Department of Cell Biology and Human Anatomy, School of Medicine, University of California, Davis
Barrilleaux, B., Knoepfler, P. Transduction of Human Cells with Polymer-complexed Ecotropic Lentivirus for Enhanced Biosafety. J. Vis. Exp. (53), e2822, doi:10.3791/2822 (2011).
Stem and tumor cell biology studies often require viral transduction of human cells with known or suspected oncogenes, raising major safety issues for laboratory personnel. Pantropic lentiviruses, such as the commonly used VSV-G pseudotype, are a valuable tool for studying gene function because they can transduce many cell types, including non-dividing cells. However, researchers may wish to avoid production and centrifugation of pantropic viruses encoding oncogenes due to higher biosafety level handling requirements and safety issues. Several potent oncogenes, including c-Myc and SV40 large T antigen, are known to enhance production of induced pluripotent stem cells (iPSC). All other known iPSC-inducing genetic changes (OCT4, SOX2, KLF4, NANOG, LIN28, and p53 loss of function) also have links to cancer, making them of relatively high safety concern as well.
While these cancer-related viruses are useful in studying cellular reprogramming and pluripotency, they must be used safely. To address these biosafety issues, we demonstrate a method for transduction of human cells with ecotropic lentivirus, with additional emphasis on reduced cost and convenient handling. We have produced ecotropic lentivirus with sufficiently high titer to transduce greater than 90% of receptor-expressing human cells exposed to the virus, validating the efficacy of this approach.
Lentivirus is often concentrated by ultracentrifugation; however, this process takes several hours and can produce aerosols infectious to human biomedical researchers. As an alternative, viral particles can be more safely sedimented onto cells by complexation with chondroitin sulfate and polybrene (CS/PB). This technique increases the functional viral titer up to 3-fold in cells stably expressing murine retrovirus receptor, with negligible added time and cost. Transduction of human dermal fibroblasts (HDFs) is maximally enhanced using CS/PB concentrations approximately 4-fold lower than the optimal value previously reported for cancer cell lines, suggesting that polymer concentration should be titrated for the target cell type of interest. We therefore describe the use of methylthiazolyldiphenyl-tetrazolium bromide (MTT) to assay for polymer toxicity in a new cell type. We observe equivalent viability of HDFs after viral transduction using either polymer complexation or the standard dose of polybrene (PB, 6 μg/ml), indicating minimal acute toxicity.
In this protocol, we describe the use of ecotropic lentivirus for overexpression of oncogenes in human cells, reducing biosafety risks and increasing the transduction rate. We also demonstrate the use of polymer complexation to enhance transduction while avoiding aerosol-forming centrifugation of viral particles.
1. Lentivirus production, harvest, and freezing
2. Transduction of human target cells with murine retrovirus receptor Slc7a1
3. Polymer complex titration to determine toxicity
4. Ecotropic transduction with polymer complexation
5. Verifying specificity of ecotropic transduction
6. Representative results:
Fig. 1A shows the lentivirus production process, and Fig. 1B shows transduction of human cells with ecotropic lentivirus, including pre-transduction with murine retrovirus receptor Slc7a1. For titering virus, we use a human rhabdomyosarcoma cell line stably transduced with Slc7a1 (Slc-hRMS). When using fluorescent control vectors to monitor transduction efficiency, we routinely achieve > 90% transduction efficiency of Slc-hRMS with ecotropic virus, as shown in Fig. 2A and 2B. Transduction rates of HDFs are generally lower because the cells have not been blasticidin-selected for receptor expression (Fig. 2C). Titers of ecotropic lentivirus are generally 10-20% of VSV-pseudotyped virus when measured on Slc-hRMS (Fig. 2D).
One freeze-thaw cycle reduces titer of ecotropic virus by 16 - 3%, equal to titer loss during a single freeze-thaw cycle of VSV-pseudotyped virus (p > 0.05). Contrary to previous reports,2 we observe no positive effect on virus titer from flash freezing in dry ice. Rather, we achieve higher post-thaw titers of either pseudotype by simply placing tubes of virus into a -80°C freezer (data not shown).
