Iozzo Renato V. Proteoglycan Protocols

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256 Handler and Iozzo

3.4. Luciferase Assay

Following the first stable transfection, neomycin-resistant clones are transiently

transfected with the plasmid pTRE-LUC. This plasmid is similar to the response

plasmid except that the luciferase gene is expressed in place of a specific cDNA under

the control of the TRE and the minimal CMV promoter. By measuring the level of

luciferase activity with a functional assay, it is possible to determine which individual

clones display the highest level of inducibility as well as the lowest background. This

allows the investigator to screen the clones quickly before performing a second stable

transfection.

1. Transiently transfect each neomycin resistant clone with 10 µg of pTRE-LUC plasmid

according to calcium phosphate protocol. We recommend using the CalPhos Maximizer

(Clontech) for maximal transfection efficiency. Although we have suggested introducing

the luciferase vector via calcium phosphate transfection, electroporation can also be uti-

lized (see Note 5).

2. Cells are grown in 35-mm dishes ± doxycycline (2 µg/mL) until 90% confluent.

3. Growth medium is removed from the cells and then rinsed twice with 1× PBS. Then 500 µL

of lysis buffer (25 mM Tris-phosphate, pH 7.8, 2 mM DTT, 2 mM 1,2-diaminocyclohexane-

N,N,N,N-tetracetic acid, 10% glycerol, 1% Triton X-100) are added to each dish and

the cells are scraped off and placed in a sterile Eppendorf tube.

4. Tubes are then spun briefly (~5 s) in a Beckman microcentrifuge at 12,000g to pellet

large debris.

5. Next, 20 µL of room-temperature cell extract is mixed with 100 µL of room-temperature

Luciferase Assay Reagent (Promega).

6. The sample is placed in a luminometer to measure the release of light as the luciferin is

being oxidized (see Note 7).

3.5. Generation of Antisense Inducible Clones

Once a clone has been selected to have low background levels and high inducibility

based on the luciferase assay results, it undergoes a second stable transfection. It

is transfected with two plasmids: a response plasmid containing specific cDNA under

the control of the TRE and minimal CMV promoter, as well as a plasmid expressing

the hygromycin resistance gene. Because the cells are already neomycin resistant, it is

necessary to use a different antibiotic resistance marker to select clones.

1. The most optimal stably transfected clone, as determined by luciferase assay, is cotransfected

with 40 µg of the pTRE construct and 2 µg of the hygromycin plasmid by electroporation

as described previously. Because the clone is already G418 resistant, hygromycin is

needed as a second antibiotic resistance marker.

2. After 48 h, 200 µg/mL hygromycin was added to the culture medium (see Note 4).

3. Then 30–40 hygromycin-resistant clones are isolated by ring cloning and expanded as

described previously.

3.6. Southern Blot Analysis

Although the clones have been selected through antibiotic resistance, it is also

important to show that the regulatory and response plasmids have in fact integrated

into the genome. This can be achieved by PCR with primers that amplify a specific

region of the regulatory and response plasmids or by Southern analysis utilizing these

Antisense Expression 257

plasmids as probes. We have found that Southern analysis is a much more reliable

although labor-intensive method. It is preferable to perform Southern analysis after the

first stable transfection as well before proceeding with the second stable transfection.

1. Extract genomic DNA from a 35-mm dish of each clone to be screened. DNA should be

phenol:chloroformed and ethanol precipitated prior to restriction digestion.

2. Separate endonuclease digestions should be performed with an enzyme(s) that will liber-

ate a distinct region within either the response plasmid and/or the regulatory plasmid.

Digest 10 µg of genomic DNA overnight at 37°C, followed by inactivation with 0.5 µL of

0.5 M EDTA, phenol:chloroform extraction and ethanol precipitation.

3. Genomic digestions are run on a 0.8% agarose gel in TAE buffer at 80 V for 4–5 h. Gel

percentage and duration of electrophoresis will depend on the size of the fragments that are

expected.

4. Plasmid DNA should be digested with the same enzymes as genomic DNA and inserts

purified as described previously.

5. After gel is finished running, it is denatured in 1.5 M NaCl and 0.5 M NaOH for 30 min at

room temperature and then neutralized in 1 M ammonium acetate and 0.5 M NaOH for

30 min at room temperature. The DNA is transferred onto a nylon membrane in 10× SSC

overnight and then UV cross-linked in a Stratalinker.

6. The plasmid probes should be labeled with

P dCTP or, if preferred, a nonradioactive

isotope such as digoxigenin, by random priming with Klenow according to the

manufacturer’s protocol.

7. Genomic DNA filters are prehybridized at 65°C for 3 h and then hybridized with the

corresponding DNA probes at 65°C overnight. Membranes are then washed and exposed to

autoradiography film. Development of the film should yield the expected results.

