MOLECULAR BIOTECHNOLOGY Volume 27, 2004

Naked Plasmid DNA Transfer 23RESEARCH PROTOCOL

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Molecular Biotechnology 2004 Humana Press Inc. All rights of any nature whatsoever reserved. 1073–6085/2004/27:1/23–31/$25.00

Abstract

*Author to whom all correspondence and reprint requests should be addressed:1Division of Clinical Nephrology and Rheumatology, NiigataUniversity Graduate School of Medical and Dental Sciences, 1-754 Asahimachi-dori, Niigata 951-8120, Japan. E-mail: hirokim@med.niigata-u.ac.jp 2Division of Stem Cell Regulation Research, G6, Osaka University Medical School, 2-2 Yamada Oka, Suita, Osaka 565-0071, Japan.

Rat Kidney-Targeted Naked Plasmid DNA Transferby Retrograde Injection Into the Renal Vein

Hiroki Maruyama,*,1 Noboru Higuchi,1 Shigemi Kameda,1 Gen Nakamura,1

Seitaro Iguchi,1 Jun-ichi Miyazaki,2 and Fumitake Gejyo1

Kidney-targeted gene transfer is expected to revolutionize the treatment of renal diseases. Recently, wedemonstrated that naked plasmid deoxyribonucleic acid (DNA) can be transferred into renal interstitial fi-broblasts near the peritubular capillaries (PTCs) in normal rats, by retrograde injection into the renal veinwith the renal vein and artery clamped. The PTC network is a main target of kidney transplant rejection andof progressive tubulointerstitial fibrosis, which typifies all progressive renal diseases. We retrogradely in-jected a lacZ expression plasmid in Ringer’s solution into the renal vein of rats using a 24-gage catheter. Wedetected lacZ expression exclusively in the interstitial fibroblasts near the PTCs of the kidney byimmunoelectron microscopy. Nephrotoxicity from the gene transfer was not apparent. We then used a raterythropoietin (Epo) expression plasmid vector pCAGGS–Epo in a reporter assay. We obtained maximalEpo expression when the DNA solution was injected within 5 s in a volume of 1.0 mL. We detected transgene-derived Epo messenger ribonucleic acid by reverse transcriptase polymerase chain reaction only in the kid-neys receiving pCAGGS–Epo. In this article, protocols for naked plasmid DNA transfer into rat kidneyusing this hydrodynamics-based transfection method and the immunoelectron microscopic technique to de-termine the lacZ gene transfer site are described in detail.

Index Entries: Hydrodynamics-based transfection; gene transfer; kidney; naked plasmid DNA; fibro-blast; Ringer’s solution; CAG promoter; rat; renal vein; catheter; peritubular capillary; interstitium.

1. IntroductionKidney-targeted gene transfer is an important

tool for expanding the treatment options for renaldiseases. Previous gene transfer methods usingnonviral vectors via renal arterial (1–5), pelvic(6,7), or ureteric (8) routes into the glomerulus(1–3), tubules (4–7), or interstitial fibroblasts (8)of the kidney have shown low efficiency andshort-term expression. The peritubular capillary(PTC) network is one of the main targets of renaltransplant rejection and of the progressive tubulo-interstitial fibrosis that typifies all progressiverenal diseases. Recently, we successfully trans-ferred naked plasmid deoxyribonucleic acid (DNA)into the kidney of normal rats by retrograde injec-

tion into the renal vein, using an erythropoietin(Epo) expression plasmid, pCAGGS–Epo, in a re-porter assay (9). Compared with previous gene-transfer methods using nonviral vectors (1–8), ourtechnique is simple, safe, and allows high-level,long-term stable expression and specific gene trans-fer to the fibroblasts near the PTC (9). Recently,Shimizu et al. demonstrated that kidney-targetedtransfer of a naked plasmid encoding the 7ND(antimonocyte chemoattractant protein-1) gene bythis technique (9) attenuates the tubulointerstitialrenal injury induced by protein-overload proteinuria(10). In this article, protocols for the transfer of na-ked plasmid DNA into the rat kidney by retrogradeinjection into the renal vein and the immunoelectron

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microscopic analysis used to determine the lacZgene transfer site are described in detail.

