БИОИМИДЖИНГ
Лекция 1
Исследования in situ and in vivo
Сергей Лукьянов
Институт биоорганической химии имени академиков М.М....
Экспрессия генов на уровне РНК
STUDYING GENE EXPRESSION
Probe for mRNA
In situ
Studying gene
expression

Northern
Hybridisation in situ or in
microarray
...
Гибридизация РНК in situ
IN-SITU HYBRIDISATION – SUMMARY

Fixed tissue section
or permeabilised
whole mount

Probe for expressed
mRNA using antisen...
Enzyme coupled immunological detection of probes
Chemoluminescence

Fluorescence Color formation
Enzymes used
Alkaline phosphatase (AP)
• At best, the most sensitive detection system
• Present in some mammalian tissues
...
RNA in situ hybridization
Whole mount in situ hybridization

Selected examples of
candidate genes from gene
expression profiling of Dll1
mouse mutan...
Whole mount in situ hybridization

Proteinase K, 3 min,
Mouse embryo, 10.5d

Chick embryo
(35h)

Proteinase K, 30 min
Mous...
Локализация белка in situ
Immunological detection of the protein
Methods:
• Immunohistochemistry (IHC),
• Immunofluorescence (IF),
• Enzyime-Linked ...
IMMUNOHISTOCHEMISTRY

Fixed tissue section
or permeabilised
whole mount

Probe for expressed
protein using primary
antibod...
Material for Immunohistochemistry and Immunofluorescence
Fresh or frozen
•
Tissue sections;
•
Cells grown on cover slips;
...
IHC and IF: overlapping terms
Direct

Indirect
or enzyme

or enzyme

Advantages
Cheap
Fast

Disadvantages
Only limited amo...
Controls
IHC
Background signal coming
from substrate

IF
Auto fluorescence

Non-specific signal coming from secondary anti...
Multuple IHC

Multiple staining can also be done with enzyme conjugated antibodies
developed with different chromogen subs...
in situ hybridization & Immunohistochemistry
Whole mount immunostaining
4 day old zebrafish embryo
labelled with SV2 and
acetylated tubulin antibodies
showing axon tra...
Whole mount fluorescence immunohistochemistry, in
situ hybridization & Optical Projection Tomography
An image created usin...
USE OF A REPORTER GENE
Engineer construct
composed of regulatory
sequence of interest
and lacZ

Inject into cells

Study e...
Флуоресцентные белки
Green Fluorescent Protein (GFP) from hydroid
jellyfish Aequorea victoria

GFP is a secondary emitter in bioluminescent sys...
Green fluorescent protein (GFP) is the first genetically
encoded fluorescent nanomarker
Crucial breakthroughs came with th...
Структура GFP

11 -слоев образуют бочонок с -спиралью в середине (238 ак). Хромофор
образуется внутри глобулы путем авто...
GFP - genetically encoded fluorescent probe
• Self-catalytic chromophore formation (the only external
O2 is required)
• Lo...
Использование GFP-подобных белков для анализа временного и пространственного
патерна экспрессии генов
GFP applications

2. Visualization of protein localization and turnover.
Enhanced GFP mutant

Absorption and emission spectral profiles of (A) wild-type A.
victoria GFP and (B) the improved S65T ...
Мутагенез

•
•
•

Изучение функции белков in vitro и in vivo
Получение белков с новыми свойствами
Разновидности мутагенеза...
Способы введения случайных мутаций
• Химический мутагенез
• Синтез ДНК с ошибками
• Случайное объединение гомологичных уча...
Случайный мутагенез
Стратегия направленной эволюции макромолекул
Направленный
мутагенез с
использованием
ПЦР и
перекрывающихся
праймеров
Введение точечных
мутаций
GFP applications
Visualization of temporal and spatial pattern of promoter activation. Organism
and cell labeling
GFP applications
Visualization of protein localization and turnover.
GFP mutants
Yellow (EYFP, Venus, …) em 528 nm
Green (EGFP, Emerald, …) em 508 nm
Cyan (ECFP, Cerulean, …) em 475 nm
Blue (...
GFP mutants