The MTT viability assay allows sensitive detection of growth arrest in target cells. Transduction with virus plus concentrations of CS/PB up to 800 μg/ml has no effect on HDF metabolism (Fig. 3A). Exposure to CS/PB without virus also has no toxic effect on cells (data not shown). FACS analysis of HDFs transduced with virus plus various concentrations of CS/PB shows enhancement of transduction compared to PB alone (Fig. 3B). The maximum enhancement occurs at 100 μg/ml CS/PB (Fig. 3C), several-fold lower than previously reported values.3 Thus, it is important to optimize conditions for any given target cell type.
Complexation with CS/PB enhances the observed titer roughly 3-fold in Slc-hRMS (Fig. 4A, p < 0.01). In practice, this yields a greater effect on transduction efficiency at low virus concentrations than at higher concentrations (Fig. 4B), which is most likely due to multiply transduced cells and receptor saturation at higher virus concentrations.
Transduction with ecotropic virus is specific for cells expressing murine retrovirus receptor. When transducing unmodified human cells with ecotropic virus, we have not observed fluorescence greater than the untransduced background whether using PB or CS/PB, as shown in Fig. 5A. Microscopically, HDFs show no transduction in the absence of receptor (Fig. 5B) while pre-transduction with receptor results in fluorescent cells (Fig. 5C).
Figure 1. Schematic view of (A) the virus production process and (B) transduction of human cells with murine retrovirus receptor followed by ecotropic lentivirus.
Figure 2. High-efficiency transduction of human cells with ecotropic lentivirus encoding GFP. Cells were pre-transduced with murine retrovirus receptor Slc7a1. (A) Human rhabdomyosarcoma cell line blasticidin-selected for Slc7a1 (Slc-hRMS), transduced (blue) or untransduced (pink) with GFP. (B) Fluorescent micrograph of ecotropic-transduced Slc-hRMS (4X). (C) HDFs transduced (blue) and untransduced (pink) with ecotropic GFP lentivirus; cells were transduced with Slc7a1 two days prior to ecotropic transduction. (D) Titer of VSV-G pseudotyped and ecotropic lentivirus, measured on Slc-hRMS.
Figure 3. Optimizing chondroitin sulfate/polybrene (CS/PB) concentrations in human dermal fibroblasts. (A) Viability of HDFs measured by MTT assay after transduction in the presence of PB only or with varied concentrations of CS/PB. (B) Enhancement of transduction efficiency in HDFs by different concentrations of CS/PB, measured by FACS. (C) Quantification of transduction enhancement, showing that maximum transduction occurs at 100 μg/ml CS/PB.
Figure 4. Effect of 400 μg/ml CS/PB on transduction of Slc-hRMS with ecotropic lentivirus. (A) Titer enhancement, and (B) fold change in transduction rate compared to 6 μg/ml polybrene alone.
Figure 5. Lack of ecotropic transduction of HDFs in the absence of murine retrovirus receptor Slc7a1. (A) FACS plot of BJ human fibroblasts, untransduced (pink) or transduced (blue) with ecotropic GFP lentivirus in both cases in the absence of receptor Slc7a1. BJ human fibroblasts transduced with ecotropic GFP lentivirus, without (B) and with (C) pre-transduction with receptor Slc7a1 (4X).
