Download How to make knockout animals?

Immunological
techniques
Wei Chen, Associate professor
Institute of Immunology
E-mail:[email protected]
http://mypage.zju.edu.cn/566 8888
Objectives
To know the main laboratory detection
method using antibody.
To know how to isolate the specific
lymphocyte population.
Be able to describe how to detect the change
of human immune function after bacteria or
virus infection.
Content
Laboratory methods using antibodies
Lymphocytes isolation
Methods for studying lymphocyte responses
Transgenic mice and gene targeting
Content
Laboratory methods using antibodies
Lymphocytes isolation
Methods for studying lymphocyte responses
Transgenic mice and gene targeting
Laboratory methods using antibodies
Quantitation of Antigen by Immunoassays
Labeling and Detection of Antigens in Cells and
Tissues
Measurement of Antigen-Antibody Interactions
Identification and Purification of Proteins
Antigen-Antibody Interactions
Traditional
Immunoassa
ys
Agglutination(aggregation) Assays:
Immunodiffusion
Complement Fixation
EIA enzyme immunoassay (IHC/ELISA/ELISPOT)
Modern
Immunoassa
ys
Immunofluorescence (IFA FACS)
ICS （Intracellular cytokine staining）
CLIA （Chemiluminescence immumoassay）
凝集反应
扩散试验
补体结合反应
Immuno-labeling techniques
免疫标记技术
a. Principle
Specific Abs (or Ags ) labelled with
fluorescein, enzymes, colloidial gold
or radioisotopes are used as probes
for the detection of Ags (or Abs).
b. Types
Immuno-labeling techniques
CD4
horseradish peroxidase
CD8
IHC
Western Blot
fluorescence
microscope
FACS
Immuno-labeling techniques
EIA (Enzyme Immunoassay)
Color Detection
Substrate
IHC
Immunohistochemistry
免疫组织化学
Secondary antibody
One conjugated 2nd antibody can
detect all primary antibodies with
same isotype, which reduce the
labor to conjugate all primary
antibodies
A secondary antibody is an antibody that binds to primary antibodies or antibody
fragments.
They are typically labeled with probes that make them useful for detection, purification or
cell sorting applications.
Specific secondary antibodies are selected according to the source of the primary
antibody, the class of the primary antibody (e.g., IgG or IgM), and the kind of label which
is preferred.
Secondary antibody
Enzyme immunoassay (EIA)
免疫酶测定法
EIA is to use enzyme-labeled Abs or Ags to
detect Ag and Ab interactions.
The enzyme converts a colorless substrate
(chromogen) to a colored product.
ELISA: Ag or Ab in solution
Enzyme immunohistochemistry: Ag in tissue
ELISA
ELISA
Indirected ELISA
ELISA
BAS（Biotin-avidin system）-ELISA
生物素-亲和素放大系统
一分子亲和素可结合四个分子生物素，敏感、快速，
生物素标记抗体、亲和素标记酶。
Biotin-avidin system -ELISA
（Enzyme-linked immuno-sorbent spot，ELISPOT)
灵敏度高，单细胞水平，可高通量筛选
Two cytokines can be detected simultaneously
Immunofluorescence免疫荧光
Immunofluorescence assay is to use a
fluorescent compound (usually fluorescein) to
detect the binding of Ag and Ab.
The Ab is labeled with the fluorescent
compound and its presence is revealed using a
fluorescence microscope.
Direct, indirect immunofluorescence and
indirect complement amplified
immunofluorescence (间接免疫荧光互补放大)
Immunofluorescence
CD4
Immunofluorescence）
CD8
Immunofluorescence
An Example
Confocal image to detect phosphorylated AKT (green)
in cardiomyocytes infected with adenovirus
Useful tools
(1) Useful diagram for fluorochomes properties
http://www.bdbiosciences.com/resear
ch/multicolor/spectrumguide/index.jsp
Useful tools
(2) Useful website tools
BD Fluorescence Spectrum Viewer:
http://www.bdbiosciences.com/research/multicolor/spectrum_viewer/index.jsp
Identification and Purification of Proteins
Immunoprecipitation.
A protein mixture is incubated
with specifi c antibody. Any
antigen–antibody complexes
that form are precipitated from
solution by the addition of
Protein A-coated beads
that bind to the antibodies and
collect at the bottom of the tube
under the force of centrifugation.
After washing, the desired
antigen is released from the
antibody-bound
beads using altered pH and/or
high salt concentration
Identification and Purification of Proteins
Affinity Chromatography
Agarose beads bearing immobilized
specifi c antibody are placed into a
column with a semi-permeable plug at
the bottom. A solution containing
antigen is passed slowly through the
column, allowing the binding of specifi
c antigen to the immobilized antibody.
Unbound entities pass through the
plug and any molecule that binds to
the beads non-specifi cally is removed
by extensive washing. A solution with
the appropriate pH and salt
concentration to disrupt Ag–Ab
binding is then passed through the
column to elute (wash off) the antigen
of interest.