Absorption (A) and emission (B) spectral profiles of the
enhanced Aequorea-GFP derivatives
Использование GFP и его мутантов

Анализ белок-белковых взаимодействий с помощью
резонансного переноса энергии флуоресценц...
Far-red fluorescence is preferable for whole body
imaging
Discovery of GFP-like fluorescent and colored
proteins in coral polyps (1999)

Yuly Labas
1933-2008

Matz et al., Nat Biot...
Evolution diversity of GFP family
Annelida

Nematoda
Crustacea

Mollusca

Arthropoda
Branchiostoma

Chordata
Echinodermata...
Разноцветные, окрашенные и бесцветные GFPподобные белки из медуз

Aequorea coerulescens
(безцветный белок)

Phyalidium sp....
Копеподы

Ланцетники
Mantis shrimp

C. H. Mazel et al., Science, 2004
Spectral diversity of GFP-like proteins
Main spectral classes, which are widely
encountered in natural GFP-like proteins
Chromophores of native fluorescent proteins
1.0

0
400
450

mTFP1

TagCFP, Cerulean

Azurite, EBFP2
TagBFP

2.0

500
550

mOrange

600

mPlum

TagRFP, TagRFP-T
mStraw...
Whole body imaging using FPs
DsRed-Exp

Katushka

White light

DsRed-Exp

Katushka

Fluorescence

Transgenic Xenopus laevi...
Cre-reporter transgenic mouse expressing the
far-red fluorescent protein Katushka

(a) Schematic structure of the transgen...
Cre-reporter transgenic mouse expressing the far-red
fluorescent protein Katushka

Detection of Katushka fluorescence in
r...
Cre-reporter transgenic mouse
expressing the far-red
fluorescent protein Katushka
In vivo assessment of pancreatic-β-cell-...
DsRed and GFP structure
In general, Anthozoa FPs have
more elliptical symmetry than
Aequorea victoria GFP
derivates
DsRed tetramer

Catalytic R96
and E222
5-color imaging with TagFPs

TagBFP-Mito
TagCFP-Actin
TagYFP-Tubulin
TagRFP-Golgi
TagFP635-H2B
mKate-Tubulin

Images were kindly provided by Michael W. Davidson (Florida State University)
mKate-EB3

Images were kindly provided by Michael W. Davidson (Florida State University)
mKate Cx43

Images were kindly provided by Michael W. Davidson (Florida State University)
mKate H2B

Images were kindly provided by Michael W. Davidson (Florida State University)
mKate-Clathrin

Images were kindly provided by Michael W. Davidson (Florida State University)
mKate2-EB3 fusion
(microtubule end-binding protein)

By Michael W. Davidson (Florida State University)
mKate2.7- alpha actinin fusion
mKate2.7- protein kinase CSRC fusion
Td-Katushka-zyxin fusion

By Michael W. Davidson (Florida State University)
Fluorescent protein’s application for drug discovery
Cell transfection with
fluorescent protein
genes linked to
genes of i...
Whole body imaging using fluorescent proteins
Prostate cancer PC-3-RFP

Glioma U87-RFP

Glioma U87-RFP and GFP

Breast can...
Glowfish и другие…

Z. Gong et al.,
BBRC 2003;
S. Ding et al., Cell,
2005
X.Y.Yin et al., Biol.
Rep., 2007
Glowfish и другие…

Z. Gong et al.,
BBRC 2003;
S. Ding et al., Cell,
2005
X.Y.Yin et al., Biol.
Rep., 2007
лекция 1 гибридизация phk in situ.
лекция 1 гибридизация phk in situ.
лекция 1 гибридизация phk in situ.
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лекция 1 гибридизация phk in situ.

