Your SlideShare is downloading. ×
лекция 1 гибридизация phk in situ.
лекция 1 гибридизация phk in situ.
лекция 1 гибридизация phk in situ.
лекция 1 гибридизация phk in situ.
лекция 1 гибридизация phk in situ.
лекция 1 гибридизация phk in situ.
лекция 1 гибридизация phk in situ.
лекция 1 гибридизация phk in situ.
лекция 1 гибридизация phk in situ.
лекция 1 гибридизация phk in situ.
лекция 1 гибридизация phk in situ.
лекция 1 гибридизация phk in situ.
лекция 1 гибридизация phk in situ.
лекция 1 гибридизация phk in situ.
лекция 1 гибридизация phk in situ.
лекция 1 гибридизация phk in situ.
лекция 1 гибридизация phk in situ.
лекция 1 гибридизация phk in situ.
лекция 1 гибридизация phk in situ.
лекция 1 гибридизация phk in situ.
лекция 1 гибридизация phk in situ.
лекция 1 гибридизация phk in situ.
лекция 1 гибридизация phk in situ.
лекция 1 гибридизация phk in situ.
лекция 1 гибридизация phk in situ.
лекция 1 гибридизация phk in situ.
лекция 1 гибридизация phk in situ.
лекция 1 гибридизация phk in situ.
лекция 1 гибридизация phk in situ.
лекция 1 гибридизация phk in situ.
лекция 1 гибридизация phk in situ.
лекция 1 гибридизация phk in situ.
лекция 1 гибридизация phk in situ.
лекция 1 гибридизация phk in situ.
лекция 1 гибридизация phk in situ.
лекция 1 гибридизация phk in situ.
лекция 1 гибридизация phk in situ.
лекция 1 гибридизация phk in situ.
лекция 1 гибридизация phk in situ.
лекция 1 гибридизация phk in situ.
лекция 1 гибридизация phk in situ.
лекция 1 гибридизация phk in situ.
лекция 1 гибридизация phk in situ.
лекция 1 гибридизация phk in situ.
лекция 1 гибридизация phk in situ.
лекция 1 гибридизация phk in situ.
лекция 1 гибридизация phk in situ.
лекция 1 гибридизация phk in situ.
лекция 1 гибридизация phk in situ.
лекция 1 гибридизация phk in situ.
лекция 1 гибридизация phk in situ.
лекция 1 гибридизация phk in situ.
лекция 1 гибридизация phk in situ.
лекция 1 гибридизация phk in situ.
лекция 1 гибридизация phk in situ.
лекция 1 гибридизация phk in situ.
лекция 1 гибридизация phk in situ.
лекция 1 гибридизация phk in situ.
лекция 1 гибридизация phk in situ.
лекция 1 гибридизация phk in situ.
лекция 1 гибридизация phk in situ.
лекция 1 гибридизация phk in situ.
лекция 1 гибридизация phk in situ.
лекция 1 гибридизация phk in situ.
лекция 1 гибридизация phk in situ.
лекция 1 гибридизация phk in situ.
лекция 1 гибридизация phk in situ.
лекция 1 гибридизация phk in situ.
лекция 1 гибридизация phk in situ.
лекция 1 гибридизация phk in situ.
лекция 1 гибридизация phk in situ.
Upcoming SlideShare
Loading in...5
×

Thanks for flagging this SlideShare!

Oops! An error has occurred.

×
Saving this for later? Get the SlideShare app to save on your phone or tablet. Read anywhere, anytime – even offline.
Text the download link to your phone
Standard text messaging rates apply

лекция 1 гибридизация phk in situ.

729

Published on

Published in: Technology
0 Comments
0 Likes
Statistics
Notes
  • Be the first to comment

  • Be the first to like this

No Downloads
Views
Total Views
729
On Slideshare
0
From Embeds
0
Number of Embeds
4
Actions
Shares
0
Downloads
0
Comments
0
Likes
0
Embeds 0
No embeds

Report content
Flagged as inappropriate Flag as inappropriate
Flag as inappropriate

Select your reason for flagging this presentation as inappropriate.

Cancel
No notes for slide

Transcript

  • 1. БИОИМИДЖИНГ Лекция 1 Исследования in situ and in vivo Сергей Лукьянов Институт биоорганической химии имени академиков М.М. Шемякина и Ю.А. Овчинникова РАН
  • 2. Экспрессия генов на уровне РНК
  • 3. STUDYING GENE EXPRESSION Probe for mRNA In situ Studying gene expression Northern Hybridisation in situ or in microarray Probe for protein In situ ‘Western’ immunohistochemistry ‘Reporter gene’ In vivo Lac Z: b-galactosidase GFP: green fluorescent protein Most methods can be adapted to either tissue sections or whole mounts
  • 4. Гибридизация РНК in situ
  • 5. IN-SITU HYBRIDISATION – SUMMARY Fixed tissue section or permeabilised whole mount Probe for expressed mRNA using antisense RNA or DNA Visualise location by fluorescence or enzyme-linked antibody
  • 6. Enzyme coupled immunological detection of probes Chemoluminescence Fluorescence Color formation
  • 7. Enzymes used Alkaline phosphatase (AP) • At best, the most sensitive detection system • Present in some mammalian tissues • Most common substrate: NBT/BCIP (nitroblue tetrazolium / 5-bromo-4-chloro-3-indolyl phosphate), dark blue colour • A fluorescent substrate: ELF-97 (Molecular Probes) Peroxidase (POD) • Variably present in mammalian tissues • Most common (and most sensitive) substrate: DAB (diaminobenzidine), brown colour
  • 8. RNA in situ hybridization
  • 9. Whole mount in situ hybridization Selected examples of candidate genes from gene expression profiling of Dll1 mouse mutant embryos verified by whole-mount in situ-hybridization.
  • 10. Whole mount in situ hybridization Proteinase K, 3 min, Mouse embryo, 10.5d Chick embryo (35h) Proteinase K, 30 min Mouse embryo, 10.5d Pax6 mRNA detected by hybridisation with digoxigenin labelled antisense RNA followed by alkaline phosphatase-coupled antibody against digoxigenin (Gilbert Fig. 4.16, A from Li et al 1994, B from Gray et al 2004)
  • 11. Локализация белка in situ
  • 12. Immunological detection of the protein Methods: • Immunohistochemistry (IHC), • Immunofluorescence (IF), • Enzyime-Linked Immunosorbent Assay (ELISA) • Western Blotting (WB), • Immunoprecipitation (IP), • Fluorescence Activated Cell Sorting (FACS) Principle of recognition primary antibody binds to specific epitope (one or several) in the protein Principle of detection primary antibody or secondary antibody that recognise primary antibody is labelled (examples: HRP for IHC and Western blotting, fluorescent dye for IF and FACS)
  • 13. IMMUNOHISTOCHEMISTRY Fixed tissue section or permeabilised whole mount Probe for expressed protein using primary antibody Visualise location using second antibody, coupled to enzyme, fluorophore or gold
  • 14. Material for Immunohistochemistry and Immunofluorescence Fresh or frozen • Tissue sections; • Cells grown on cover slips; • Cells sedimented on object glass • using cytospin centrifuge Advantages Antigens are in a good shape, and most of primary antibodies can be used Intracellular localization studies are possible even in tissue sections Disadvantages Limited time of storage Retrospective analysis is not possible Paraffin embedded • Tissue sections Advantages Extremely long storage time, Retrospective analysis can be done on archive material Disadvantages Antigen-retrieval has to be designed individually for most of antigens Only limited amount of labeled primary antibodies recognize retrieved antigen
  • 15. IHC and IF: overlapping terms Direct Indirect or enzyme or enzyme Advantages Cheap Fast Disadvantages Only limited amount of labeled primary antibodies are available commercially Advantages Wide range of labeled secondary antibodies are available commercially It is always possible to design combination for double and triple staining Disadvantages Takes more time, sometimes is more expensive Additional control for the background staining is absolutely necessary
  • 16. Controls IHC Background signal coming from substrate IF Auto fluorescence Non-specific signal coming from secondary antibody alone Non-specific signal coming form primary antibody. Solution Isotype control for monoclonal antibodies or preimmune serum for polyclonal antibodies has to be used
  • 17. Multuple IHC Multiple staining can also be done with enzyme conjugated antibodies developed with different chromogen substrates to produce the end products of different colors
  • 18. in situ hybridization & Immunohistochemistry
  • 19. Whole mount immunostaining 4 day old zebrafish embryo labelled with SV2 and acetylated tubulin antibodies showing axon tracts(green) and neuropil (red) viewed from lateral (top) and dorsal (bottom) orientations.
