Jens W Fischer Michael G Kinsella David Hasenstab Alexander W Clowes and Thomas N Wight

1. Introduction

Replication-defective retroviral systems for transduction of genes into target eukaryotic cells have grown recently in popularity. In general, the use of a retroviral packaging system involves the preparation and transfection by standard molecular biological techniques of the retroviral vector into packaging cell lines for the production of replication-defective retrovirus (1,2). Virus-laden medium from packaging cells is then used to infect target cells. Retroviral transduction of cDNA sequences has distinct advantages and disadvantages when compared to other gene transfer technologies. Foremost among the advantages of viral gene delivery approaches are the high cellular infection rates. This efficiency allows the preparation and rapid selection of large pools of transduced cells without the added step of cell cloning. This is critical in the preparation of sufficient numbers of low-passage primary cells for both characterization of cellular phenotype and expression of the transgene in vitro, as well as for experimental use in vivo. Moreover, because pools of transduced cells can be used rather than clonal cell lines, clonal variability unrelated to transgene expression is not a major issue in interpretation of experimental results. A major advantage in the use of the retroviral delivery system is that the transduced gene is rapidly incorporated into the host genome of dividing cells, thus ensuring stable expression of the gene product, although typically a single copy of the target gene is inserted. However, because the insertion of the viral sequences requires mitosis of the target cell, retroviral vectors cannot be used effectively to transfer genes to nondividing cells. Thus, retroviral trans-duction is usually performed on cells in culture, and gene transfer in animal models can be performed by implantation of cells that express the transgene, a process referred to as cell-mediated gene transfer. One advantage of the cell-mediated gene transfer approach is that long-term expression of the transgene in vivo can be achieved (3). Moreover, local expression of the gene can be induced in an adult animal without concern that adaptive processes that compromise function or viability might occur

From: Methods in Molecular Biology, Vol. 171:Proteoglycan Protocols Edited by: R. V. Iozzo © Humana Press Inc., Totowa, NJ

Fig. 1. Flow diagram of protocols involved in cell-mediated proteoglycan gene transfer. In step 1 the dark panel indicates the gene of interst.

during development, such as is possible in transgenic and knockout models. However, the preimplantation culture of the target cells in vitro may induce a phenotype that differs from that of endogenous cells. The approach of cell-mediated gene transfer has been used both to model disease processes and to evaluate therapeutic agents in vivo. For example, the role of tissue factor in the thrombotic occlusion of restenotic vessels has been examined by local overexpression of tissue factor by cell-mediated gene transfer (4). Retrovirally transduced cells that overexpress the human granulocyte colony stimulating factor (G-CSF) gene have been used to examine the effects of the systemic delivery of that factor on circulating neutrophil levels (5). Several studies have used cell-mediated gene transfer to express locally potential therapeutic agents in vivo. For example, tissue inhibitor of metalloproteinases has been assessed as an approach to limiting vessel aneurysms in an animal model (6), and the local expression of decorin induced by cell-mediated gene transfer has been assessed as an agent to limit the growth of the neointima (7). We include protocols that we have used for cell-mediated gene transfer to the injured rat carotid artery as examples of this approach (Fig. 1).

2. Materials

2.1. Retroviral Transfection of Target Cells

1. PE501 cells.

2. PA317 cells.

4. LXSN vector (~20 ^g/transfection).

6. 2x HEPES buffered saline (2x HBS): 50 mM HEPES, 280 mM NaCl, 1.5 mM Na2HPO4, pH 7.2.

7. Polybrene (hexadimethrine bromide) stock solution, 4 mg/mL, store at 4°C. Polybrene is added to the media of cultures to be infected in order to increase infection rates.

8. G-418 (Geneticin) stock solution, 50 mg/mL in PBS. G-418 is a neomycin analog used for cell selection after transduction of vector.

