Retroviral vector–mediated transfer and expression of human tissue plasminogen activator gene in human endothelial and vascular smooth muscle cells Daryoush.

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Retroviral vector–mediated transfer and expression of human tissue plasminogen activator gene in human endothelial and vascular smooth muscle cells Daryoush Ekhterae, PhD, James C. Stanley, MD Journal of Vascular Surgery Volume 21, Issue 6, Pages 953-962 (June 1995) DOI: 10.1016/S0741-5214(95)70223-7 Copyright © 1995 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 1 MFG tPA is retroviral-based vector containing human tPA cDNA. This vector consists of Moloney murine leukemia virus LTRs and human tPA cDNA. Important restriction sites are Kpn I, Cla I, Nhe I. SD, splice donor sequence; Ψ, retroviral packaging sequence; Kb, kilobases; bp, base pair. Fragment Cla I to NheI was excised, labeled, and used as probe for Southern and Northern analyses. Journal of Vascular Surgery 1995 21, 953-962DOI: (10.1016/S0741-5214(95)70223-7) Copyright © 1995 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 2 Representative Southern blot analysis of human EC transduced with MFG-tPA vector. Genomic DNA, 10 μg, from nontransduced (C) and transduced cells (E) were electrophoresed on 1% agarose. Kpn I digest 17.5 pg of plasmid representing one copy per cell was also electrophoresed into same gel. Blot was hybridized to 32p labeled "LTR" probe, which represents 172 bp of retroviral vector; Cla I to Nhe I fragment. Lanes in blot were quantitated by use of Betascope 630 blot analyzer. C, nontransduced control; EC lanes E1 to E4, human iliac artery EC. Lane E1 and E2, EC transduced 3 and 4 days after plating (harvested at day 14); lane E3 and E4, EC transduced 3 and 4 days after plating (harvested at day 28). Lane 5 and 6, saphenous vein EC transduced 3 and 5 days after plating (harvested at day 14). Lane E7 and E8, saphenous vein EC transduced 3 and 5 days after plating (harvested at day 28). Journal of Vascular Surgery 1995 21, 953-962DOI: (10.1016/S0741-5214(95)70223-7) Copyright © 1995 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 3 Representative Northern blot analysis of human iliac and saphenous EC transduced with MFG-tPA. Total cellular RNA was extracted, 7 μg were fractionated on 1.2% formaldehyde agarose gels, blotted and hybridized to a 32P-labelled "LTR" probe (A), PAI-1 probe (B), or GAPDH probe (C). Lane C1, human iliac artery EC, control, harvested at day 14. Lane C2, human saphenous vein EC, control, harvested at day 14. Lanes E1, E3, E5, E7, E8, iliac artery EC, transduced at 3, 4, and 5 days after plating, harvested at day 14 or transduced at 4 and 5 days after plating, harvested at day 28, respectively. Lanes E2, E4, E6, represent saphenous vein EC, transduced at 3 and 5 days after plating and harvested at day 14 or transduced at 5 days after plating and harvested at day 28, respectively. Intensity of band for tPA was normalized to GAPDH: E1, 2.5; E3, 1.4; E5, 0.32; E2, 2.4; E4, 2.1. Average intensity of two bands for PAI was normalized to GAPDH: C1 = 0.125, E1 = 0.125; C2 = 0.18, E2 = 0.17. Journal of Vascular Surgery 1995 21, 953-962DOI: (10.1016/S0741-5214(95)70223-7) Copyright © 1995 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 4 Representative Northern blot analysis of human iliac artery SMC transduced with MFG-tPA. Total cellular RNA 10 μg was electrophoresed and probed with "LTR" probe (A) or PAI-1 (B), or GAPDH probe (C). Lanes C1, C2, C3, and C4 represent nontransduced control for 30 minutes or 14 hours, harvested at day 14 and day 28, respectively. Lanes E1, E2, E3, and E4 represent transduced for either 30 minutes, or 14 hours, harvested at day 14 and day 28, respectively. Intensity of band for tPA was normalized to GAPDH:E1, 0.22; E2, 1.1; E3, 0.29; E4, 2.3. Average intensity of two bands for PAI was normalized to GAPDH: C1 = 0.07, E1 = 0.07; C2 = 0.07, E2 = 0.07; C3 = 0.07, E3 = 0.08; C4 = 0.13, E4 = 0.15. Journal of Vascular Surgery 1995 21, 953-962DOI: (10.1016/S0741-5214(95)70223-7) Copyright © 1995 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions