Cyto-Tracers™: Novel Lentiviral-Based Molecular Imaging Tools

Cyto-Tracers™: Novel Lentiviral-Based Molecular Imaging Tools

Introduction

Fluorescent protein technology has revolutionized cell biology by permitting visualization of a wide range of molecular events within living cells (1). With the continued expanding stem cell research and developmental biology, there is greater demand for a real-time monitoring protein expression in relation to the dynamics of reprogramming and lineage commitment. Currently, most plasmid-based probes in this class are ineffective due to narrowed tissue expressions and poor transfection efficiency (1, 2). To overcome these limitations, System Biosciences (SBI) has created a line of lentiviral-based Cyto-Tracers™ (Table 1) that are the most effective vectors for transducing and stably expressing fluorescent fusion proteins in any mammalian cells, including dividing and nondividing cells and whole model organisms. Using Cyto-Tracers™, cellular structures can be specifically visualized in spatial and temporal manner by fluorescence microscopy. In addition, our Cyto-Tracers™ also enable researchers to monitor the dynamic movement of a protein-of-interest in relationship to a given subcellular compartment by labeling the protein-of-interest with different fluorescent proteins (Table 1). Therefore, theCyto-Tracers™could provide unique mechanistic insights into cellular processes that cannot be otherwise resolved in standard biochemical or immunological assays.

Materials and Methods

Construction of Cyto-Tracers™

Each construct encodes a fusion protein consisting of a signaling peptide and the copGFP (Figure 1). The signaling peptide directs the copGFP to the appropriate subcellular location. SBI also offers a GFP untagged vector for researchers who would like to create their own tag for subcellular locations not currently available.

CopGFP is a natural green monomeric GFP protein derived from copepod (Pontellina sp.). The copGFP has optimized human codons for a high level of expression of the fluorescent protein. CopGFP is a non-toxic, non-aggregating protein with fast maturation, high stability at a wide range of pH (pH4-12), and does not require any additional cofactors or substrates. The copGFP protein has very bright fluorescence that exceeds at least 1.3 times the brightness of EGFP, the widely used Aequoreavictoria GFP mutant. The copGFP is an excellent fluorescent marker that can be used instead of EGFP for monitoring molecular dynamics.

Results and Discussion

How Cyto-Tracers™ work

The newly synthesized proteins have intrinsic signals, named “signal peptide” or “address tags", which govern their transport and localization in the cell. By fusing specific signal peptides with fluorescent proteins, we have built a collection of Cyto-Tracers™ that can light up various subcellular compartments, organells, secretory vesicles, or structural/functional molecules (Table 1). In addition, some of our Cyto-Tracers™ are able to capture a number of critical biological events with additional natural or artificial sensor sequences, such as BAX-GFP or luciferase circulized with a caspase-3 substrate peptide. The migration of BAX-GFP from cytosol to mitochondria or activation of the inactive cyclic luciferase upon cell apoptosis allows a real-time monitoring of cell death (3, 4). To our knowledge, SBI has built the most comprehensive collections of GFP-fusion tracers. Table 1 summarizes the partial collection of SBI’sCyto-Tracers™.

Performing the transient transfection experiment with Cyto-Tracers™

The Cyto-Tracers™ plasmid DNA can be transfected into target cells for use in pilot experiments. As shown in Figures 1 A and B, human HEK293 cells are transfected with either nucleus or mitochondria Cyto-Tracers™. 24 hours after transfection, the nuclei or mitochondria of transfected HEK293 cells appear green as the Cyto-Tracers™ express fusion-GFP that are targeted specifically to these organelles.

