title: angiogenesis
status: Public on Nov 25 2003
contact.city: Adelaide
contact.first: Anna
contact.last: Tsykin
contact.email: anna.tsykin@adelaide.edu.au
contact.state: SA
contact.address:  Frome Road
contact.department: Hanson Institute
contact.country: Australia
contact.institute: SA Pathology
contact.phone: 61 8 8222 3479
organism: Homo sapiens
description: Cell culture - In vitro capillary tube model:
  HUVEC were isolated as described previously[Gamble,1993] and cultured in gelatin-coated flasks in M199 medium with Earles balanced salts and 0.68 mM glutamine, 20 mM Hepes, 20% FCS, 15 ug/ml endothelial cell growth supplement (ECGS, BD Biosciences, Bedford, MA, USA), 50 U/ml penicillin, 50 U/ml streptomycin and 15 ug/ml heparin (Sigma, St. Louis, MO, USA).
  The capillary tube formation assay was performed as previously described[Gamble,1993]. Briefly, 6.4 x 104 cells/160 ul  HUVEC medium were plated onto 100 ul gelled bovine type I collagen (Celtrix Laboratories, Palo Alto, CA, USA) in 96-well flat-bottomed microtitre plates. Capillary tube formation was stimulated by the addition of 20 ng/ml phorbol myristate acetate (PMA) and an antibody against A2B1-integrin (RMAC11) which promotes the formation of complex multicellular tubes.
  Generation of subtracted cDNA libraries:
  HUVEC from 3 different umbilical cords were plated onto 100ul of bovine type I collagen in 96-well microtitre plates (15 wells/cord). At times 0, 0.5, 3, 6 and 24 hours after stimulation with PMA and RMAC11, total RNA was isolated using the TRIZOL
  procedure (Invitrogen, Carlsbad, CA, USA). These times were chosen because they approximate time points at which distinct morphological events are known to occur[Gamble,1993;Meyer,1997]. Yields of 7-13ug of total HUVEC RNA were obtained. To achieve enough mRNA for subtraction experiments, the "Switching Mechanism At the 5' end of RNA Transcript" (SMART) PCR cDNA synthesis procedure (Clontech, Palo Alto, CA, USA) was used to convert the mRNA to cDNA and to preferentially amplify full length
  cDNAs. The PCR Select? cDNA Subtraction Kit (Clontech) was then used to perform suppression subtractive hybridisation between each adjacent time point in both the forward and reverse orientation. Four forward and four reverse libraries were then generated by digesting the subtracted cDNAs with EagI and cloning into the NotI site in pBluescriptIISK+ (Stratagene, La Jolla, CA, USA). These libraries were used to transform XL-10 Gold? ultracompetent bacteria (Stratagene).
  Generation and probing of microarrays:
  Generation of microarrays was performed by the Australian Genome Research Facility (AGRF, Melbourne, Australia). Colonies from the forward subtracted libraries (0-0.5h 3200 colonies; 0.5-3h; 3000 colonies, 3-6h, 2800 colonies; 6-24h, 1000 colonies) were robotically picked and glycerols stocks generated. A total of 10,000 clones were picked. Each clone was then PCR amplified using primers to the T7 and T3 promoters in the vector that flank the cloning site. The PCR products were microarrayed
  onto glass slides in duplicate.
  
  The cDNAs used in this microarray were identified as part of collaborative project between the IMVS and Bionomics Limited. The project aims were to identify genes up-regulated during in vitro capillary tube formation as targets for angiogenesis-based therapeutics.
  
  Any requests for further information regarding the data generated from this collaboration should be directed to Bionomics Limited (www.bionomics.com.au) at the following address:
  
  Bionomics Limited
  31 Dalgleish Street
  Thebarton, South Australia
  Australia, 5031
  Phone: 618 8354 6104
  Fax: 618 8354 6199
  Email: busdev@bionomics.com.au
  Keywords = in-vitro
  Keywords = HUVEC
  Keywords = capillary tube formation