2. Materials2.1. Experimental Animals

1. Rats. We use 8-wk-old male Wistar rats pur-chased from Charles-River Japan Inc. (Tokyo,Japan). Rats of other ages or strains may betreated similarly.

2. Anesthetic (diethyl ether). Desiccator with lidand porcelain plate.

2.2. Plasmid DNA1. Plasmid vector (pCAGGS). The plasmid vec-

tor must include an expression unit that is ac-tive in the kidney. We have successfully usedthe pCAGGS vector (11). To assess the site ofgene transfer, we used pCAGGS–lacZ (12),which was constructed by inserting the Es-cherichia coli lacZ gene into the unique EcoRIsite between the cytomegalovirus immediate-early enhancer/chicken β-actin hybrid (CAG)promoter and the 3'-flanking sequence of therabbit β-globin gene of pCAGGS (see Note 1).To assess the efficiency of the gene transfer,we used pCAGGS–Epo (13), which was con-structed by inserting the rat Epo complemen-tary DNA (cDNA) into the unique XhoI site ofthe pCAGGS, as described previously (13). ThepCAGGS and pCAGGS–Epo vectors can beprovided by J. Miyazaki and by H. Maruyama,respectively, on request (see Note 1).

2. Competent cells. The pCAGGS plasmid isbased on pUC 13, a high-copy-number plas-mid, and is easily grown in E. coli DH 5α, orother strains.

3. Endofree Plasmid Giga Kit (Qiagen, Hilden,Germany).

4. Ringer’s solution (Ohtsuka, Tokushima, Ja-pan): 147 mEq/L Na, 4 mEq/L K, 5 mEq/L Ca,156 mEq/L Cl, pH 5.0–7.5.

2.3. Retrograde InjectionInto the Renal Vein

1. 24-gage SURFLO® iv catheter (Terumo, To-kyo, Japan), consisting of a 24-gage catheter19-mm long and 0.67 mm in diameter, with a27-gage needle 0.47 mm in diameter (Fig. 1A–C) (see Note 2).

2. 2.5-mL capacity syringe (Terumo).3. 50-mL capacity polypropylene conical tube

(Falcon; Becton Dickinson, Franklin Lakes, NJ).4. Angled-type Diethrich bulldog clamps, 45-mm

total length (cat. no. 18039-45; MuromachiKikai, Tokyo, Japan).

5. Scissors, 14-cm long with Keisei-micrins razoredges (cat. no. PR5014C-R; Keisei Medical In-dustrial, Tokyo, Japan).

6. Retractor.7. Curved-type mosquito hemostatic forceps,

12.5-cm long (cat. no. 09-230-02; MizuhoIkakogyo, Tokyo, Japan).

8. Mouse-tooth forceps, 13-cm long (cat. no. 09-100-00; Mizuho Ikakogyo).

9. Dry-type cotton (Oneshot; Hakujuji MedicalProducts, Tokyo, Japan).

10. ELP sterile poly pack needled suture (cat. no. F25-30B1; Akiyama Medical MFG, Tokyo, Japan).

11. Needle holder, 19-cm long (cat. no. N-184D;Keisei Medical Industrial).

12. Mikron autoclip, 9 mm, applier (Becton Dick-inson, Sparks, MD) for skin suture.

13. Mikron autoclip, 9 mm (Becton Dickinson) forskin suture.

2.4. Assessment of Gene Transfer1. Preparation of pCAGGS–lacZ using the Endo-

free Plasmid Giga Kit (Qiagen).2. 0.1M phosphate buffer (PB), pH 7.4. Mix one

volume of 0.1M NaH2PO4 · 2H2O (Wako PureChemical Industries, Osaka, Japan) and 3 volof 0.1M Na2HPO4 ·12H2O (Wako Pure Chemi-cal Industries), and adjust the pH to 7.4.

3. 4% paraformaldehyde in phosphate-bufferedsaline (PBS), pH 7.4 (Gibco, Invitrogen, GrandIsland, NY).

4. 40 mM 5-bromo-4-chloro-3-indolyl-β-D-galac-toside (X-gal; Takara, Shiga, Japan) in dim-ethyl sulfoxide. Dilute this to 1 mM in PBSbefore using it for staining.

5. Tissue-Tek OCT compound (Sakura Finetech-nical, Tokyo, Japan).

6. Dry ice and acetone.7. Cryostat.8. Glass slides coated with 3-aminopropyltri-

ethoxysilane (Matsunami Glass, Osaka, Japan).9. 1.5% glutaraldehyde (Nacalai Tesque, Kyoto,

Japan) in 0.1M PB.

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10. Nuclear fast red (Kernechtrot stain sol.: MutoPure Chemicals, Tokyo, Japan).

2.5. Assessment of Gene Transfer Site1. Periodate-lysine-paraformaldehyde (PLP) fixa-

tive. 0.075M L (+)-lysine hydrochloride, 2%paraformaldehyde, 0.01M sodium metaperio-date, 0.037M PB.

a. 100 mL of 0.1M L (+)-lysine hydrochloridesolution:1.827 g L (+)-lysine hydrochloride(MW, 182.65, Wako Pure Chemical Indus-tries); 50 mL distilled H2O. Adjust the pHto 7.4 with 0.1M Na2HPO4. Add 0.1M PB,pH 7.4, up to 100 mL.

b. 100 mL of 8% paraformaldehyde solution:8 g paraformaldehyde (Nacalai tesque),100µL 1N NaOH (see Note 3), distilled H2O to100 mL. Dissolve using stirrer at 60°C.

c. 120 mL of PLP fixative: 90 mL of 0.1M L(+)-lysine hydrochloride solution, 30 mL of8% paraformaldehyde solution, 256.8 mgsodium metaperiodate (MW, 213.90, PureChemical Industries).

2. Curved-type mosquito hemostatic forceps 12.5-cm long (cat. no. 09-230-02; Mizuho Ikakogyo).

3. 18-gage SURFLO iv catheter (Terumo).4. TERUFUSION® solution administration set

(Terumo), which comes equipped with a con-stant flow-rate clamp and small-bore tube.

5. Plastic bag containing 200 mL of isotonic so-dium chloride solution. Discard the solutionand infuse the bag with PBS or PLP.

6. Rabbit polyclonal anti-E. coli β-galactosidaseantibody (Biogenesis, Poole, UK).

7. Goat antirabbit Ig EnVision+ Peroxidase Rab-bit (1:10 dilution, DAKO, Carpinteria, CA).

8. 0.02% diaminobenzidine (DAB) (Dojindo,Kumamoto, Japan).

9. 0.01% H2O2 (Mitsubishi Gas Chemical, Tokyo,Japan).

10. 2.5% glutaraldehyde (Nacalai Tesque).11. 1% OsO4 (Structure Probe, West Chester, PA).12. Epok 812 (Oken, Tokyo, Japan), which is equiv-

alent to Epon.13. A razor blade Feather-S (FAS-10; Feather,

Osaka, Japan).14. A Reichert-Nissei Ultracut N ultramicrotome

(Hitachi High-Technologies, Tokyo, Japan).

15. An H-600A electron microscope (Hitachi,Ibaragi, Japan).

2.6. Blood Analyses1. A 23-gage needle connected to a 2.5-mL ca-

pacity syringe (Terumo).2. Blood-collecting tubes containing ethylene-

diaminetetraacetic acid (EDTA).3. Blood-diluent Cellpack (Sysmex) for red blood

cell analyses.4. Blood-collecting 1.8-mL capacity Sumilon

tubes (Sumitomo Bakelite, Tokyo, Japan) withscrew caps.

5. Separapid tube S mini tubes (Sekisui Chemi-cal, Tokyo, Japan) for serum separation.

6. Serum rat Epo levels are determined using aRecombigen EPO kit (Iatron, Chiba, Japan),which is a radioimmunoassay with a rabbitpolyclonal antibody against Epoetin α. This kithas a linear range of measurement between 3.0and 200 mU/mL of human Epo with a detec-tion threshold of 3.0 mU/mL.

7. Reticulocytes are determined using a SysmexR-3500 (Sysmex, Hyogo, Japan).

8. Red blood cell, hematocrit, and hemoglobin aremeasured using a Sysmex SE-9000 electroniccounter (Sysmex).

3. MethodsWe describe the method of gene transfer into

the left kidney of 8-wk-old rats by retrograde in-jection into the renal vein. It will be necessary tomodify this method to apply it to rats of a differ-ent size, according to their kidney volume.