  1. 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. 2. Экспрессия генов на уровне РНК
  3. 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. 4. Гибридизация РНК in situ
  5. 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. 6. Enzyme coupled immunological detection of probes Chemoluminescence Fluorescence Color formation
  7. 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. 8. RNA in situ hybridization
  9. 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. 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. 11. Локализация белка in situ
  12. 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. 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. 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. 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. 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. 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. 18. in situ hybridization & Immunohistochemistry
  19. 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. 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. 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. 22. Флуоресцентные белки
  23. 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. 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. 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. 26. GFP - genetically encoded fluorescent probe • Self-catalytic chromophore formation (the only external O2 is required) • Low toxisity • High stability • Monomeric state
  27. 27. Использование GFP-подобных белков для анализа временного и пространственного патерна экспрессии генов
  28. 28. GFP applications 2. Visualization of protein localization and turnover.
  29. 29. Enhanced GFP mutant Absorption and emission spectral profiles of (A) wild-type A. victoria GFP and (B) the improved S65T derivative.
  30. 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. 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. 32. Случайный мутагенез
  33. 33. Стратегия направленной эволюции макромолекул
  34. 34. Направленный мутагенез с использованием ПЦР и перекрывающихся праймеров Введение точечных мутаций
  35. 35. GFP applications Visualization of temporal and spatial pattern of promoter activation. Organism and cell labeling
  36. 36. GFP applications Visualization of protein localization and turnover.
  37. 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. 38. GFP mutants Absorption (A) and emission (B) spectral profiles of the enhanced Aequorea-GFP derivatives
  39. 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. 40. Far-red fluorescence is preferable for whole body imaging
  41. 41. Discovery of GFP-like fluorescent and colored proteins in coral polyps (1999) Yuly Labas 1933-2008 Matz et al., Nat Biotechnol. 1999
  42. 42. Evolution diversity of GFP family Annelida Nematoda Crustacea Mollusca Arthropoda Branchiostoma Chordata Echinodermata Hydrozoa Anthozoa Cnidaria Typhlocoela Ctenophora Porifera
  43. 43. Разноцветные, окрашенные и бесцветные GFPподобные белки из медуз Aequorea coerulescens (безцветный белок) Phyalidium sp. (желтый белок) Antomedusae (хромобелок)
  44. 44. Копеподы Ланцетники
  45. 45. Mantis shrimp C. H. Mazel et al., Science, 2004
  46. 46. Spectral diversity of GFP-like proteins Main spectral classes, which are widely encountered in natural GFP-like proteins
  47. 47. Chromophores of native fluorescent proteins
  48. 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. 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. 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. 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. 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. 53. DsRed and GFP structure In general, Anthozoa FPs have more elliptical symmetry than Aequorea victoria GFP derivates
  54. 54. DsRed tetramer Catalytic R96 and E222
  55. 55. 5-color imaging with TagFPs TagBFP-Mito TagCFP-Actin TagYFP-Tubulin TagRFP-Golgi TagFP635-H2B
  56. 56. mKate-Tubulin Images were kindly provided by Michael W. Davidson (Florida State University)
  57. 57. mKate-EB3 Images were kindly provided by Michael W. Davidson (Florida State University)
  58. 58. mKate Cx43 Images were kindly provided by Michael W. Davidson (Florida State University)
  59. 59. mKate H2B Images were kindly provided by Michael W. Davidson (Florida State University)
  60. 60. mKate-Clathrin Images were kindly provided by Michael W. Davidson (Florida State University)
  61. 61. mKate2-EB3 fusion (microtubule end-binding protein) By Michael W. Davidson (Florida State University)
  62. 62. mKate2.7- alpha actinin fusion
  63. 63. mKate2.7- protein kinase CSRC fusion
  64. 64. Td-Katushka-zyxin fusion By Michael W. Davidson (Florida State University)
  65. 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. 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. 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. 68. Glowfish и другие… Z. Gong et al., BBRC 2003; S. Ding et al., Cell, 2005 X.Y.Yin et al., Biol. Rep., 2007

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