Gamma-Retroviral Vector Guide

Gamma-retroviruses are a subtype of retrovirus and belong to the Retroviridae family. Gamma-retrovirus infection is associated with many serious illnesses. Common types include murine leukemia virus (MLV), murine sarcoma virus (MSV), and feline leukemia virus (FeLV).

Retroviruses are partially characterized by their ability to integrate their genomes into hosts in order to continue their lifecycle and replicate. This feature can be harnessed for stable delivery of various genes, mutations, or treatments into cells, and has been widely used in the research community for decades.

Wildtype gamma-retroviruses have been engineered into gamma-retroviral vectors that can be safely used in laboratory settings. These engineered gamma-retroviral vectors have many aspects that make them useful research tools in a variety of cell types and models, such as stable long-term expression in a host and low immunogenicity.

This guide contains many viral vector-specific terms and acronyms, so if you're new to viral vectors or simply need a refresher, we've included a glossary at the end!

Browse retroviral plasmids available at Addgene.

Gamma-Retrovirus Versus Lentivirus

Lentiviruses and gamma-retroviruses fall under the Retroviridae family. The genome of retroviruses is made of RNA. This retroviral RNA is reverse transcribed into DNA before being integrated in the genome of a host. Going from RNA to DNA is the reverse of what you would expect in biology — hence the use of “retro”. Retroviruses have simpler genomes, containing only the necessary packaging genes, while lentiviruses also contain accessory genes specific to each virus type.

From an experimental standpoint, the main difference between lentiviruses and gamma-retroviruses is that lentiviruses are capable of infecting non-dividing and actively dividing cell types, whereas gamma-retroviruses can only infect mitotically active cell types. This means that lentiviruses can infect a greater variety of cell types than retroviruses.

Both lentiviruses and gamma-retroviruses use the same packaging genes. However, they are different viruses and require different isoforms of these packaging components. Therefore, gamma-retroviruses may not be efficiently packaged by lentiviral packaging systems, and vice versa. Gamma-retroviral vectors do not have defined “generations” of plasmids, as lentiviral vectors do.

For more about lentiviruses, see Addgene's lentiviral vector guide.

Gamma-Retroviral Vectors

The genome of gamma-retroviruses ranges from 9–11 kb, encoded on RNA (Figure 1). This RNA is reverse transcribed into the provirus, the cDNA that will be integrated into a host genome. This single strand of RNA contains packaging genes and the long-terminal repeats (LTRs) necessary for integration. Wildtype gamma-retroviruses contain the gag, pol, and env genes necessary for gamma-retroviral production, and scientists have made changes to these components for increased safety when producing viral vectors in a laboratory setting.

Figure 1: Wild-type gamma-retrovirus genome. Created with BioRender.com.

All gamma-retroviral vectors use the common packaging genes gag, pol, and env. The LTRs flank all of these required genes, and anything in between will be integrated into the host genome. To produce gamma-retroviral vectors, these required components have been separated into distinct plasmids for safety to reduce the chance of creating replication-competent retroviruses. For a summary of all gamma-retroviral plasmid components, see the Gamma-Retroviral Plasmid Elements table.

In order to produce gamma-retroviral vectors, you need three plasmids (Figure 2):

• Transfer plasmid — contains transgene, sgRNA, or shRNA of interest flanked by LTRs; ~8 kb packaging capacity
• Packaging plasmid — contains packaging genes gag and pol
• Envelope plasmid — contains packaging gene env; usually VSV-G due to wide infectivity

VSV-G (vesicular stomatitis virus G protein) is the most common envelope gene, due to a wide range of infectivity for different cell types (known as tropism).

Figure 2: Gamma-retroviral plasmids. Created with BioRender.com.

Gamma-Retroviral Vector Production

Cloning

Cloning your transgene, gRNA, or shRNA of interest into the transfer plasmid can be done with most standard cloning methods, including restriction enzyme, Gibson Assembly, or Gateway. Some transfer plasmids may have limited restriction sites or may only be compatible with certain cloning methods (such as a Gateway destination vector), so be sure to confirm your chosen plasmid is compatible with cloning methods available in your lab.

When cloning your plasmids, be sure to use recombination-deficient bacteria strains, such as NEB Stable cells. These strains reduce the frequency of homologous recombination of unstable regions, like the LTRs found in retroviral plasmids. This will ensure that the repeats will be maintained and often results in a greater yield of DNA. However, if the plasmid contains a Gateway cassette containing the ccdB gene, a ccdB-resistant strain is necessary.

For more information on cloning and working with plasmids, visit Addgene’s Molecular Biology Reference.