Recombinant ecotropic gammaretrovirus based on Moloney murine leukemia virus (MLV) and its receptor mSlc7a1 are well studied and widely available, having been used for over 20 years to deliver transgenes to murine cells. Ecotropic gammaretrovirus has also been used more recently to deliver oncogenes to human cells; in the context of cellular reprogramming, the use of mSlc7a1 to avoid generation of amphotropic virus harboring human oncogenes is well established.4,5 However, lentivirus provides significant advantages over gammaretrovirus in transducing refractory cell populations,6 including primary cells that are often desirable targets for reprogramming, because the lentiviral pre-integration complex allows transduction of non-dividing cells.7
Lentiviruses have been produced with dozens of different pseudotypes, including MLV, in an effort to alter virus tropism, toxicity, and other properties.8 MLV-pseudotyped ecotropic lentivirus has been used to transduce mouse cells,9 but has seldom been used on human cells.10 We therefore propose the use of MLV-pseudotyped ecotropic lentivirus as a safe, cost effective, and highly efficient vehicle to deliver known or suspected oncogenes, including cellular reprogramming factors, to human cells.
It is critical to note that this protocol does not entirely eliminate the need to produce and use pantropic lentivirus; rather, this protocol separates the oncogene(s) from the pantropic virus, insulating researchers from potential self-inoculation with cancer-related viruses. The protein mSlc7a1 and its human homologue hSlc7a1 are ubiquitously expressed amino acid transporters with no known tumorigenicity or ability to confer a selective growth advantage on recipient cells,11 making mSlc7a1 of relatively low risk for incorporation into amphotropic virus. This added step may be particularly useful in laboratories lacking dedicated virus or tissue culture facilities of the required biosafety level.
In some situations it may be possible to completely eliminate the use of pantropic virus by transfecting the Slc7a1 plasmid directly into target cells; however, many cells that would be useful targets of this technique are also refractory to transfection. As an alternative, the ability to isolate Slc7a1-transduced cells by blasticidin selection means that VSV-G pseudotyped lentivirus can be used once to generate a stock of receptor-expressing cells, after which ecotropic virus can be used routinely to transduce these cells for many experiments. Researchers should always follow their institutional safety guidelines for working with any lentivirus, regardless of its tropism.
Titers of ecotropic virus achieved with this protocol are moderately lower than VSV-pseudotyped virus, generally 10-20% of the pantropic virus titer, in agreement with previous studies.9 These titers were measured in human cells stably transduced with Slc7a1, so the lower observed titer for ecotropic virus may be partly due to varied expression of the receptor gene in target cells. Nonetheless, the viral titers attained in our protocol are more than sufficient for most applications and lead to transduction of the majority of cells, in some cases > 90% of cells.
Centrifugation-based techniques are commonly used to concentrate viral particles. However, centrifugation requires several hours, generates infectious aerosols, and can result in significant loss of viral particles.12 As an alternative, complexation with CS/PB can be used to enhance transduction without altering viral tropism.3 This method is rapid (5 minutes) and inexpensive ($0.03 per 10 ml virus), while roughly tripling the observed titer. No special equipment or proprietary reagents are required, and we observed minimal acute toxicity after exposure of HDFs to CS/PB, in agreement with previous work in other cell lines.13 One potential drawback of this protocol is that some microscopically visible polymer complexes adhere to the cells for several days in culture. We cannot rule out the possibility that these complexes, while not overtly toxic to the cells, may have more subtle effects on cellular processes.
Here we have described a methodology for safely and efficiently transducing human cells with oncogenic factors. This approach should be of great utility to researchers studying oncogenes and stem cell biology including iPS cells.
No conflicts of interest declared.
Funding for this project was provided by the California Institute for Regenerative Medicine.
|pLenti6/UbC/mSlc7a1||Addgene||17224||Murine MLV receptor|
|pCMV-dR8.91||Addgene||D. Trono lab14
Packaging plasmid (equivalent plasmid psPax2 is available from Addgene, Cat. Number 12260)
|FUGW||Addgene||14883||GFP control plasmid|
|Fugene HD||Roche Group||04 709 705 001|
|Hexadimethrine bromide (Polybrene)||Sigma-Aldrich||H9268|
|Chondroitin sulfate sodium salt from shark cartilage||Sigma-Aldrich||C4384|
|Blasticidin S||Fisher Scientific||BP 2647100|
|Methylthiazolyldiphenyl-tetrazolium bromide (MTT)||Sigma-Aldrich||M2128|