9. Standard tissue culture supplies.

10. Culture medium: Dulbecco's Minimal Essential Medium (DMEM) with 10% fetal bovine serum.

2.2. Internal Seeding of Retrovirally Transfected Rat Smooth Muscle Cells into the Injured Rat Carotid Artery

The following is an example of how cell mediated gene transfer, using retrovirally transduced smooth muscle cells (3,8), can be used to overexpress proteoglycans in the vessel wall and to investigate their effect on neointima formation (7). For this purpose smooth muscle cell from Fischer 344 rats were retrovirally transduced using the protocol described above. Since Fischer 344 rats are an inbred rat strain, Fischer 344 rat smooth muscle cells can be transferred into any Fischer 344 rat without inducing an immune response (see Note 1).

1. Fischer 344 rats, male, 250-300 g.

2. Anesthetic: 50 mg/kg ketamine, 5 mg/kg xylazine, 1mg/kg acepromazine.

4. 2F Fogarty balloon embolectomy catheter (Baxter) filled with water, attached to 1-cm2 syringe filled with lactate ringer's solution.

5. 1-cm3 syringe attached to silicone elastomer medical grade tubing (0.012 in. id x 0.025 in. od).

6. Lactated Ringer's solution.

7. Gauze sponges.

8. Cotton-tipped applicators.

9. 4.0 silk sutures.

10. Scalpel.

11. Ethanol, betadine.

12. Instruments: fine point (watchmaker's) forceps; blunt-curved forceps, fine curved forceps, retractor, 3 mosquito clamps, arterial (vanna) scissors, regular scissors.

2.3. Peri-adventitial Seeding on Decellularized Carotids

In some circumstances, it may be advantageous not to have endogenous cells present in the media and neointima and to repopulate a vessel entirely with exog-enously seeded cells. Rapid freeze/thaw cycles can be used to decellularize the artery before seeding the cells in the peri-adventitial region. Transduced smooth muscle cells can be cultured in 5'-Bromo-2'-deoxyuridine (BrdU)(Roche, 24 h in 0.06 ^g/mL), which will be incorporated in the DNA and serve as a genomic tag for identifying seeded cells at later times. Smooth muscle cells seeded in the peri-adventitial region will migrate through the media and form a neointima by 2 wk. This protocol is well suited for measuring the effects of gene expression on migration in vivo (4,10).

1. As under Subheading 2.2.1.

2. Liquid nitrogen.

4. Teflon sheet.

5. Anti-BrdU antibodies.

6. Secondary antibody and standard supplies for immunocytochemistry (ICC).

3. Methods

3.1. Retroviral Transfection of Target Cells

1. Insert the gene of interest into the LXSN vector using standard procedures (see Note 2).

2. Transfection of primary ecotropic (PE501) cells with the retroviral vector (LXSN and L gene of interest SN) and transduction of secondary amphotropic (PA317) packaging cells with the replication-defective retrovirus (see Note 3):

Day 1: Plate PE501 cells at 5 x 105 cells/60mm dish, using 4 mL of medium.

Day 2: Prepare tube A with 20 ^g of vector DNA, and 50 ^L of 2.5 M CaCl2. Add H2O to make 500 ^L. Prepare tube B with 500 ^L of 2x HBS solution. Add contents of tube A dropwise to tube B, mixing by constant moderate aeration of tube B with a Pasteur pipet. Incubate at ambient temperature for 30 min (a precipitate will form). Add 1 mL of the mixture to the medium in each plate and incubate cells overnight at 37°C in the incubator.

Day 3: Change medium of transfected PE501 cells. Plate secondary packaging cell line (PA317) at 5 x 105 cells/60-mm dish; include an extra dish with PA317 cells to monitor the G-418 selection (see Note 4). Note: After transfection, cells, media and associated culture materials must be considered Biohazardous!

Day 4: Collect 16-h-conditioned medium from PE501 cells. Centrifuge collected culture supernatant for 10 min at 4800g (see Note 5). Prepare medium for infection of PA317 cells by mixing fresh medium 1/1 with the conditioned medium of PE501 cells and add Polybrene from 1000x stock at final concentration of 4 ^g/mL. Replace the culture medium of the target PA317 cells with the freshly prepared viral infection medium and incubate overnight.

