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Speaker ： Jethro Chen Date ： 2008.06.26

Reporter Vector. Speaker ： Jethro Chen Date ： 2008.06.26. Introduction. 1. Construction. 2. Reporter gene. 3. 4. Common reporter genes. Application. 5. Addition. 6. Contents. Promoter. Promoter. Structural Gene. Structural Gene. Insert. Reporter Gene. S. P. Vector. S. P.

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Speaker ： Jethro Chen Date ： 2008.06.26

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  1. Reporter Vector Speaker：Jethro Chen Date：2008.06.26

  2. Introduction 1 Construction 2 Reporter gene 3 4 Common reporter genes Application 5 Addition 6 www.themegallery.com Contents

  3. Promoter Promoter Structural Gene Structural Gene Insert Reporter Gene S P Vector S P Reporter Vector R Transcription Translation www.themegallery.com Introduction Insert Reporter Gene Light…Color…

  4. www.themegallery.com Construction • Constructive Biology ：

  5. www.themegallery.com Construction • Method： Insert source (1) Restriction Site (2) Purification (3) Ligation vector

  6. Reporter Gene Promoter Structural Gene Insert www.themegallery.com Construction • Types： A. Ex. 將一段 DNA 序列接到一個具有 GFP基因的 vector 上面，而接入的位置，剛好在 GFP 基因的前面。當 接好的載體，送入細胞後並經過培養，在特定位置 具有螢光蛋白的細胞就表示 insert 有成功接入 vector

  7. Insert Reporter Gene Promoter Structural Gene www.themegallery.com Construction B. Ex.將一段 DNA 序列接到一個具有 lacZ基因的 vector 上面，而接入的位置，會把 lacZ 基因從中切斷。當 接好的載體，送入菌體中並養在含有 X-gal (當 lacZ 基因表現的時候，X-gal 會代謝出藍色的物質) 的培 養基上，觀察會發現有些菌落呈現藍色 (表示 vector 自接，所以 lacZ 仍會表現)，有些菌落呈現白色 (表 示 insert 接入切位，所以使得 lacZ 無法表現) (Blue-white assay)

  8. www.themegallery.com Reporter Gene • History： 基因表達在細胞內有時很難適時被偵測，研究者為了避 開這些阻礙，設計了報導基因 (reporter gene) 於有興 趣的基因後面 • Property： 有非常容易被測知的表型，靈敏、可定量、可一再分析 的優點，甚至可作為 selectable marker

  9. www.themegallery.com Reporter Gene • Application： (1)定出promoter與 enhancer的強度 (2)定出 transcription / translation factor 的角色 (3)定出 transfection之效率 (4)藉由將報導蛋白質與欲研究蛋白質融合，可知蛋白質的 運行方向，鑑定蛋白質間的交互作用

  10. www.themegallery.com Reporter Gene (5)訊號傳遞相關研究，如發炎現象、細胞增殖、細胞循 環、PKC 及 Ca 的訊息傳遞等，從以下圖示，可知報導 基因篩選系統的應用領域非常廣泛

  11. www.themegallery.com Common reporter genes Luciferase (luc) β-galatosidase (β-gal) Green fluorescent proteins (GFP) Secreted alkaline phosphatase (SEAP) β-glucuronidase (GUS) Chloramphenicol acetyltransferase (CAT)

  12. www.themegallery.com Common reporter genes • 1.Green fluorescent proteins (GFP) : (1)自水母衍生而來，在UV 照射下即發出螢光，是理想 的 in vivo 基因表達指標，不需要基質，不怕熱、蛋 白質脢等 (2)可用螢光顯微鏡或 FACS (fluorescence-activated cell sorting) 進行分析

  13. www.themegallery.com Common reporter genes (3)由於 GFP 不是酵素，不具放大反應的效果，所以許 多廠商即利用 GFP 的突變使其更明亮，或產生不同顏 色，可分佈於不同細胞組成中

  14. www.themegallery.com Common reporter genes (4) pCMV-GFP-LpA： A CMV-driven vector for expression of GFP in eukaryotic cells. Constructed by Rein Aasland, EMBL Heidelberg

  15. www.themegallery.com Common reporter genes • 2.Luciferase (luc): (1)為一生物螢光酵素，從北美螢火蟲中所分離出來，可 催化 luciferin的反應；另一種結構不同的 Luciferase 則來自珊瑚蟲，其反應基質為 coelenterazine (2)因 Luciferase 的分析半衰期短，目前開發許多使半衰 期增加穩定性的配方 Clarity™ Luminescence Microplate Reader

  16. www.themegallery.com Common reporter genes (3)圖解原理：

  17. www.themegallery.com Common reporter genes (4) pBV-Luc： The vector features a transcription blocker and convenient multiple cloning sites upstream a minimal promoter. Constructed by Bert Vogelstein lab

  18. www.themegallery.com Common reporter genes • 3.β-galactosidase (β-gal): (1)Lac Operon： E.coli chromosome X X inducer

  19. www.themegallery.com Common reporter genes (2)利用 OPNG (0-nitrophenyl-β-D-galactoside) 或 CPRG (chlorophenol red β-D-galactoside) 水解 後產生有色物質，可用來定量分析 乳糖操縱子的誘導物 不會被 β-gal 所分解

