Title of Experiment: Author, Institute and address:

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Neil Randell Warren
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1 Title of Experiment: Trsfetion of murine mrophge RAW264.7 ells with METAFECTENE PRO. Author, Institute n ress: Ptrizi Pellegtti n Frneso Di Virgilio. Deprtment of Experimentl n Dignosti Meiine, Setion of Generl Pthology, University of Ferrr, Borsri, Ferrr; Phone: +39 (0) ; Fx: +39 (0) ; E-mil: fv@unife.it

efore trnsfetion, so tht were pproximtely 70% onfluent t the time of

The y of trnsfetion the pegfp-n1 plsmi (4 µg) (Clonteh Lortories) ws

Metfetene-PRO (from 4 to 8 µl) ws gently mixe efore use, then ilute in 30

. The ilute DNA ws omine with Metfetene-PRO fter 5 minutes of inution t

4 hours prior to hnge the meium with 1 ml of fresh serum-ontining meium

Gene tivity ws teste 36 hours fter The rtios DNA/metfetene-PRO use were

2 Trnsient trnsfetion: RAW264.7 ells (1.2 x 10 5 ells/well) were seee in 1 ml of DMEM/F12 in 24-well ulture one y efore trnsfetion, so tht were pproximtely 70% onfluent t the time of trnsfetion. The y of trnsfetion the pegfp-n1 plsmi (4 µg) (Clonteh Lortories) ws ilute in 30 µl of Opti-MEM I (Invitrogen) Meium without serum n ntiiotis; Metfetene-PRO (from 4 to 8 µl) ws gently mixe efore use, then ilute in 30 µl of Opti-MEM I.. The ilute DNA ws omine with Metfetene-PRO fter 5 minutes of inution t room temperture, without ny mixture proeure n inute for nother 20 minutes t room temperture. The mixture ws e rop y rop to the ells n inute t 37 C in CO 2 inutor for 4 hours prior to hnge the meium with 1 ml of fresh serum-ontining meium (DMEM/F12). Gene tivity ws teste 36 hours fter trnsfetion. The rtios DNA/metfetene-PRO use were respetively: 4µg:4µl; 4µg:6µl; 4µg:8µl (Figure 1). Trnsmitte light Fluoresene light Figure 1.. RAW264.7 wil type;. RAW264.7 trnsiently trnsfete with 4 µg of pegfp-n1 n 4 µl of metfetene-pro;. RAW264.7 trnsiently trnsfete with 4 µg of pegfp-n1 n 6 µl of metfetene-pro;. RAW264.7 trnsiently trnsfete with 4 µg of pegfp-n1 n 8 µl of metfetene-pro.

Stle trnsfetion: 3.5 x 10 6 RAW264.7 ells were plte in 10 m petri ishes n inute overnight.

4) ws use s omprison) were resuspene in 700 µl of Opti-MEM I respetively. After 5 min inution t room temperture, they were mixe n re-inute for other 20 min.

8 mg/ml of G418 (Cliohem) ws e to selete trnsfete lones. The mount of G418 ws reue to 0.2 mg/ml fter 20 ys of trnsfetion n stle lones were mintine in ulture meium ontining G418 t the sme onentrtion.