3.7. Northern Blot Analysis

Northern analysis is needed to determine that the tTA or rtTA and the antisense

cDNA are expressed. This will confirm expression of both the regulatory and response

transcripts. Proper controls include extraction of total RNA in both the presence and

absence of doxycycline. Once again, Northern analysis should ideally also be per-

formed after the first stable transfection.

1. Total RNA is extracted from 35-mm confluent dishes of the clones to be screened by

commonly used guanidium thiocyanate protocols (e.g., TRI-Reagent, Sigma) (see Note

8).

2. Then 20 µg of total RNA are run on a 1% agarose/formaldehyde denaturing gel in MOPS

buffer at 70 V for 4–5 h.

3. Total RNA is transferred onto a nylon filter and cross-linked with a UV Stratalinker. Filters

are prehybridized at 42°C for 3 h and hybridized at 42°C overnight with the probes labeled

the same as described for Southern analysis.

4. Membranes are then washed and exposed to autoradiography film. Development of the film

should yield the expected results.

3.8. Western Immunoblotting Analyses

Western analysis is necessary to determine that there is a decrease in specific pro-

tein expression upon addition or removal of doxycycline. Which system is being used

(Tet-On or Tet-Off) will dictate whether expression should be blocked with or without

doxycycline. It is also possible to perform functional assays for the protein as well.

258 Handler and Iozzo

1. Antisense transfected clones are expanded into 35-mm dishes and then incubated for 48 h

in serum-free defined media with or without 2 µg/mL doxycycline.

2. Serial dilutions of the conditioned media from each clone are vacuum blotted onto a nylon

filter (Schleicher and Scheull).

3. The filter is then blocked in 3% milk for 3 h and then incubated overnight at room tempera-

ture with a primary antibody to Protein X.

4. After several washes, the blot is incubated with an appropriate horseradish peroxidase-conju-

gated secondary antibody.

5. Enhanced chemiluminescence is performed according to the manufacturer’s protocol

(Amersham).

6. Autoradiographs are then scanned by laser densitometry and quantitated to determine the

relative protein levels.

4. Notes

1. When working with tissue cultures, sterility is of concern. Although antibiotics are added

to the culture media, be sure to wipe down the hood with 70% ethanol before and after

use, and wear gloves throughout the procedure.

2. Any DNA to be transfected into cells should be of highest quality. That is, after plasmid

preparation, DNA should be phenol:chloroformed and ethanol precipitated under a sterile

tissue culture hood. Also, as a final check, it is a good idea to run an aliquot on an agarose

gel prior to transfection.

3. Be sure to determine the correct capacitance and voltage for the cell line and electroporator.

4. Due to variations in potency between different lots, it may be necessary to titrate the proper

concentrations of G418 and hygromycin for each cell line.

5. It is important to mention that certain cell lines can alter the effectiveness of the tetracycline

controlled gene expression (11). The type of transfection method as well as the amount of

plasmid that is transfected can also alter considerably the background levels and inducibility

of each clone. Therefore, it may be necessary to test several experimental parameters for

each cell line and screen many clones in order to find one that is highly inducible but also

gives very low levels of background expression. Although labor intensive, the results will

be much more trustworthy.

6. When ring cloning, we have found that plastic rings work better than metal rings and require

less vacuum grease to attach to the plate. Also, while picking clones, try to work rapidly,

because the plates can dry out quickly and the cells will die.

7. We have noticed that often there are extremely high levels of background luciferase expres-

sion in many of the selected clones. Therefore, it may be necessary to select more than the

recommended number of clones until one of low background can be isolated.

8. Be certain when working with RNA that gloves are always worn and are changed fre-

quently. RNA is very susceptible to degradation from RNases that are found on most

surfaces, including skin.

References

1. Gossen, M. and Bujard, H. (1992) Tight control of gene expression in mammalian cells by

tetracycline-responsive promoters. Proc. Natl. Acad. Sci. (USA) 89, 5547–5551.

2. Gossen, M., Bonin, A. L., and Bujard, H. (1993) Control of gene activity in higher eukary-

otic cells by prokaryotic regulatory elements. TIBS 18, 471–475.

3. Gossen, M., Freundlieb, S., Bender, G., Muller, G., Hillen, W., and Bujard, H. (1995)

Transcriptional activation by tetracyclines in mammalian cells. Science 268, 1766–1769.

Antisense Expression 259

4. Baron, U., Freundlieb, S., Gossen, M., and Bujard, H. (1995) Co-regulation of two gene

activities by tetracycline via a bidirectional promoter. Nucleic Acids Res. 23, 3605–3606.

5. Baron, U., Gossen, M., and Bujard, H. (1997) Tetracycline-controlled transcription in

eukaryotes: novel transactivators with graded transactivation potential. Nucleic Acids Res.

25, 2723–2729.