Day 5: Replate infected and control PA317 cells at various dilutions (1/10, 1/100, 1/1000) into 100-mm culture dishes with medium containing 800 ^g of G-418/mL for selection of virally transduced cells (see Note 4).

3. Selection and cloning of secondary packaging cell line (PA317): Replace selection medium every 2-3 d until all cells in the control dish die (approximately 7-10 d). Pick clones with the use of cloning rings following standard procedures and pass individual clones into 60-mm plates. Continue growth in selection media for 3 d more. Freeze clones back before you determine the virus titer of PA317 clones (step 4).

4. Titer virus produced by PA317 clones. In this step, the relative number of virus produced by the different clones is determined, in order to select the PA317 clone that produces the highest titer for transduction of target cell lines.

Day 1: Plate cells from the PA317 clone to be titered at 5 x 105 cells/60 -mm dish.

Day 2: Replace medium of the PA317 cells with 4 mL of fresh medium and plate one dish of NIH 3T3 TK- cells at 5 x 105 cells/60 -mm dish for each clone to be titered. Plate one additional dish to use as a selection control.

Day 3: Collect 16-h-conditioned medium of PA317 cells, centrifuge for 10 min at 4800g, prepare medium for infection of NIH 3T3 TK- cells by mixing fresh medium with 10 ^L of the virus-containing medium of PA317 cells and add Polybrene at a final concentration of 4 ^g/mL. Replace the medium of the NIH 3T3 TK- cells with the freshly prepared infection medium. Add fresh medium only to the selection control dish. Incubate overnight at 37°C.

Day 4: Trypsinize 3T3 TK- cells and replate three different dilutions (1/20, 1/100, 1/500) of the cells into 6-well plates in medium containing G-418 (600 ^g/mL). Grow the cells for 7-10 d until the cells in the selection control dish have died and the colonies in the titering plates are big enough to be counted. To count clones, remove media, rinse with PBS, fix for 2 min with 100% methanol, rinse with distilled water, and stain cells with 0.1% methylene blue in 50% ethanol for 5 min. After staining, dishes are rinsed with water until excess stain is removed and clones are visible, then dishes are air-dried, and the colonies counted by visual inspection. The number of clones and the dilution factors allow the calculation of the number of virus particles produced by the different clones. For example, 10 clones at the 1/500 dilution equals 5000 infectious units (cfu) in 10 ^L of medium or 5 x 105 cfu/mL.

After virus-titering, selected clones are expanded and frozen back under liquid nitrogen.

5. Infection of the target cells (see Note 6).

Day 1: Plate the PA317 clone with the highest titer at 5 x 105 cells/60-mm dish (see Note 7).

Day 2: Change medium of PA317 cells (4 mL), plate target cells at 5 x 105 cells/ 60-mm dish. Plate a control dish of target cells to monitor the G-418 selection.

Day 3: Collect 16-h-conditioned medium from PA317 cells (see Note 7) and centrifuge for 10 min at 4800g. Prepare infection medium for the target cells by mixing fresh medium 1/1 with the conditioned medium of PA317 cells and adding Polybrene at 4 ^g/mL. Remove medium from the target cells and apply the freshly prepared infection medium.

Day 4: Replate cultures of transduced target cells into 100-mm dishes and add G-418 at the previously determined concentration for selection.

Days 5-14: Continue selection in G-418, with medium changes every 3 d, until all of the cells in nontransduced control cultures die. Target cells are now stably transduced and can be passed and frozen back following standard tissue culture procedures.

6. Characterization of the transduced cells: The characterization procedures typically include Northern and Western blot analysis to demonstrate mRNA and protein expression of the transgene. The DNA sequences that encode the gene of interest and the neomycin phosphotransferase gene are expressed in a single contiguous mRNA. Therefore, the mRNA for the transgene is ~3.1 kb larger than that of the endogenous gene, allowing the retrovirally expressed gene product to be easily distinguished from the endogenous mRNA. Further characterization after the Western blotting depends largely on the gene of interest. If the target gene is a proteoglycan, it is important to determine the extent to which glycosaminoglycan chains are added and elongated on the core protein. The appropriate procedures are described elsewhere.