  • 20. Whole mount fluorescence immunohistochemistry, in situ hybridization & Optical Projection Tomography An image created using Optical Projection Tomography has won a Special Award at the 2008 Wellcome Image Awards. Image shows wildtype E10.5 mouse embryo visualised using whole mount fluorescence immunohistochemistry. Green shows the staining pattern of an antibody against neurofilament and blue highlights the expression of the HNF3β protein. Red indicates the heart.
  • 21. USE OF A REPORTER GENE Engineer construct composed of regulatory sequence of interest and lacZ Inject into cells Study expression of LacZ • b-galactosidase (E.coli) cleaves the substrate XGAL to release a coloured insoluble product • Fluorescent proteins can be used as an alternative and have the advantage of being observable in vivo
  • 22. Флуоресцентные белки
  • 23. Green Fluorescent Protein (GFP) from hydroid jellyfish Aequorea victoria GFP is a secondary emitter in bioluminescent system 1962: discovery (Shimomura et al.) 1992: cloning (Prasher et al.) 1994: First application (Chalfie et al.) 2008: Nobel Prize (O. Shimomura, M. Chalfie, and R. Tsien)
  • 24. Green fluorescent protein (GFP) is the first genetically encoded fluorescent nanomarker Crucial breakthroughs came with the cloning of the gene by Prasher et al. (1992) and the demonstration by Chalfie et al. (1994) that expression of the gene in other organisms creates fluorescence. Chalfie et al. Science 1994
  • 25. Структура GFP 11 -слоев образуют бочонок с -спиралью в середине (238 ак). Хромофор образуется внутри глобулы путем автокаталитической циклизации остова трех аминокислот (Ser65-Tyr66-Gly67)
  • 26. GFP - genetically encoded fluorescent probe • Self-catalytic chromophore formation (the only external O2 is required) • Low toxisity • High stability • Monomeric state
  • 27. Использование GFP-подобных белков для анализа временного и пространственного патерна экспрессии генов
  • 28. GFP applications 2. Visualization of protein localization and turnover.
  • 29. Enhanced GFP mutant Absorption and emission spectral profiles of (A) wild-type A. victoria GFP and (B) the improved S65T derivative.
  • 30. Мутагенез • • • Изучение функции белков in vitro и in vivo Получение белков с новыми свойствами Разновидности мутагенеза: 1. Создание коллекции делеционных инсерционных мутантов 2. Направленный мутагенез 3. Случайный мутагенез 4. Транспозоны – инактивация генов
  • 31. Способы введения случайных мутаций • Химический мутагенез • Синтез ДНК с ошибками • Случайное объединение гомологичных участков генов (DNA shuffling) • Удлинение ДНК с переменой матриц (Staggered Extension Process) • Рекомбинирование фрагментов генов, независимое от гомологии
  • 32. Случайный мутагенез
  • 33. Стратегия направленной эволюции макромолекул
  • 34. Направленный мутагенез с использованием ПЦР и перекрывающихся праймеров Введение точечных мутаций
  • 35. GFP applications Visualization of temporal and spatial pattern of promoter activation. Organism and cell labeling
  • 36. GFP applications Visualization of protein localization and turnover.
  • 37. GFP mutants Yellow (EYFP, Venus, …) em 528 nm Green (EGFP, Emerald, …) em 508 nm Cyan (ECFP, Cerulean, …) em 475 nm Blue (EBFP, Azurite, …) em 448 nm Wavelength, nm
  • 38. GFP mutants Absorption (A) and emission (B) spectral profiles of the enhanced Aequorea-GFP derivatives
  • 39. Использование GFP и его мутантов Анализ белок-белковых взаимодействий с помощью резонансного переноса энергии флуоресценции (Foster Resonance Energy Transfer, FRET) Передача энергии от молекулы донора к молекуле акцептора происходит посредством диполь–дипольного взаимодействия.
  • 40. Far-red fluorescence is preferable for whole body imaging
  • 41. Discovery of GFP-like fluorescent and colored proteins in coral polyps (1999) Yuly Labas 1933-2008 Matz et al., Nat Biotechnol. 1999
  • 42. Evolution diversity of GFP family Annelida Nematoda Crustacea Mollusca Arthropoda Branchiostoma Chordata Echinodermata Hydrozoa Anthozoa Cnidaria Typhlocoela Ctenophora Porifera
  • 43. Разноцветные, окрашенные и бесцветные GFPподобные белки из медуз Aequorea coerulescens (безцветный белок) Phyalidium sp. (желтый белок) Antomedusae (хромобелок)
  • 44. Копеподы Ланцетники
  • 45. Mantis shrimp C. H. Mazel et al., Science, 2004
  • 46. Spectral diversity of GFP-like proteins Main spectral classes, which are widely encountered in natural GFP-like proteins
  • 47. Chromophores of native fluorescent proteins
  • 48. 1.0 0 400 450 mTFP1 TagCFP, Cerulean Azurite, EBFP2 TagBFP 2.0 500 550 mOrange 600 mPlum TagRFP, TagRFP-T mStrawberry mRuby mCherry mRaspberry mKate2 mKO Venus, TagYFP, Citrine, Topaz, SYFP2, etc. EGFP, EmGFP, AcGFP1, Wasabi, TagGFP2, etc. Sirius Fluorecence, a.u. Fluorescent proteins: wide color palette 650 700 Wavelength, nm