Performing transduction experiment with Cyto-Tracers™

Whether you’re using a hard-to-transfect mammalian cell line, an animal model, or simply want efficient gene delivery, the lenti-based Cyto-Tracers™ are the most effective vehicles for transducing various cells. To produce a high titer of replication-incompetent lentiviral particles, a transient transfection of HEK 293 cells with an optimized mixture of three plasmids (pPACKH1TM, SBI) with our Cyto-Tracers™ plasmid DNA was conducted. After 48 hr, the supernatant containing psudoviral particles was collected and viruses were subsequently precipitated using lentiviral precipitation solution (PEG-itTM, SBI). The precipitated viruses were then used to infect HEK293 cells at MOI of 5 and allowed to recover for 48 hours. The cells were subsequently switched to selection medium containing 5 ng/ml of puromycin. Because of the integration nature of lenti-virus, the Cyto-Tracers™ stable cell lines can be established as little as 10 days. As shown in Figures 1C-E, the CD63-GFP (an exosome marker) expressing stables can specifically light up the secretory visicles and some of the secreted exosomes as observed in the living HEK239 cells.

Lighting up pluripotent stem cells with Cyto-Tracers™

Psudotyped lentiviruses are effective tools to transduce embryonic stem (ES) and induced pluripotent stem (iPS) cells. However, expression of transgenes in stem cells varies in a promoter-dependent manner (2, 3). In consistent with published reports (2), we observed an inactivation of CMV-driven GFP transgene in a human ES line (H9) as shown in Figures 2A and a. To determine whether the incactivation of CMV-driven GFP was coupled with the pluripotent status, we monitored the expression of GFP in primary human foreskin fibroblasts (HFF) and during the reprogramming of the HFF. As shown in Figures 2C and c, CMV-driven GFP in HFF remained positive when HFF were failed to be fully reprogrammed, however, the GFP expression was lost (Figures 2D and d)when HFF were reprogrammed to stem cell status. In contrast, murine stem cell virus promoter (MSCV)-driven GFP expressed strongly in stem cells (Figures 2E and e). These data strong support that a selective inactivation of CMV-driven transgenes occurs in stem cells and during reprogramming. Therefore, MSCV promoter is particularly useful for driving transgene expression in stem cells.

Advantages of using the lenti-viral based Cyto-Tracers™

The Cyto-Tracers™ are built on an HIV-based lentiviralvector system to enable broad tropism for delivery, stable expression via integration, and robust expression. To ensure the expression of these constructs in various cells, optimized vectors are built with two extensively-tested promoters, the cytomegalovirus (CMV) and the murine stem cell virus (MSCV) promoters. While the most popular CMV promoter can be used for most cell types, the MSCV promoter is particularly useful for stem cells and hematopoietic cell types. Functioning as a regular plasmid, the Cyto-Tracers™ can be simply used to perform fast plasmid-based transfection experiments. As needed, Cyto-Tracers™ can also be packaged into lentiviral particles and used for transduction into difficult-to-transfect cell-types or tissues to allow long-term and in depth studies both in vitro and in vivo.

Conclusion

To address this critical and growing need of reliable and high-throughput image tools, we have developed a lentiviral-based Cyto-Tracers™ platform that can accurately capture various molecular events in living cells. The combined powers of a lentiviral delivery system with our robust fluorescent fusion protein technology enable long-term and in-depth studies in virtually any cell types. For reliable expression in stem cells, our MSCV-driven Cyto-Tracers™ will meet the growing need for stem cell researches. More information on the Cyto-Tracers™ can be found online: http://www.systembio.com/cyto-tracers/or call SBI at 650-968-2200.

Acknowledgement

This work is supported by a Small Bussiness Innovation Research grant (1R43GM087116-01) from the National Institutes of Health. We thank Dr. Joseph Huang for critical comments.

References

1. Muzzey D, van OudenaardenA. Quantitative time-lapse fluorescence microscopy in single cells. Annu Rev Cell Dev Biol. 2009; 25:301-27.
2. Xia XF, Zhang YS, Zieth CR, and Zhang SC. Transgene delivered by lentiviral vector are suppressed in a promoter-dependent manner. Stem Cells Dev. 2007; 16:167-176
3. Hotta A, Cheung AY, et al. Isolation of human iPS cells using EOS lentiviral vectors to select for pluripotency.Nat Methods. 2009;6(5):370-6.