  20. www.themegallery.com Common reporter genes (3)藍白測試： 水解 X-gal (5-bromo-4-chloro-3-indoyl-β-D- galactopyranoside) 產生藍色沉澱物

  21. www.themegallery.com Common reporter genes (4) pCMV Mammalian lacZ Vector： This vector expresses the full- length -galactosidase gene under the control of the CMV IE (cytomegalovirus immediate early gene) promoter. Constructed by Marker Gene Technologies, lnc

  22. www.themegallery.com Common reporter genes • 4.β-glucuronidase (GUS): 大腸菌的水解酵素，可水解glucuronide，大部份用 在研究病毒性的植物病源菌、植物、酵母菌、真菌等 無內生性來源的物種 β-glucuronidase staining of the ILR3 promoter-GUS fusion in an Arabidopsis flower

  23. www.themegallery.com Common reporter genes • 5. Chloramphenicol acetyltransferase (CAT): (1)細菌的 CAT 酵素將 acetyl-CoA 的乙醯基轉移到 Chloramphenicol 抗生素上，使去毒性

  24. www.themegallery.com Common reporter genes (2)藉由放射線或螢光的標示可加以定量分析，因真 核細胞無 CAT，且其穩定性佳，適合研究累積的 表現量

  25. www.themegallery.com Common reporter genes • 6. Secreted alkaline phosphatase (SEAP): (1)為人類胎盤鹼性磷酸脢(PLAP : placental alkaline phosphatase) (2)最大優點為可被分泌出細胞外，所以可以進行非破壞 性的活性分析，分析完畢尚可做其他研究用途

  26. www.themegallery.com Common reporter genes (3)偵測方式有螢光與化學光， 基質分別為 CDP-Star與 CSPD，靈敏度很高

  27. www.themegallery.com Common reporter genes (4) pSEAP2M-HTFR： Construction of a vector containing the extracellular domain of the human transferrin receptor fused with a secretory form of human placental alkaline phosphatase Constructed by Valentina DUBLJEVIC, Adnan SALI and James W. GODING

  28. www.themegallery.com Application The most typical use of a reporter gene for our purposes is to analyze “ How a gene is regulated? ”. 可將報導基因接於研究目標基因調控區之下游， 以了解研究其基因如何調控

  29. www.themegallery.com Application Gene Expression assays Promoter and enhancer assay Transformation and transfection assay Two-hybrid screening

  30. www.themegallery.com Application • Promoter assays ： Reporter genes can be used to assay for the activity of a particular promoter in a cell or organism

  31. www.themegallery.com Application • Using the CAT gene as a reporter for promoter activity assays Inactive Active+est TLC (Thin Layer Chromatography)：液相薄膜層析法

  32. www.themegallery.com Application • Enhancer assays ： Insertion of the enhancer-trap vector close to the enhancer of gene X will lead to the transcription and translation of the lacZ reporter contain enhancer B A B A

  33. www.themegallery.com Reporter Gene pre-gene expression assays • Reporter geneis directly attached to the gene of interest to create a gene fusion；the two genes are under the same promoter

  34. www.themegallery.com Application • Gene Expression assays ： Reporter genes can be used to assay for the expression of the gene of interest, which may produce a protein that haslittle obvious or immediate effect on the cell culture or organism Expression pattern of a GFP fusion to the peroxisomal multifunctional protein (MFP) in onion epidermal cells

  35. www.themegallery.com Application • Transformation assay： Transformation - changing the properties of a bacterial cell as a result of uptake of pure DNA Competent cell Ex. ECOS101

  36. www.themegallery.com Application The selectable-marker reporters such as CAT, the transfected population of bacteria can be grown on a substrate that contains chloramphenicol Only those cells that have successfully taken up the construct containing the CAT gene will survive and multiply under these conditions

  37. www.themegallery.com Application • Transfection assay： Transfection - a way of expressing a foreign or modified gene in an organism Ex. Lipofecamine

  38. www.themegallery.com Reporter Gene • Primary human hepatocytes expressing GFP after transfection using the pCMVEGFPLuc

  39. www.themegallery.com Application • Two-hybrid screening： A more complex usage of reporter genes on a large scale is in two-hybrid screening, which aims to identify proteins that natively interact with one another in vivo(the study of protein-protein interactions)

  40. www.themegallery.com Application A.Gal4 transcription factor gene produces two domain protein (BD and AD) which is essential for transcription of the reporter gene (LacZ)

  41. www.themegallery.com Application B,C. Two fusion proteins : Gal4BD+Baitand Gal4AD+Prey None of them is usually sufficient to initiate the transcription (of the reporter gene) alone.

  42. www.themegallery.com Application D. When both fusion proteins are produced and Bait part of the first interact with Prey part of the second, transcription of the reporter gene occurs interaction

  43. www.themegallery.com Addition • http://www.promega.com/ • http://www.panomics.com/ • http://www.clontech.com/ • http://www.promokine.de/index.php#top • http://www.ambion.com/index.html

  44. Bye!! Thank You

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