirus can be produced at very high titer (>10⁹ TU/ml) which allows for very efficient transduction of susceptible target cells.

Adeno-associated virus

AAV is another non-integrating, episomal virus usually producing transient gene expression. Unlike adenovirus, AAV has very low immunogenicity and is almost entirely nonpathogenic in vivo. A major practical advantage is that AAV can in most cases be handled in biosafety level 1 (BSL1) facilities. This viral vector has broad tropism, toward both dividing and non-dividing cells, and the relatively high titer of most AAV preparations makes this an efficient gene delivery system.

References

1. Warnock JN, Daigre C, Al-Rubeai M. (2011) Introduction to viral vectors. Methods Mol Biol. 737:1-25

2. Walther W and Stein U. (2000) Viral vectors for gene transfer: a review of their use in the treatment of human diseases. Drugs 60:249-71

For single-color experiments, green-emitting fluorescent proteins (FP) are the most common choices. Although EGFP is the most popular green FP, EmGFP (Emerald GFP) is a better choice for most applications due to its superior folding. If a red FP is preferred, mCherry is a very good choice for most experiments. The brighter dTomato works well in situations where dimerization of the FP is acceptable.

For multicolor experiments, researchers must carefully consider the spectral properties of FPs. Care must be taken that the FPs (and dyes used in the experiments) are distinguishable using the microscope filters or other hardware that will be used for detection. Frequently, three-color experiments will make use of a red and a green FP (e.g. mCherry + EGFP), together with a DNA dye such as DAPI. Another effective multicolor scheme would include a red, a yellow, and a cyan FP (mCherry + YPet + CyPet). These FP combinations are easily separable on most fluorescence microscopes or flow cytometers.

Recommendations: Single Color: mCherry or dTomato or EmGFP or EGFP

Three-color (Red, Green, Blue): mCherry + EmGFP or EGFP + DAPI (DNA dye)

Three-color (Red, Yellow, Cyan): mCherry + YPet + CyPet

Special Applications

Förster resonance energy transfer (FRET) is a specialized application of FPs that is highly dependent on experimental details, such as an appropriate FP pair and relative positioning of the FPs within the protein structure. CyPet and YPet are optimized FPs developed for use as a donor/acceptor pair, and we recommend this pair as a starting point for FRET experients.

Fluorescent Protein Properties

Notes: * In practice, many factors can influence brightness in the context of an experiment (e.g. FP maturation, pH, photobleaching). This value is based on experimental measurements of purified FPs under idealized conditions.

References

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3. Cormack BP, Valdivia RH, Falkow S. FACS-optimized mutants of the green fluorescent protein (GFP). Gene. 1996;173(1 Spec No):33-8.

4. Ward WW, Cormier MJ. An energy transfer protein in coelenterate bioluminescence. Characterization of the Renilla green-fluorescent protein. J Biol Chem. 1979 Feb 10;254(3):781-8.

5. Strack RL, Strongin DE, Bhattacharyya D, Tao W, Berman A, Broxmeyer HE, Keenan RJ, Glick BS. A noncytotoxic DsRed variant for whole-cell labeling. Nat Methods. 2008 Nov;5(11):955-7.

6. Shaner, N.C. et al., Improved monomeric red, orange, and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat Biotechnol, 2004.

7. Campbell RE, Tour O, Palmer AE, Steinbach PA, Baird GS, Zacharias DA, Tsien RY. A monomeric red fluorescent protein. Proc Natl Acad Sci U S A. 2002 Jun 11;99(12):7877-82.

8. Nguyen AW, Daugherty PS. Evolutionary optimization of fluorescent proteins for intracellular FRET. Nat Biotechnol. 2005 Mar;23(3):355-60.