Retro-X FAQs

Wild-type moloney murine leukemia virus (MMLV) contains ~8.2 kb of genome, including both LTRs. Artificially creating a genome larger than 8.2 kb will result in unstable viral particles and a dramatic drop in viral titer. For recombinant retroviruses such as those generated using Retro-X systems, much of the viral genome has been replaced with other useful sequences such as selection markers or fluorescent proteins, but enough space remains for cloning transgenes. Since each pRetro-X and pRetro-Q vector contains different useful sequences, the available space for cloning your transgene varies.

To determine the maximum recommended transgene size, consult your retroviral vector map and determine the position of the end of the 3' LTR (the end of the 5' LTR is always at position 1), then subtract that from 8.2 kb. For example:

• The 3' LTR of pQCXIP ends at 4.3 kb, so ~3.9 kb of space remains for you to clone in your gene.
• The 3' LTR of pRetroX-IRES-ZsGreen1 ends at 3.3 kb, so ~4.9 kb of space remains for you to clone in your gene.

Approximately 12–17%, depending on the vector backbone. pLXRN contains 11.9% of the wild-type MMLV genome, and the MSCV vectors contain 16.9% of the wild-type MMLV genome.

No. The GOI or selection marker expression cassette in the retroviral vector should not include a poly(A) signal or any other transcription termination signal, since it is located in the 3' LTR of the retroviral vector. A transcription termination sequence in the middle of a retroviral construct can lead to the premature cleavage of viral genomic mRNA, resulting in the loss of downstream sequences that are essential for efficient packaging and transduction of the target cells.

Bicistronic lentiviral vectors contain an optimized internal ribosome entry site (IRES) that permits your gene of interest and a second gene* to be coexpressed from a single mRNA transcript. Although translation initiation occurs almost exclusively at the 5' cap of eukaryotic mRNAs, the IRES attracts ribosomes to begin translation at a second, internal location. The result is that two proteins are expressed simultaneously from a single bicistronic mRNA transcript.

*This is often the gene for a fluorescent protein or drug selection marker.

Typically, expression of the gene downstream of the IRES is optimal only if the gene cloned upstream of the IRES is 0.5–1.5 kb in size. Therefore:

• You can expect reduced expression of the downstream gene when a gene larger than 2 kb is cloned upstream of the IRES.
• It is better to clone a stuffer into the vector than to use the empty vector as a control.

Takara Bio's retroviral packaging cell lines are routinely tested to ensure the absence of replication-competent virus, using control NIH 3T3 pLAPSN stable cells. Please see each packaging cell line's Certificate of Analysis (located under the Documents tab on the Retroviral Packaging Systems product page) for more information.

Our MMLV-based retroviral vectors are compatible with MMLV-based retroviral packaging systems. However, we cannot guarantee the performance or biosafety of retroviral packaging systems from sources other than ours.

Yes, Takara Bio's retroviral packaging cell lines and retroviral systems are compatible with MMLV-based retroviral vectors from other sources.

Viral envelopes are classified by the receptors they use to enter host cells. Your virus' range of infectivity (cell tropism) is determined by its envelope, which is in turn conferred by the packaging cell line or system. You should choose your packaging cell line or system based on your target cells.

* Available as part of the Retro-X Universal Packaging System and the Pantropic Retroviral Expression System.

** The GP2-293 Cell Line requires cotransfection of one of several types of envelope proteins and can produce virus with various tropisms.

Yes, as long as the cells are mitotically active. For quiescent or slowly dividing cells, we recommend lentiviral-mediated gene delivery.

Yes. You can use a retroviral vector as a regular plasmid, via transfection. However, this negates one of the advantages of retroviral transduction: reproducible integration of individual copies of your transgene. During stable integration, plasmid vectors break randomly and integrate into the cellular genome nonspecifically with respect to the original vector backbone. By contrast, proviral DNA from a retrovirus is integrated into the transduced cell's genome precisely via the 5' and 3' LTRs, ensuring the integrity and linearity of the integrated expression construct.

SIN vectors have a 5' LTR that is active in transfected cells but inactive in transduced cells. During packaging, the 5' LTR is active and generates multiple copies of viral genomic mRNA molecules, which are packaged into retroviral particles. When cells are infected with the packaged virus, reverse transcription transfers a U3 deletion from the 3' LTR to the 5' LTR, thus inactivating it in the integrated proviral DNA.

The GOI and drug resistance expression cassettes of the Retro-X Q Vectors in transduced cells are driven from internal promoters only.

If a Retro-X Q Vector is introduced into the cells via transfection, its 5' LTR will be active.

Yes. The Retro-X Concentrator can be used to concentrate a lentivirus according to the Lenti-X Concentrator Protocol-At-A-Glance. 

The Retro-X Concentrator can concentrate retroviral supernatants created with all commonly used envelopes. Fold concentration varies for retroviruses pseudotyped with various envelopes.

Retro-X Concentrator can concentrate retroviral supernatants created with all commonly used envelopes. A retrovirus expressing DsRed2 was packaged with different retroviral envelopes using the Retro-X Universal Packaging System (Cat. # 631530), and harvested at 72 hr posttransfection. Retro-X Concentrator was used to concentrate dualtropic (10A1), amphotropic (Ampho), ecotropic (Eco), and VSV-G (pantropic) pseudotyped retrovirus samples from 10 ml down to 100 µl. Crude and concentrated viral stocks were then titrated on HT1080 or NIH 3T3 cells and analyzed via flow cytometry at 72 hr postinfection to determine the percentage of positive cells.

No. The Retro-X qRT-PCR Titration Kit is not compatible with MSCV-based retroviruses. It does, however, work with all MMLV-based retroviral vectors.

MOI (multiplicity of infection) is the number of infectious viral units per cell at the time of infection.

IFU (infectious units) indicate the ability of the virus to infect cells and express protein. You can determine the infectious viral titer (IFU/ml) by measuring the expression of a reporter or a specific protein by flow cytometry, immunostaining, etc.

CFU (colony forming units) determine the virus' ability to transduce cells and express protein. Cells harboring integrated proviral DNA and carrying an antibiotic resistance gene survive the presence of the drug and form cell colonies. Lentiviral titer in CFU/ml is determined by counting drug-resistant cell colonies.