6. Freundlieb, S., Baron, U., Bonin A.L., Gossen, M., and Bujard, H. (1997) Use of tetracy-

cline-controlled gene expression systems to study mammalian cell cycle. Meth. Enzymol.

283, 159–173.

7. Furth, P. A., Onge, L. S., Boger, H., Gruss, P., Gossen, M., Kistner, A., Bujard, H., and

Henninghausen, L. (1994) Temporal control of gene expression in transgenic mice by a

tetracycline-responsive promoter. Proc. Natl. Acad. Sci. (USA) 91, 9302–9306.

8. Kistner, A., Gossen, M., Zimmermann, F., Jerecic, J., Ullmer, C., Lubbert, H., and Bujard,

H. (1996) Doxycycline-mediated quantitative and tissue-specific control of gene expression

in transgenic mice. Proc. Natl. Acad. Sci. (USA) 93, 10,933–10,945.

9. Baron, U., Schnappinger, D., Helbl, V., Gossen, M., Hillen, W., and Bujard, H. (1999)

Generation of conditional mutants in higher eukaryotes by switching between the expression

of two genes. Proc. Natl. Acad. Sci. (USA) 96, 1013–1018.

10. Bonin, A. L., Gossen, M., and Bujard, H. (1994) Photinus pyralis luciferase: vectors that

contain a modified luc coding sequence allowing convenient transfer into other systems.

Gene 141, 75–77.

11. Gossen, M. and Bujard, H. (1995) Efficacy of tetracycline-controlled gene expression is

influenced by cell type: commentary. BioTechniques 19, 213–216.

Cell-Mediated Transfer of PG Genes 261

261

From:

Methods in Molecular Biology, Vol. 171: Proteoglycan Protocols

Edited by: R. V. Iozzo © Humana Press Inc., Totowa, NJ

Cell-Mediated Transfer of Proteoglycan Genes

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 produc-

tion 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 dis-

tinct advantages and disadvantages when compared to other gene transfer technolo-

gies. 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 character-

ization 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 re-

ferred to as cell-mediated gene transfer. One advantage of the cell-mediated gene trans-

fer 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

262 Fischer et al.

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 expres-

sion 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 ap-

proach (Fig. 1).

2. Materials

2.1. Retroviral Transfection of Target Cells

1. PE501 cells.

2. PA317 cells.

3. NIH 3T3 TK– cells.

4. LXSN vector (~20 µg/transfection).

5. 2.5 M CaCl

, pH 7.2.

Fig. 1. Flow diagram of protocols involved in cell-mediated proteoglycan gene transfer. In

step 1 the dark panel indicates the gene of interst.

Cell-Mediated Transfer of PG Genes 263

6. 2× HEPES buffered saline (2× HBS): 50 mM HEPES, 280 mM NaCl, 1.5 mM Na

HPO

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 proto-

col 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.

3. Heparin, 10U/kg.

4. 2F Fogarty balloon embolectomy catheter (Baxter) filled with water, attached to 1-cm

syringe filled with lactate ringer’s solution.

5. 1-cm

syringe attached to silicone elastomer medical grade tubing (0.012 in. id × 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 µg/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.

264 Fischer et al.

3. 5'-Bromo-2'-deoxyuridine (BrdU).

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 × 10

cells/60mm dish, using 4 mL of medium.

Day 2: Prepare tube A with 20 µg of vector DNA, and 50 µL of 2.5 M CaCl

. Add H

to make 500 µL. Prepare tube B with 500 µL of 2× 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 × 10

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 asso-

ciated culture materials must be considered Biohazardous!

Day 4: Collect 16-h-conditioned medium from PE501 cells. Centrifuge collected cul-

ture 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 1000× stock at final concentration of 4 µg/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 µg 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 me-

dium 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 × 10

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 × 10

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 µL of the virus-containing medium of PA317 cells and add Polybrene at a final con-

centration of 4 µg/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. Incu-

bate overnight at 37°C.

Cell-Mediated Transfer of PG Genes 265

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 µg/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 µL of medium or 5 × 10

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 × 10

cells/60-mm dish

(see Note 7).

Day 2: Change medium of PA317 cells (4 mL), plate target cells at 5 × 10

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 centri-

fuge 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 µg/mL. Remove medium from the target cells and apply the freshly prepared infec-

tion 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 in-

clude 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 appro-

priate 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 × 10

cells/mL in serum-free DMEM. The estimated amount of

cell suspension per carotid is 40 µL; to allow for waste, prepare 150 µL per seeded carotid

(see Note 10).

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

266 Fischer et al.

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 µL into the 1cm

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 morpho-

logical examination.

3.3. Peri-adventitial Seeding on Decellularized Carotids

1. Cells are prepared as described previously except that 24 h before trypsinization, 0.06 µg/mL

of BrdU are added to the culture medium. This dose should be determined for each cell