3 Stle trnsfetion: 3.5 x 10 6 RAW264.7 ells were plte in 10 m petri ishes n inute overnight. The next y, 24 µg of pmeluc DNA ( DNA engineere in our lortory) n 40 µl of Metfetene-PRO (Figure 2) (the sme mount of LIPOFECTAMINE 2000 (Invitrogen) (Figure 3) n ARREST IN (Open iosystems) (figure 4) ws use s omprison) were resuspene in 700 µl of Opti-MEM I respetively. After 5 min inution t room temperture, they were mixe n re-inute for other 20 min. The mix ws e to the ish ropwise n gently swirle. The meium ws hnge fter 4 hours of inution t 37 C in CO 2 inutor. On the seon y post-trnsfetion, 0.8 mg/ml of G418 (Cliohem) ws e to selete trnsfete lones. The mount of G418 ws reue to 0.2 mg/ml fter 20 ys of trnsfetion n stle lones were mintine in ulture meium ontining G418 t the sme onentrtion. Conlusions: For trnsient trnsfetion there is signifint inrese in the intensity of the fluoresene of the RAW264.7 trnsfete with Metfetene-PRO, ut t 4µg:8µl rtio, the polytioni liposoml eome toxi for the ells. For stle trnsfetion, Metfetene-Pro hs signifitive higher tnsfetion effiieny thn Arrest- In, ut the sme effiieny of Lipofetmine 2000 in trnsfeting RAW264.7 ells. Arrest-In hs isplye low toxiity espite Metfetene-Pro n Lipofetmine 2000, ut no stle lones were otine from the polymer. METAFECTENE-PRO Figure 2.. RAW264.7 efore trnsfetion;. RAW y fter trnsfetion with METAFECTENE PRO;. RAW264.7 uner G418 seletion (15 ys fter trnsfetion);. RAW264.7 stle lone (1 month fter trnsfetion).

7 1 y fter trnsfetion with LIPOFECTAMINE

4 LIPOFECTAMINE 2000 Figure 3.. RAW264.7 efore trnsfetion;. RAW y fter trnsfetion with LIPOFECTAMINE 2000;. RAW264.7 uner G418 seletion (15 ys fter trnsfetion);. RAW264.7 stle lone (1 month fter trnsfetion). ARREST IN Figure 4.. RAW264.7 efore trnsfetion;. RAW y fter trnsfetion with ARREST IN;. RAW264.7 uner G418 seletion (15 ys fter trnsfetion);. All ells ie following G418 seletion (1 month fter trnsfetion).

Anlysis of pmeluc/mrna Expression - Totl RNA ws extrte from RAW 264.

Helthre) s esrie y the mnufturer. Primers for pmeluc were esigne, se on sequene t: forwr 5'- ATATGTGGATTTCGAGTCGTC-3' n reverse 5'-GATGGATTCCAATTCAGCGGG-3' (PCR prout, 597 p).

5 Anlysis of pmeluc/mrna Expression - Totl RNA ws extrte from RAW pmeLUC stle lone (otine with METAFECTENE PRO n LIPOFECTAMINE 2000 methos) n HEK293-pmeLUC (HEK293 stly expressing pmeluc lrey present in our lortory, s ontrol) using RNAspin Mini Isoltion Kit (GE Helthre) s esrie y the mnufturer. Primers for pmeluc were esigne, se on sequene t: forwr 5'- ATATGTGGATTTCGAGTCGTC-3' n reverse 5'-GATGGATTCCAATTCAGCGGG-3' (PCR prout, 597 p). To otin DNA, 200 ng of totl RNA were trnsrie with Aess RT-PCR System (Promeg). RT-PCR ws performe using 10 µl of uffer AMV/Tfl Retion uffer, 30 µl H 2 O DNAse/RNAse free, 2 µl of eh primer (finl onentrtion 20 piom eh), n 2 µl of MgSO 4 (stok, 25 mm). Retrotrnsriptions onitions were: 2 min t 95 C, then 1 yle of 48 C for 55 min n 2 min t 95 C; followe 35 yles of mplifition: 94 C for 1 min, 56 C for 1 min, 72 C for 2 min) n finl elongtion t 72 C for 8 min. PCR prouts were resolve y eletrophoresis on 1% grose gel. LIPOFECTAMINE HEK293-pmeLUC 2. HEK293 wil type 3. RAW264.7-pmeLUC 4. RAW264.7 wil type 5. Mrker 600 p 500 p METAFECTENE PRO HEK293 wil type 2. HEK293-pmeLUC 3. RAW264.7-pmeLUC 4. RAW264.7 wil type 5. Mrker 564 p

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