Production Using HEK293T

Gamma-retroviral vectors can be packaged directly in human embryonic kidney 293T (HEK293T) cells (Figure 3A), similar to lentiviral packaging methods. The three plasmids described above (envelope, packaging, and transfer) are co-transfected into the HEK293T packaging cell line. This system provides the greatest flexibility to pseudotype gamma-retrovirus using different envelopes to modify tropism, as the env gene can easily be changed.

The HEK293T cells will produce the virus and release it into the supernatant. After an initial media change, the supernatant containing the viral particles is removed and stored or centrifuged to concentrate. Crude or concentrated virus can then be used to transduce the cells of interest, either with or without determining the viral titer.

For more detailed protocols, see Addgene’s viral vector protocols.

Production Using Packaging Cell Lines

Gamma-retroviral vectors can be packaged using helper-free cell lines (Figure 3B). These cell lines have been engineered to stably express gag-pol and/or env, eliminating the need to deliver these genes on separate plasmids (in trans). This method, therefore, reduces the number of plasmids that are required in the transfection step of the viral packaging cell line.

For example, Phoenix™ is a popular second-generation gamma-retroviral packaging cell line developed by Garry Nolan at Stanford. These cell lines contain gag-pol and either an ecotropic envelope, Phoenix-ECO (for infection of mouse and rat cells), or an amphotropic envelope, Phoenix-AMPHO (for the infection of mammalian cells). Using this system, viral vectors are produced in just a few days. Another variant, Phoenix-gp, contains only gag-pol and enables additional flexibility in pseudotyping.

Figure 3: Gamma-retroviral vector packaging methods. Created with BioRender.com.

Viral Vector Integration

Much like lentiviral vectors, gamma-retroviral vectors integrate at random locations throughout the genome. Gamma-retroviral vectors tend to prefer transcription start sites, such as promoters and enhancers. Many times, integration sites are found in proto-oncogenes. This can lead to activation of the oncogene and possible development of cancer.

Pseudotyping

Tropism dictates which types of host cells the gamma-retroviral vector will infect. This can be altered by changing the envelope gene, a process called pseudotyping. VSV-G is by far the most common envelope gene, as it has a wide range of infectivity across many cell types and enhances the stability of the viral particles. However, VSV-G is toxic under long-term, constitutive expression. In order to make stable cell lines, VSV-G is often put under an inducible promoter. Depending on the cell type, there are other envelope gene options, including:

• Gibbon Ape Leukemia Virus (GALV) — T and B cell transduction
• Murine Leukemia Virus (MuLV) and HIV glycoprotein — CD4+ cell transduction

Pseudotyped retroviral vectors are commonly used in gene therapy applications to more specifically direct delivery of gene therapies.

Common Uses of Gamma-Retroviral Vectors

Due to their integration, gamma-retroviral vectors are useful research and clinical tools thanks to their long-term expression of a transgene.

Stable Cell Lines

Gamma-retroviral vectors are often used to make stable cell lines. Like their popular lentiviral counterparts, gamma-retroviral vectors offer stable, long-term expression of a transgene due to their integration into a host’s genome. The process of generating stable cell lines with gamma-retroviral vectors is essentially the same as lentiviral vectors.

Many gamma-retroviral vectors have selectable markers, such as the puromycin resistance gene, conferring antibiotic resistance to infected host cells. When these antibiotics are added to the growth medium of the host cells, they kill off any cells that have not incorporated the retroviral genome and the cells that survive can be expanded to create stable cell lines. The cell lines will have the gamma-retroviral genome and the genetic information encoded by that genome.

Antibiotic resistance is not the only type of selectable marker. Fluorescent reporters, such as GFP, are another common selection marker. Instead of selection by an antibiotic, fluorescence-activated cell sorting (FACS) is used to sort cells expressing GFP and later, sorted cells are expanded into a cell line.

You can find more information in Addgene’s stable cell line protocol. Addgene’s protocol is for lentiviral vectors, but a similar method can be applied when using gamma-retroviral vectors.

Gene Therapy

Gamma-retroviral vectors are a popular choice in gene therapy applications. There are many approved gamma-retroviral gene therapies to treat disease like cancer or immunodeficiency disorders. Delivery of chimeric antigen receptor T cell (CAR-T) therapy is a particularly popular application of gamma-retroviral vectors.

Gamma-retroviral vectors have been shown to be safe delivery vehicles and provide stable integration of the intended therapeutic. While similar to lentiviral vectors, they offer the benefit of having well-established protocols for fast and efficient generation of large-scale, high-titer preps. This is thanks to established retroviral production cell lines that are able to keep up with the demand of clinical studies and commercial purposes. The use of helper-free production cell lines also saves time in downstream purification, as extraneous plasmid DNA will not have to be removed.