3.2. Internal Seeding Of Retrovirally Transfected Rat Smooth Muscle Cells into the Injured Rat Carotid Artery

1. Preparation of cells for the seeding procedure (see Note 9): After characterization of the transduced smooth muscle cells in vitro, grow cells nearly to confluence and trypsinize. Suspend the cells in DMEM/10% FCS to inactivate the trypsin, centrifuge cells at 960g (1000 rpm) and wash with DMEM without serum, repeat the washing step once, count the cells, and resuspend at 2.5 x 106 cells/mL in serum-free DMEM. The estimated amount of cell suspension per carotid is 40 ^L; to allow for waste, prepare 150 ^L per seeded carotid (see Note 10).

2. Anesthetize and weigh animals (see Note 11).

3. Shave throat.

4. Restrain rat lying on its back (make sure that the tongue does not obstruct the airway).

5. Disinfect the shaved throat area.

6. Make a midline incision from breast bone to chin bone.

7. Blunt dissect until the carotid artery is visible.

8. Set retractor to open neck cavity.

9. Dissect the common carotid artery from chin bone (distal to the bifurcation) to approx 1 cm proximal of the bifurcation.

10. Put a double, temporary loop around the common carotid artery as far proximally as possible.

11. Install a single loop around the external carotid just distal of the bifurcation.

12. Ligate the external carotid as distal as possible.

13. Put a double, temporary loop around the internal carotid.

14. Attach hemostats to all ties for better control.

15. Shut off blood flow by tightening the double loop around the proximal part of the common carotid artery.

16. Make an arteriotomy between the distal tie and the single temporary loop aroung the external carotid artery (step 11). Use tips of the Vanna scissors at a 45° angle, close and insert the tips of the scissors into the arteriotomy, and carefully open the scissors to spread the arteriotomy, then close the scissors again before pulling it out.

17. Hold the arteriotomy open with the cut tip of a 30-g needle and insert tip of the catheter filled with lactated Ringer's and flush to remove blood.

18. Insert balloon catheter and perform balloon injury to the carotid artery (Clowes et al. [1983], Lab Invest 49, 208), afterwards tighten loop to prevent blood flow.

19. Flush again with lactated Ringer's.

20. Resuspend cells thoroughly (no clumps) and draw about 150 ^L into the 1cm3 syringe, attach the catheter, remove air, and insert the catheter into the ateriotomy, proximal of the middle tie at the bifurcation. Tie the catheter in with the middle loop (make sure that it seals) and instill the cells into the carotid artery while pulling the catheter back toward the arteriotomy. Be careful not to pull the catheter tip out. Invert the rat for 2 min, then return the rat to its original position for 13 min (cover the wound with lactated Ringer's-soaked gauze).

21. To close, remove the catheter, loosen the proximal tie of the common carotid artery and flush the artery with blood to remove the cells that did not adhere to the denuded vessel wall.

22. Ligate the middle loop around the external carotid artery around the bifurcation.

23. Restore blood flow by loosening the proximal tie around the common carotid artery.

24. Remove the loop on the internal carotid artery, and check blood flow through the internal and common carotid artery.

25. Cut the ends of the ties; leave the proximal loop loosely around the common carotid artery as a marker.

26. Close the incision.

27. At the end of the experimental period, seeded carotid arteries can be harvested as fresh tissue for further examination by methods of molecular biology or protein and proteoglycan biochemistry, or the animal can be perfusion fixed at physiological pressures for morphological examination.

3.3. Peri-adventitial Seeding on Decellularized Carotids

1. Cells are prepared as described previously except that 24 h before trypsinization, 0.06 ^g/mL of BrdU are added to the culture medium. This dose should be determined for each cell type to ensure that it does not decrease proliferation and gives complete labeling as detected by immunocytochemistry.