3.1. Preparation of Naked Plasmid DNA1. Naked plasmid DNA is extracted and purified

using the Endofree Plasmid Giga Kit (Qiagen)(see Note 4).

2. Dissolve the DNA in Tris-EDTA buffer, andassess its quantity and quality by optical den-sity at 260 and 280 nm. Store the plasmid DNAat –20°C until use.

3. Immediately before injection, dilute the plas-mid DNA with Ringer’s solution (Ohtsuka) atroom temperature to its final concentration.

4. This injection system has approx 70 µL of deadvolume. The dead space is produced by the gapbetween the catheter or tip of the syringe cylin-

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der and the tip of the gasket. Thus, to inject thenet volume (1.0 mL) into the kidney, the injec-tion is performed using a syringe containing1070 µL.

3.2. Naked Plasmid DNA InjectionInto the Renal Vein

We injected various doses of naked plasmidDNA in various volumes of Ringer’s solution(Ohtsuka) through the 24-gage SURFLO iv cath-eter, connected to a 2.5-mL capacity syringe, intothe renal vein, using an injection time of 5 s orless (see Note 5). Our three-person team canachieve gene transfer at a rate of approx 20 ratsper h (see Note 6).

1. Anesthetize the rats with diethyl ether in a des-iccator fitted with a lid and porcelain plate.

2. Place the rat supine on the board.3. To access the renal vein easily, perform an in-

cision in the median section of the abdomen.4. To obtain a good view of the whole left kidney,

move the organs covering it (see Note 7). Inparticular, moving the stomach and spleen tothe right side is a very effective way to obtain agood view.

5. Penetrate the peritoneum and scoop up the re-nal vein and artery with curved-type mosquitohemostatic forceps (Mizuho Ikakogyo), with-out occluding the adrenal vein.

6. Clamp the left renal vein and artery with angled-type Diethrich bulldog clamps (MuromachiKikai), without occluding the adrenal vein.

7. Insert the 24-gage SURFLO iv catheter (Terumo)into the renal vein approx 1/2 of the vein lengthfrom the hilum of the kidney (Fig. 1A–C). Dur-ing insertion of the catheter we feel two pointsof penetration resistance. The first resistance isowing to penetration of the inner needle intothe vein, and the second is owing to the pen-etration of the outer catheter into the vein. Af-ter feeling the second resistance, do not insertthe catheter any farther (see Note 8).

8. Maintaining the outer catheter in the renal vein,extract the inner needle, remove the air in thelumen of the outer catheter by displacing it withblood, and connect the 2.5-mL capacity syringecontaining the DNA solution. Inject the solu-

tion containing the naked plasmid DNA into thevein.

9. No incubation time is used in this procedure.Reestablish the blood flow immediately afterthe injection.

10. At the same time, apply pressure to achieve he-mostasis at the injection site using dry cotton(Hakujuji Medical Products) for approx 5 s (seeNote 9).

11. Hold ELP sterile poly pack needled suture(F25-30B1; Akiyama Medical MFG) usingneedle holder, 19-cm long (N-184D; KeiseiMedical Industrial) and suture the abdominalmuscles using.

12. Suture the skin by Mikron autoclip, 9 mm,applier (Becton Dickinson) loaded with Mikronautoclip, 9 mm (Becton Dickinson).

3.3. Assessment of Gene TransferBefore introducing the gene of interest by ret-

rograde injection into the renal vein, it is impor-tant to test the effectiveness of the experimentalprocedures with a reporter gene. For this purpose,we use a plasmid carrying pCAGGS–lacZ, whichexpresses β-galactosidase. To this end, we alsouse the plasmid pCAGGS–Epo in a reporter assay,because we can easily monitor its physiological ef-fects by measuring serum Epo and performing redblood cell analyses (14).

3.3.1. LacZ Gene TransferTo clarify the transgene expression site, we de-

liver pCAGGS–lacZ into the renal vein (5).

3.3.2. β-Galactosidase StainingX-gal staining is performed according to previ-

ously described methods (9,15).

1. Inject 100 µg of pCAGGS–lacZ in Ringer’s so-lution into the renal vein of anesthetized rats,as described in Subheading 3.2.