Viral Vector Safety

The two main safety concerns surrounding the use of gamma-retroviral vectors are:

• The potential for generation of replication-competent gamma-retroviral vectors
• The potential for oncogenesis

The potential for generation of replication-competent gamma-retroviral vectors is very low thanks to the design of the plasmids. The genes used for viral vector production are separated into three different plasmids containing the transfer, envelope, or packaging components. The transfer plasmid encodes the gene of interest and contains the sequences that will be incorporated into the host cell genome but cannot produce functional viral particles without the genes encoded in the envelope and packaging plasmids. Unless multiple recombination events occur between the packaging, envelope, and transfer plasmids, and that resulting construct is packaged into a viral particle, it is not possible for viral vectors to replicate and produce more virus after the initial infection.

Some retroviral transfer plasmids that have been deposited with Addgene are self-inactivating (SIN). These plasmids have a deletion in the 3' LTR of the viral genome that is transferred into the 5' LTR after one round of reverse transcription. After incorporation into a host cell, this deletion prevents further transcription of the full-length virus.

The potential for oncogenesis is largely based on whether or not the specific insert contained within the retroviral transfer plasmid is an oncogene. Additional consideration for oncogenes should be made on a case-by-case basis. Retroviruses carry a slightly elevated risk of integrating into proto-oncogenes.

As with most experiments, infection risks occur with contact to mucous membranes or broken skin. Needle sticks and ripped gloves are common points of entry. Biosafety should always be considered with respect to the precise nature of experiments being performed. Your biosafety office can provide more information on your institution's best practices with regard to gamma-retroviral-related research.

See Addgene’s Biosafety Resource Guide for more information and resources on viral safety.

Resources and References

Resources on addgene.org

• Gamma-retroviral Plasmids Collection
• Viral Vectors 101 eBook
• Addgene's Biosafety Resources

Resources on Addgene's blog

• Popular Retroviral Vectors and Their Uses in Scientific Research
• Gamma-retroviral Packaging Systems
• The Retroviral Lifecycle
• Types of Viruses
• Viral Vector Elements
• More Viral Vectors 101 topics
• More Retroviral Vector topics

Addgene protocols

• Generating Stable Cell Lines Using Lentiviral Vectors
• Addgene's Viral Protocols

References

Coffin, J. M., Hughes, S. H., & Varmus, H. E. (Eds.). (1997). Principles of Retroviral Vector Design. In Retroviruses. Cold Spring Harbor Laboratory Press. NIH Bookshelf

De Ravin, S. S., Su, L., Theobald, N., Choi, U., Macpherson, J. L., Poidinger, M., Symonds, G., Pond, S. M., Ferris, A. L., Hughes, S. H., Malech, H. L., & Wu, X. (2014). Enhancers are major targets for murine leukemia virus vector integration. Journal of Virology, 88(8), 4504–4513. https://doi.org/10.1128/jvi.00011-14 PMID: 24501411

Gilroy, K. L., Terry, A., Naseer, A., De Ridder, J., Allahyar, A., Wang, W., Carpenter, E., Mason, A., Wong, G. K., Cameron, E. R., Kilbey, A., & Neil, J. C. (2016). Gamma-Retrovirus integration marks cell Type-Specific cancer genes: a novel profiling tool in cancer genomics. PLoS ONE, 11(4), e0154070. https://doi.org/10.1371/journal.pone.0154070 PMID: 27097319

LaFave, M. C., Varshney, G. K., Gildea, D. E., Wolfsberg, T. G., Baxevanis, A. D., & Burgess, S. M. (2014). MLV integration site selection is driven by strong enhancers and active promoters. Nucleic Acids Research, 42(7), 4257–4269. https://doi.org/10.1093/nar/gkt1399 PMID: 24464997

Maetzig, T., Galla, M., Baum, C., & Schambach, A. (2011). Gammaretroviral vectors: Biology, technology and application. Viruses, 3(6), 677–713. https://doi.org/10.3390/v3060677 PMID: 21994751

Mekkaoui, L., Tejerizo, J. G., Abreu, S., Rubat, L., Nikoniuk, A., Macmorland, W., Horlock, C., Matsumoto, S., Williams, S., Smith, K., Price, J., Srivastava, S., Hussain, R., Banani, M. A., Day, W., Stevenson, E., Madigan, M., Chen, J., Khinder, R., . . . Pule, M. (2022). Efficient clinical-grade gamma-retroviral vector purification by high-speed centrifugation for CAR T cell manufacturing. Molecular Therapy — Methods & Clinical Development, 28, 116–128. https://doi.org/10.1016/j.omtm.2022.12.006 PMID: 36620071