2. The carotid is injured as described previously but not recannulated for seeding (omit step 20) (see Note 12).

3. A small Teflon sheet (1.5 x 3.0 cm) is placed underneath the carotid to protect surrounding tissue. Loose connective tissue surrounding the carotid artery should be removed.

4. The carotid artery is decellularized by gently touching liquid nitrogen-cooled forceps to the carotid artery until frozen (several seconds). The vessel is allowed to thaw and the cycle repeated a total of three times.

5. The Teflon sheet is removed, blood flow restored, and the decellularized carotid artery immersed in the suspension of BrdU-labeled cells (1.25-2.5 x 106 cells/mL in 400 ^L). The cells are allowed to incubate in the peri-adventitial region of the carotid for 15 min and then the neck incision is closed.

6. Seeded cells are identified in histological sections using anti-BrdU antibodies (Roche) and standard immunocytochemical stains (see Note 13).

4. Notes

1. Non-species-matched genes can be used in the procedure as well. However, the development of antibodies against the gene of interest is to be expected and might abrogate the biological function (11).

2. In the protocols described above, we reference the LXSN vector, developed in the laboratory of A. D. Miller, Fred Hutchinson Cancer Research Center, Seattle, WA, USA, although procedures for transduction of related replication-defective retroviruses are similar. This vector uses the viral long terminal repeat (L) promoter to drive constitutive expression of the gene of interest, which is inserted into the multicloning site of the vector (X). Expression of the neomycin phosphotransferase gene (N) is driven by an internal SV40 promoter (S) and is responsible for the survival of the cells in the presence of the neomycin analog, G-418. After insertion of the cDNA for the gene of interest, the orientation of the insert in the LXSN vector should be determined by PCR or restriction digestion. Related vectors with different promoter elements are available, including retroviral vectors that are designed to express the gene of interest under control of a regulatable promoter.

3. PE501 and PA317 virus packaging cells (12) are grown in Dulbecco's Minimal Essential Medium supplemented with 10% fetal bovine serum. We suggest the sequential use of both the mouse 3T3 cell-derived ecotropic packaging line (PE 501), which is designed for packaging of virus that will efficiently infect mouse cells, such as the amphotropic packaging line, PA317. Amphotropic virus packaging lines allow the production of virus that will infect cells from a wide variety of different species. Well-designed packaging cell lines are capable of synthesizing the retroviral proteins necessary for the assembly of infectious virus, but do not release replication-competent virus.

4. Control dishes that are not infected with the virus should always be included to monitor the selection of cells expressing neomycin phosphotransferase by G-418. In advance of attempting viral transductions, each cell type to be used as a target must be titered against different concentrations of G-418 to determine the concentration that will kill the cells during the course of 10-14 d. Note that the sensitivity to G-418 varies substantially among cells of different tissue origin or species and that, moreover, different lots of G-418 contain different concentrations of active inhibitor. Note also that cells must divide in order to incorporate the retroviral sequences into their genome. Passage of cells to relatively low density after transduction also aids in selection, as rapidly growing cells are more readily killed by the inhibition of protein synthesis by G-418. During passage, be careful not to over-trypsinize the PA317 cells.

5. Centrifugation of the conditioned media is a precaution to avoid transfer of the virus-producing cells (PE501 or PA317) to the next cell type. Alternatively (or additionally), a 0.45-^m filter can be used to process virus-laden medium. Media of retrovirally transduced PA317 and PE501 cell cultures contains virus particles and, although the virus is modified to be replication-defective, all culture products must be considered Biohazardous.

6. Use the target primary cell lines at as a low passage number as is feasible. Passage 3-4 has been appropriate for endothelial cells and aortic smooth muscle cells. Typically, transfection efficiency of >80% can be achieved, and the resultant pools of transduced cells can be used without cloning. The high transduction efficiency is an advantage of this method because it allows the establishment of transfected cell lines before cells transform or become senescent. For experimental controls, it is always desirable to infect the target cell line with vector alone, and to passage this control cell line in parallel with the target cells carrying the gene of interest.