Fig. 1. (opposite page) (A). Rats were anesthetizedwith diethyl ether, and an incision in the median sec-tion of the abdomen was performed: left adrenal vein(asterisk); left adrenal gland (arrow). (B). Immedi-ately before injecting the DNA solution, the left renalvein was clamped with angled-type Diethrich bulldogclamps (Muromachi Kikai), without occlusion of the

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Fig. 1. (continued) adrenal vein. (C). Retrograde injec-tion of the solution containing naked plasmid DNA so-lution into the renal vein through a 24-gage SURFLOiv catheter (Terumo) connected to a 2.5-mL capacitysyringe.

Fig. 2. (A, B). Rat kidney section stained for X-gal1 d after injection with pCAGGS–lacZ. Original mag-nification: (A) ×60, (B) ×250. (C) Immunoelectronmicroscopic analysis of ultrathin sections of rat kid-neys 1 d after injection of pCAGGS–lacZ. Originalmagnification, ×4000. The fibroblasts have long cyto-plasmic processes. C, peritubular capillary lumen; F,fibroblast; PT, proximal tubule. Arrows indicate thearea of lacZ expression.

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2. The following day, sacrifice the rats with di-ethyl ether in a desiccator fitted with a lid andporcelain plate.

3. Harvest the kidney for X-gal staining, removethe capsule of the kidney, embed the kidney inTissue-Tek OCT compound (Sakura Finetech-nical), and freeze it in a mixture of dry ice andacetone.

4. Cut serial sections (5-µm thick) with a cryostatand place them on glass slides coated with 3-amino-propyltriethoxysilane (Matsunami).

5. Fix the slices in 1.5% glutaraldehyde in PBS,pH 7.4, at room temperature for 10 min, thenwash them three times in cold PBS (5 min/wash) (see Note 10).

6. Incubate the slices in X-gal staining solutioncontaining 1 mg/mL X-gal, 2 mM MgCl2, 5mM K4Fe(CN)6, 5 mM K3Fe(CN)6, and 0.5%Nonidet P-40 in PBS, at 37°C‚ for 3 h.

7. Counterstain the slices with nuclear fast red(Muto Pure Chemicals) at room temperature for4 min, then dry them, and clear them three timesin xylene. Use a mounting medium, for example,malinol (Muto Pure Chemicals), to affix thecover slips over the stained tissue sections.

8. Observe the sections with a light microscopefor X-gal staining. We observed β-galactosi-dase expression in the interstitium of the kid-ney on d 1 (Fig. 2A).

3.3.3. Immunoelectron Microscopyto Determine the LacZ Gene Transfer Site

To determine the transgene expression site atthe ultrastructural level, we analyze the kidneyinjected with pCAGGS–lacZ as described previ-ously (9).

1. Inject 100 µg of pCAGGS–lacZ in Ringer’ssolution into the renal vein of anesthetized rats.

2. One day after the injection, place the rats undergeneral anesthesia with diethyl ether, and makean abdominal incision.

3. Clamp the upper portion of abdominal aortafrom where the left renal artery starts, usingcurved-type mosquito hemostatic forceps(Mizuho Ikakogyo). Insert an 18-gage SURFLOiv catheter into the lower portion of the abdomi-nal aorta where the left renal artery starts, andcut the left renal vein to create an exit for theperfusion solution.

4. Perfuse the left kidney through the catheterwith 50 mL of PBS followed by 50 mL of PLPfixative using a gravity-feed system at a rate of200 mL/h.

5. Harvest the kidney, and remove the capsule ofthe kidney.

6. Fix the kidney in PLP fixative for 4 h at 4°C,immerse it in 10% sucrose in PBS for 1 h, em-bed it in Tissue-Tek OCT compound (SakuraFinetechnical), and freeze it in hexane at –80°C.

7. Cut sections (4-µm thick) with a cryostat andincubate them with rabbit polyclonal anti-E.coli β-galactosidase antibodies (1:800 dilution,Biogenesis) for 30 min at room temperature(see Note 11).

8. Wash the sections in PBS, then incubate themwith the goat antirabbit Ig, EnVision+ Peroxi-dase Rabbit (1:10 dilution, DAKO, Carpinteria,CA) for 1 h.