Mirow, M., Schwarze, L. I., Fehse, B., & Riecken, K. (2021). Efficient pseudotyping of different retroviral vectors using a novel, CoDOn-Optimized gene for chimeric GALV envelope. Viruses, 13(8), 1471. https://doi.org/10.3390/v13081471 PMID: 34452336

Schnierle, B. S., Stitz, J., Bosch, V., Nocken, F., Merget-Millitzer, H., Engelstädter, M., Kurth, R., Groner, B., & Cichutek, K. (1997). Pseudotyping of murine leukemia virus with the envelope glycoproteins of HIV generates a retroviral vector with specificity of infection for CD4-expressing cells. Proceedings of the National Academy of Sciences, 94(16), 8640–8645. https://doi.org/10.1073/pnas.94.16.8640 PMID: 9238030

Wu, X., Li, Y., Crise, B., & Burgess, S. M. (2003). Transcription start regions in the human genome are favored targets for MLV integration. Science, 300(5626), 1749–1751. https://doi.org/10.1126/science.1083413 PMID: 12805549

Gamma-Retroviral Plasmid Elements

Plasmid Type	Element	Delivery relative to transgene	Purpose
Transfer plasmid	LTR	in cis	Long terminal repeat; comprised of a U3-R-U5 structure and are found on each side of the provirus. The U3 (unique 3’) contains sequences necessary for activation of viral genomic RNA transcription. R is the repeat region.
	U3	in cis	Unique 3’; contains sequences necessary for activation of viral genomic RNA transcription. Removal of this region in the 3’ LTR creates self-inactivating viral vectors.
	R	in cis	Repeat region with repeated sequence.
	U5	in cis	Unique 5'; in some newer plasmids, this region is removed in 5’ LTRs and replaced with a heterologous promoter (usually CMV or RSV).
	5' LTR	in cis	Acts as an RNA pol II promoter; the transcript begins at the beginning of R, has a 5' cap structure, and proceeds through U5 and the rest of the provirus. Some newer plasmids use a hybrid 5' LTR with a constitutive promoter such as CMV or RSV.
	3' LTR	in cis	Terminates transcription started by 5' LTR by the addition of a polyA tract just after the R sequence.
	WPRE	in cis	Woodchuck hepatitis virus post‐transcriptional regulatory element; stimulates the expression of transgenes via increased nuclear export.
	Psi (Ѱ)	in cis	RNA packaging signal; recognized by nucleocapsid proteins and essential for efficient viral packaging.
	cPPT	in cis	Central polypurine tract; recognition site for proviral DNA synthesis. Increases transduction efficiency and transgene expression.
Packaging plasmid	gag	in trans	Precursor structural protein of the retroviral particle containing matrix, capsid, and nucleocapsid components.
	pol	in trans	Precursor protein containing reverse transcriptase and integrase components.
Envelope plasmid	env	in trans	The viral envelope gene; typically vesicular stomatitis virus G glycoprotein (VSV-G), an envelope protein with broad tropism used to pseudotype most gamma-retroviral vectors.

Glossary

Term	Definition
Gamma-retrovirus	A retrovirus from the Retroviridae family. Characterized by long incubation and the eventual cause of serious diseases.
Immunogenicity	The ability of a molecule or substance to induce an immune response in the body.
in cis	In the context of viral vector production, in cis refers to genetic elements located in the same plasmid as the gene of interest.
in trans	In the context of viral vector production, in trans refers to genetic elements provided outside of the plasmid containing the gene of interest (i.e., other plasmids, packaging cell line).
Infection	The natural process of entry and multiplication of a pathogen, like viruses or bacteria, within a host organism, leading to disease.
Provirus	The genetic material (cDNA for retroviruses) that will be integrated into a host cell’s genome.
Pseudotyping	The production of viral vectors (or viruses) using viral envelope proteins from different species. Used to alter infectivity and tropism.
Replication-competent	The ability of a virus (or viral vector) to replicate and reproduce within host cells. In viral vectors, can occur from a spontaneous crossover event between the transfer plasmid and genes provided in trans.
Transduction	The process of artificially introducing foreign DNA into eukaryotic cells using viral methods.
Transformation	The process where bacteria take up DNA from their environment.
Tropism	The specific types of cells and tissues that a virus can infect and replicate in.
Viral vector	The modified form of a wild-type virus used to deliver genetic material into different host cells. Engineered for safety and efficiency.

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Content last reviewed on 9 June 2025.