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Overexpression of Epidermal Growth Factor Like Domain 7 Using an Inducible Piggybac Vector

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Overexpression of Epidermal Growth Factor Like Domain 7 Using an Inducible Piggybac Vector Overexpression of Epidermal growth factor like domain 7 using an inducible PiggyBac vector Jakob Jakobsson Líf- og umhverfisvísindadeild Háskóli Íslands 2017i Overexpression of Epidermal growth factor like domain 7 using an inducible PiggyBac vector Jakob Jakobsson 15 eininga ritgerð sem hluti af Baccalaureus Scientiarum gráðu í sameindalíffræði Leiðbeinandi Guðrún Valdimarsdóttir Faculty of life and environmental sciences School of engineering and Natural sciences University of Iceland Reykjavík, May 2017 i Overexpression of Epidermal growth factor like domain 7 using an inducible PiggyBac vector 15 eininga ritgerð sem hluti af Baccalaureus Scientiarum gráðu í sameindalíffræði Höfundarréttur © 2017 Jakob Jakobsson Öll réttindi áskilin Líf- og umhverfisvísindadeild Verkfræði- og náttúruvísindasvið Háskóli Íslands Askja, Sturlugötu 7 101 Reykjavík Sími: 525 4000 Skráningarupplýsingar: Jakob Jakobsson, 2017, Overexpression of Epidermal growth factor like domain 7 using an inducible PiggyBac vector, BS ritgerð, Líf- og umhverfisvísindadeild, Háskóli Íslands, 23 bls. ii Útdráttur Æðamyndun er ferli sem að æðaþelsfrumur fara í gegnum til að mynda nýjar æðar útfrá þeim eldri. Óvenjuleg æðamyndun á þátt í fjölda sjúkdóma og í æxlisvexti krabbameina. Rannsóknir á æðamyndun og æðaþelsfrumum auka skilning á þessum flóknu ferlum sem gefur leið að nýjum meðferðum gegnsúk dómum. Epidermal growth factor like domain 7 (EGFL7) er þekkt sem mikilvægur þáttur í æðamyndun en virkni hans er ekki skilin að fullu. Markmið þessa verkefnis er að meta hvort að hægt sé að innskeyta EGFL7-PiggyBac genaferju í æðaþelsfrumur úr mönnum (HUVEC) og hvort að virkni genaferju gæti verið stjórnað með cumate títrunum. Annað markmið var að rannsaka hver áhrif yfirtjáningar EGFL7 væri á æðamyndun og á genatjáningu nokkura þátta tengdu utanfrumuefninu. Til að ná genaferju inn í frumurnar þurfti að nota nokkrar ólíkar aðferðir áður en loksins tókst að tjá EGFL7. Þrátt fyrir að tekist hafi að koma genaferju inn og að tjá genið tókst ekki að stjórna því ennfrekar með cumate titrunum. Engar breytingar sáust á tjáningu VE-cadherin en bæling varð á tjáningu elastins með yfirtjáningu á EGFL7 og við örvun með BMP9. Yfirtjáning EGFL7 jók einnig á fjarlægð milli greina æða. Abstract Angiogenesis is the process where endothelial cells form new blood vessels from existing vessels. Aberrant angiogenesis is a cause of many diseases as well as being involved in cancer growth. Research on angiogenesis and endothelial cells increases our understanding of these complex processes and gives new insights on possible treatments for diseases related to angiogenesis. Epidermal growth factor like domain 7 (EGLF7) is known to be a key factor in angiogenesis but much of its exact function is still to be uncovered. The aim of the project was to evaluate whether or not an unproven EGFL7-PiggyBac vector could be used to successfully transfect HUVECs and if its expression could be controlled using cumate titrations. A secondary aim was to study what effects EGFL7 overexpression had on angiogenesis and certain ECM related factors. To achieve this several transfection attempts were made using different reagents and protocols, until successful induction of EGFL7 expression in transfected cells was achieved. However, EGFL7 expression could not be controlled upon tritration of cumate. No changes were seen in the expression of VE-cadherin but the downregulation of the ECM gene elastin was seen in cells overexpressing EGFL7 and those treated with BMP9. The effect of EGFL7 and BMP9 on angiogenesis was studied using a tube-like formation assay. The overexpression of EGFL7 and BMP9 treatment did increase the branching interval in tubes. iii iv Fyrir Elísu v Table of contents Figures ................................................................................................................................ viii Tables ................................................................................................................................... ix Abbreviations ....................................................................................................................... x Acknowledgements ............................................................................................................. xi 1 Introduction ..................................................................................................................... 1 1.1 The vascular system and blood vessel formation .................................................... 1 1.1.1 Vasculo and angiogenesis .............................................................................. 1 1.1.2 The role af blood vessel formation in tumorigenesis ..................................... 2 1.2 The TGF β signaling pathway ................................................................................. 2 1.2.1 TGF-β subfamilies .......................................................................................... 3 1.2.2 The BMP subfamily ....................................................................................... 3 1.3 The extracellular matrix and cell junctions ............................................................. 4 1.3.1 Elastin and Collagen ....................................................................................... 4 1.3.2 Cell-Cell and Cell-Matrix adhesion ................................................................ 5 1.4 Epidermal growth factor-like domain 7 ................................................................... 5 1.4.1 EGFL7 and angiogenesis ................................................................................ 6 1.4.2 EGLF7 and the Notch pathway ...................................................................... 6 1.4.3 EGFL7 and cancer .......................................................................................... 7 2 Aim of the project ............................................................................................................ 8 3 Materials and methods ................................................................................................... 8 3.1 Cell culture ............................................................................................................... 8 3.1.1 Cell stimulation ............................................................................................... 9 3.2 Transfections ............................................................................................................ 9 3.2.1 Transpass D1 ................................................................................................ 10 3.2.2 Lipofectamine 3000 ...................................................................................... 10 3.2.3 Fugene HD .................................................................................................... 10 vi 3.3 Nucleic acids ............................................................................................................. 10 3.3.1 RNA extraction ............................................................................................. 10 3.3.2 cDNA synthesis ............................................................................................ 10 3.3.3 Polymerase chain reaction ............................................................................ 10 3.4 Western Blot .............................................................................................................. 11 3.4.1 Protein preperation ........................................................................................ 11 3.4.2 SDS page ...................................................................................................... 11 3.4.3 Transfer ......................................................................................................... 12 3.4.4 Antibody staining.......................................................................................... 12 3.5 Tube-like formation assay and immunofluorescence ............................................... 13 3.5.1 Tube-like formation assay .......................................................................... 13 3.5.2 Immunofluorescent staining ....................................................................... 13 4 Results ............................................................................................................................ 14 4.1 EGFL7 overexpression .............................................................................................. 14 4.1.1 Transfection .................................................................................................... 14 4.1.2 Confirmation of cumate induced gene expression .......................................... 15 4.2 Effects of EGFL7 and BMP9 on gene expression .................................................... 16 4.3 Tube-like formation assay ......................................................................................... 17 4.3.1 Analysis of tube-like formation .................................................................... 18 5 Discussion ....................................................................................................................... 20 Bibliography ....................................................................................................................... 22 vii Figures Figure 1: Schematic representation of he processes of vasculo and angiogenesis. ............... 2 Figure 2: A simplified overview of the TGF-β family .........................................................
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