9. Wash the sections in PBS and incubate them in0.02% DAB in 0.05 M Tris buffer (pH 7.6) con-taining 0.01% H2O2 for 3 min.

10. Wash the sections in PBS, and fix them with2.5% glutaraldehyde for 3 min at room tem-perature.

11. Wash the sections in PBS, postfix them with1% OsO4 in 0.1 M PB, pH 7.2, for 3 min; washthem in distilled H2O, dehydrate them in agraded ethanol series, and flat-embed them inEpok 812 (Oken, Tokyo, Japan), which isequivalent to Epon.

12. Allow the Epok 812 to polymerize. Then ex-amine the sections by light microscopy, clip outfields of interest with a razor blade Feather-S(Feather), then cut them with a Reichert-NisseiUltracut N ultramicrotome (Hitachi High-Technologies) into ultrathin (100-nm) sections.

13. Examine the sections for DAB staining with anH-600A electron microscope (Hitachi). We ob-served the DAB product exclusively through-out the cytoplasm of fibroblasts near the PTC(Fig. 2B).

3.3.4. Epo Gene TransferWe evaluated the time-course of Epo expres-

sion using a 5-s injection time and a vol of 1.0mL. Rats were assigned to two groups: pD8ØGS–Epo 400 µg (n = 3) and pCAGGS 400 µg (n = 3).

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1. The rats are placed under general anesthesia withdiethyl ether, then blood samples (2 mL) are ob-tained from the heart according to Ohwada’smethod for mice (16). We modified this proce-dure for rats by using a 23-gage needle con-nected to a 2.5-mL capacity syringe (Terumo)(15). The rat is placed supinely on the boardand its neck is extended fully. The needle is in-serted into the jugular notch, moved horizon-tally under the sternum, and blood is obtained.The rats can easily survive this procedure. Ourteam of three persons is able to perform bloodsampling at a rate of approx 60 rats per h (seeNote 12).

2. The blood is divided for Epo measurements andred blood cell analyses as follows. A portion ofthe blood (> 200 µL) is put into a blood-col-lecting tube containing EDTA. A portion (200µL) of this blood is then transferred into aSumilon tube (Sumitomo Bakelite) with 800µL of Cellpack (Sysmex). Red blood cell analy-ses are performed within 24 h. The rest of theblood (the remaining EDTA-treated blood andthe non-EDTA-treated blood) is transferred intoa Separapid tube S mini tube (Sekisui Chemi-cal), left for 1 h at room temperature, and spun at1710g for 10 min at room temperature. The se-rum is decanted into a tube and stored at –80°Cuntil it is used for measurements.

3. The serum samples are assayed for Epo using aRecombigen EPO kit (Iatron), according to thesupplier’s instructions (Fig. 3A).

4. Reticulocytes are measured using a Sysmex R-3500 (Sysmex) (Fig. 3B). Red blood cells, he-matocrit, and hemoglobin, are measured usinga Sysmex SE-9000 electronic counter (Sysmex)(Fig. 3C).

5. All rats survived the gene transfer. pCAGGS–Epo transfer continuously produced biologicallyactive Epo for more than 24 wk (Fig. 3A), re-sulting in a significant elevation of the reticulo-cyte (Fig. 3B) and hematocrit levels (Fig. 3A).

4. Notes1. To assess the efficiency of the gene transfer,

we used pCAGGS–Epo in a previous study(9), which was constructed by inserting the ratEpo cDNA into the unique XhoI site of thepCAGGS vector, as described previously (13).

Fig. 3. The time-course after pCAGGS–Epo trans-fer by retrograde injection into the renal vein: *, p <0.05; **, p < 0.01; ***, p < 0.001; and ****, p <0.0001 for comparisons between pCAGGS–Epo andpCAGGS rats (comparisons were made at each time-point). Blood samples were obtained 1 wk before and1 wk after the injection of naked DNA. A, serum epolevels; B, reticulocyte count; C, hematocrit; n = 3 ineach group. Error bars are not shown for some datapoints because of low variability.

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The pCAGGS and pCAGGS–Epo vectors canbe provided by J. Miyazaki (e-mail: jimiyaza@nutri.med.osaka-u.ac.jp) and by H. Maruyama(e-mail: hirokim@med.niigata-u.ac.jp), respec-tively, on request.

2. Leakage of the DNA solution from the injec-tion site during the injection should be avoidedfor successful gene transfer. We found thatrapid injection with a needle was often accom-panied by leakage. A needle is not suitable forthis method in rats.

3. Paraformaldehyde is not easily dissolved. 1NNaOH 100 µL is added to promote its dissolu-tion.

4. Endotoxin-free plasmid DNA should be pre-pared. Contaminating endotoxin may cause lo-cal immunologic reactions, which can lead toan early loss of gene expression or affect theexperimental results. The use of Endofree Plas-mid Giga Kits (Qiagen) is recommended.

5. To obtain the approximate injection volumethat could be accommodated by a rat kidney,we measured the kidney capacity as follows.We resected the kidney of an 8-wk-old maleWistar rat and placed it inside a 50-mL gradu-ated cylinder containing 30 mL of water. Weestimated that the increase in volume was equalto the kidney capacity. The mean kidney capac-ity was 1.0 ± 0.02 mL (n = 10).

6. Prior to the actual experiments, we recommendthat you confirm you can reproducibly performthe injection into the renal vein of anesthetizedrats using Ringer’s solution.

7. Because the left renal vein of 8-wk-old oryounger Wistar rats is not covered by fat, wecan see the full length of the vein under theperitoneum. Wistar rats older than 10 wk havefat around the renal vein, and sometimes wecannot see its full length in these rats.

8. Injection through the tip of the catheter imme-diately next to the kidney may cause the deliv-ery of DNA solution into just part of the kidneyand increase the resistance against rapid injec-tion. Do not place the tip of the catheter imme-diately next to the kidney.

9. To avoid the occurrence of chemical peritonitisfrom alcohol, be careful not to soak the cottonin alcohol.

10. In X-gal staining it is important to avoid back-ground staining attributable to the endogenousβ-galactosidase. In all but a few specializedeukaryotic cells, lysosomal enzymes that cata-lyze the hydrolysis of β-galactosidic linkagesare active only under acidic conditions and areinactive at neutral pH. However, E. coli β-ga-lactosidase is active at the neutral pH (17).Therefore, we use PBS, pH 7.4, in the assay forlacZ.

11. A rabbit polyclonal anti-E. coli β-galactosidaseantibody produced by Biogenesis works wellin both the immunoelectron microscopic analy-sis and immunofluorescence staining (9).

12. It is much harder to perform safe blood sam-pling using a needle with a diameter larger thanthat of a 23-gage needle owing to the high riskof cardiac tamponade. Awakening of the ratfrom the anesthesia during blood sampling cancause rupture of the heart. Therefore, adequateanesthesia is important. The position of the ratis another important factor for smooth bloodsampling. If the first attempt fails, you shouldextract the needle from the rat and perform thesampling again. Trying again without extract-ing the needle after the first attempt can in-crease the risk of rupture of the heart (16).

References1. Tomita, N., Higaki, J., Morishita, R., et al. (1992) Di-

rect in vivo gene introduction into rat kidney. Biochem.Biophys. Res. Commun. 186, 129–134.

2. Isaka, Y., Fujiwara, Y., Ueda, N., Kaneda, Y., Kamada,T., and Imai, E. (1993) Glomerulosclerosis induced byin vivo transfection of transforming growth factor-betaor platelet-derived growth factor gene into the rat kid-ney. J. Clin. Invest. 92, 2597–2601.

3. Arai, M., Wada, A., Isaka, Y., et al. (1995) In vivotransfection of genes for renin and angiotensinogeninto the glomerular cells induced phenotypic changeof the mesangial cells and glomerular sclerosis.Biochem. Biophys. Res. Commun. 206, 525–532.

4. Boletta, A., Benigni, A., Lutz, J., Remuzzi, G., Soria,M. R., and Monaco, L. (1997) Nonviral gene deliveryto the rat kidney with polyethylenimine. Hum. Gene.Ther. 8, 1243–1251.

5. Foglieni, C., Bragonzi, A., Cortese, M., et al. (2000)Glomerular filtration is required for transfection ofproximal tubular cells in the rat kidney following in-jection of DNA complexes into the renal artery. GeneTher. 7, 279–285.

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