7. The procedures can be modified for the infection of larger quantities of target cells.

8. Use only freshly prepared the virus and the infection media. It is possible to repeat the infection of target cells several times in sequence if transduction efficiency for single infections is low. Note, however, that target cells must continue to divide.

9. Cells stay viable for a couple of hours at room temperature. Ensure that cells are resus-pended before they are instilled into the balloon-injured rat carotid artery.

10. Note that only one carotid per rat can be seeded with cells, due to vagus nerve damage, which results in breathing difficulties for the animal.

11. The application of heparin directly before surgery can be helpful to reduce the risk of thrombosis associated with the ballooning and cell seeding procedure.

12. Injury is not required for peri-adventitial cells to migrate through the media, but balloon injury increases the rate of migration.

13. Cell divisions will decrease the intensity of BrdU staining.

References

1. Miller, A. D. and Rosman, G. J. (1989) Improved retroviral vectors for gene transfer and expression. Biotechniques 7, 980-990.

2. Linial, M. L. and Miller, A. D. (1990) Retroviral RNA packaging: sequence requirements and implications. In Current Topic in Microbiology and Immunology. Vol. 157 Retrovirues: Strategies of Replication. (Swanstrom, R. and Vogt, P. K., eds.), SpringerVerlag, Berlin, pp.125-152.

3. Lynch, C. M., Clowes, M. M., Osborne, W. R. A., Clowes, A. W., and Miller, D. A. (1992) Long-term expression of human adenosine deaminase in vascular smooth muscle cells of rats: a model for gene therapy. Proc. Natl. Acad. Sci. (USA) 89, 1138-1142.

4. Hasenstab, D., Lea, H., Hart, C. E., Lok, S., and Clowes, A. W. (2000) Tissue factor overexpression in rat arterial neointima models thrombosis and progression of advential atherosclerosis. Circulation, 101, 2651-2657.

5. Bonham, L., Palmer, T., and Miller, A. D. (1996) Prolonged expression of therapeutic levels of human granulocyte colony-stimulating factor in rats following gene transfer to skeletal muscle. Hum. Gene Ther. 7, 1423-1429.

6. Allaire, E., Forough, R., Clowes, M., Starcher, B., and Clowes, A. W. (1998) Local overexpression of TIMP-1 prevents aortic aneurysm degeneration and rupture in a rat model. J. Clin. Invest. 102, 1413-1420.

7. Fischer, J. W., Kinsella, M. G., Clowes, M. M., Lara, S., Clowes, A. W., and Wight, T. N. (2000) Local expression of bovine decorin by cell-mediated gene transfer reduces neointima formation after balloon injury in rats. Circ. Res., 86, 676-683.

8. Clowes, M. M., Lynch, C. M., Miller, A. D., Miller, D. G., Osborne, W. R. A., and Clowes, A. W. (1994) Long-term biological response of injured rat carotid artery seeded with smooth muscle cells expressing retrovirally introduced human genes. J. Clin. Invest. 93, 644-651.

11. Miller, A. D., and Buttimore, C. (1986) Redesign of retrovirus packaging cell lines to avoid recombination leading to helper virus production. Mol. Cell. Biol. 6, 2895-2902.

11. Forough, R., Hasenstab, D., Koyama, N., Lea, H., Clowes, M., and Clowes, A. W. (1996) Generating antibodies against secreted proteins using vascular smooth muscle cells transduced with replication-defective retrovirus. Biotechniques 20, 694-701.

12. Mason, D. P., Kenagy, R. D., Hasenstab, D., Bowen-Pope, D. F., Seifert, R. A., Coats, S., Hawkins, S. M., and Clowes, A. W. (1999) Matrix metalloproteinase-9 overexpression enhances vascular smooth muscle cell migration and alters remodeling in the injured rat carotid artery. Circ. Res. 85, 1179-1185.

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