Breaking the Kubernetes Kill Chain: Host Path Mount
лекция 1 гибридизация phk in situ.
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
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
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
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
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)
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
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.
22. 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
24. 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)
25.
26.
27. 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
28. 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)
29. GFP - genetically encoded fluorescent probe
• Self-catalytic chromophore formation (the only external
O2 is required)
• Low toxisity
• High stability
• Monomeric state
32. Enhanced GFP mutant
Absorption and emission spectral profiles of (A) wild-type A.
victoria GFP and (B) the improved S65T derivative.
33. 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
34. 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
42. 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.
52. 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.
53. 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
54. 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.
55. 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
56. DsRed and GFP structure
In general, Anthozoa FPs have
more elliptical symmetry than
Aequorea victoria GFP
derivates
68. 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
69. 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
70. Glowfish и другие…
Z. Gong et al.,
BBRC 2003;
S. Ding et al., Cell,
2005
X.Y.Yin et al., Biol.
Rep., 2007
71. Glowfish и другие…
Z. Gong et al.,
BBRC 2003;
S. Ding et al., Cell,
2005
X.Y.Yin et al., Biol.
Rep., 2007