  • 49. Whole body imaging using FPs DsRed-Exp Katushka White light DsRed-Exp Katushka Fluorescence Transgenic Xenopus laevis expressing Katushka or DsRed-Express under the control of cardio-actin promoter. Shcherbo et al., Nat Methods 2007.
  • 50. Cre-reporter transgenic mouse expressing the far-red fluorescent protein Katushka (a) Schematic structure of the transgenic construct. The transgene contains the CMV early enhancer/chicken β-actin promoter (CAG), a transcription STOP cassette (STOP) flanked by loxP sites (black triangles), the Katushka cDNA and the rabbit β-globin polyadenylation signal (pA). (b) Cre-mediated transgene recombination in a head-to-tail multicopy integration event (a 2 tandem copies integration is depicted as an example). Diéguez-Hurtado et al., 2010
  • 51. Cre-reporter transgenic mouse expressing the far-red fluorescent protein Katushka Detection of Katushka fluorescence in reporter mice after Cre-mediated recombination. a: In vivo whole-body direct fluorescence analysis of N1 progeny (newborns) from crosses of Tg(CAGLSL-KFP) with Tg(CMV-Cre) mice. Only the double transgenic pup is visible and exhibits ubiquitous and strong expression of Katushka. b: Fluorescence image of seven littermates obtained after mating a germ-line Cre-recombined reporter male with a wild-type female. Diéguez-Hurtado et al., 2010 c: Katushka fluorescence in isolated organs of germ-line recombined reporter adult mice (center panel). The two panels at the left show the bright field (BF) and red fluorescence (RF) images of a wild-type mouse as control of tissue autofluorescence, while the two panels at the right show those of a Tg(CAG-LSL-KFP) mouse carrying the intact floxed reporter transgene.
  • 52. Cre-reporter transgenic mouse expressing the far-red fluorescent protein Katushka In vivo assessment of pancreatic-β-cell-specific, Cre-mediated expression of Katushka in adult mice. a: IVIS Spectrum (Xenogen Co.) whole body fluorescence images of a single transgenic Tg(CAGLSL-KFP) mouse (left) and a double transgenic Tg(RIP-Cre);Tg(CAG-LSL-KFP) mouse (right). Katushka fluorescence is observed only in the region where the pancreas is positioned. The abdominal region has been previously depilated to avoid signal attenuation by the hair. b: IVIS fluorescence image of the isolated pancreas from mice shown in panel a. c: Confocal images of cryosections of the pancreas shown in panel b after 4% paraformaldehyde fixation and OCT embedding. A white dashed line has been traced around the pancreatic islets. Blue channel shows nuclei stained with DAPI. Katushka fluorescence (red channel) is detected only in the double transgenic islet. Background autofluorescence is indistinguishable between single and double transgenic mice. d: Confocal image of a cryosection of the pancreas from the double transgenic mouse shown in panel c (top). Katushka expression is restricted to the βcells of the pancreatic islets. Diéguez-Hurtado et al., 2010
  • 53. DsRed and GFP structure In general, Anthozoa FPs have more elliptical symmetry than Aequorea victoria GFP derivates
  • 54. DsRed tetramer Catalytic R96 and E222
  • 55. 5-color imaging with TagFPs TagBFP-Mito TagCFP-Actin TagYFP-Tubulin TagRFP-Golgi TagFP635-H2B
  • 56. mKate-Tubulin Images were kindly provided by Michael W. Davidson (Florida State University)
  • 57. mKate-EB3 Images were kindly provided by Michael W. Davidson (Florida State University)
  • 58. mKate Cx43 Images were kindly provided by Michael W. Davidson (Florida State University)
  • 59. mKate H2B Images were kindly provided by Michael W. Davidson (Florida State University)
  • 60. mKate-Clathrin Images were kindly provided by Michael W. Davidson (Florida State University)
  • 61. mKate2-EB3 fusion (microtubule end-binding protein) By Michael W. Davidson (Florida State University)
  • 62. mKate2.7- alpha actinin fusion
  • 63. mKate2.7- protein kinase CSRC fusion
  • 64. Td-Katushka-zyxin fusion By Michael W. Davidson (Florida State University)
  • 65. Fluorescent protein’s application for drug discovery Cell transfection with fluorescent protein genes linked to genes of interest Transfer of visible targets to mice Stably transfected tumor cells Discovery and evaluation of candidate drugs Target visualization Drug treatment control control drug1 drug2 treated
  • 66. Whole body imaging using fluorescent proteins Prostate cancer PC-3-RFP Glioma U87-RFP Glioma U87-RFP and GFP Breast cancer MDA-MB-435-GFP Pancreas cancer MIA-PaCa-2-RFP Colon cancer HCT-116-RFP
  • 67. Glowfish и другие… Z. Gong et al., BBRC 2003; S. Ding et al., Cell, 2005 X.Y.Yin et al., Biol. Rep., 2007
  • 68. Glowfish и другие… Z. Gong et al., BBRC 2003; S. Ding et al., Cell, 2005 X.Y.Yin et al., Biol. Rep., 2007

×