Western blotting
Western Blot
Western Blot
Content
Laboratory methods using antibodies
Lymphocytes isolation
Methods for studying lymphocyte responses
Transgenic mice and gene targeting
Isolation of lymphocytes
Magnetic cell sorting (MACS)
Three basic steps
1) Target cells are labeled with antibodyconjugated magnetic particles.
2) The labeled cells are placed within a magnetic field.
3) The labeled cells are retained in the magnetic field while
the unlabeled cells are washed away.
Cell subsets Purification
FACS separation(流式细胞术分离)
The basic principle of FACS is immunofluorescence and
therefore flow cytometers can be considered to be
specialized fluorescence microscopes.
The modern flow cytometer consists of a light source,
collection optics, electronics and a computer to translate
signals to data
Isolation of different cell populations by FACS relies on the
different expression of surface Ags.
Identification of cell subsets by FACS
Type of cell
CD markers
stem cells
CD34+,CD31-
all leukocyte groups
CD45+
Granulocyte
CD45+,CD15+
Monocytes
CD45+,CD14+
T lymphocyte
CD45+,CD3+
T helper cell
CD45+,CD3+,CD4+
Cytotoxic T cell
CD45+,CD3+,CD8+
B lymphocyte
CD45+,CD19+ or
CD45+,CD20+
Thrombocytes
CD45+,CD61+
Natural killer cell
CD16+,CD56+,CD3-
B cell
T cell
CD4+ T cell
CD8+ Tcells
Tregs
(CD4+CD25+)
Immune Cell types and subtypes defined by surface markers (CDs) Conventional
Basics of Flow
Cytometry
•Fluidics
•Cells in suspension
•flow in single
•Optics
•scatter light and
emit fluorescence
•that is collected,
filtered
•Electronics
*Collection
*Drop charged
and collected
•converted to digital
values
•Stored & analyzed
on a computer
FACS
Flow cytometry or Fluorescence Activated Cell Sorter (FACS)
Quantitative, multiparameter analysis of
large numbers of
individual cells
Cell surface markers
Intracellular proteins
Ca++ mobilization
12 colors and 15
parameters
Sorting: 70,000
cell/second
FCM
Content
Laboratory methods using antibodies
Lymphocytes isolation
Methods for studying lymphocyte responses
Transgenic mice and gene targeting
T Lymphocyte Function Assays
Function Measurement
--Proliferation
-- CTL killing
-- DTH
--Apoptosis
--Cytokine
--HLA detection
T cell proliferation
Morphology
3H-thymidine labeling
MTT
FACS-CFSE staining
T cell proliferation Morphology
Lymphoblast
(morphological features):
Lymphoblasts are 12-20 µm in
diameter with a round to oval
nucleus. The periphery of both
the nucleus and the cell may be
irregular in outline.
The fine, highly dispersed
nuclear chromatin stains a light
reddish-purple, and one or two
pale blue or colorless large
nucleoli are visible. The
cytoplasm is usually basophilic,
with marginal (peripheral)
intensity a common
characteristic.
T cell proliferation
3H-thymidine labeling
T cell proliferation -MTT
Mitochondria enzyme catalyze the reduction of MTT – Turn blue
T cell proliferation
FACS-CFSE staining
CTL Assay
CTL cytotoxicity mechanism
1. Cytolysis (necrosis) ------ three stages:
a. contact phase: recognition of antigen in the context of MHC class I molecules
b. Polarization phase: TCR, co-stimulatory molecules, cytolytic granules
c. lysis phase: release perforin and granzymes, osmotic death
CTL Assay
Supernatant
51Cr
Release Assay
Controls: Natural release well – medium;
Maximal release well – Detergent Lysed
No antigen control
Control release ratio (》5%): average value
Control cpm– Natural release
Maximal release – Natural release
Specific release ratio(》10%):
Specific cpm– Natural release
Maximal release – Natural release
Natural release ratio(《15%)
CTL – DIOC/PI staining
Tetramer
MHC tetramers can be used to quantitate numbers
of antigen-specific T cells (especially CD8+ T cells).
Biotinylated recombinant class I molecules folded
with the peptide of interest and β2M and
tetramerized by a fluorescently labeled
streptavidin (Streptavidin binds to four biotins per
molecule.) .
This tetramer reagent will specifically label T cells
that express T cell receptors that are specific for a
given peptide-MHC complex.
Antigen specific responses can be measured as
CD8+, tetramer+ T cells as a fraction of all CD8+
lymphocytes.
Tetramer
Tetramers can
bind to three
TCRs at once,
allowing
specific binding
in spite of the
low (10-6
molar) affinity
of the typical
class I-peptideTCR interaction.
Utility of Tetramer
CD8 response related(now CD4 too) and known epitope
Infectious Disease
HIV-AIDS, EBV, CMV, HPV, HBV, HCV, Influenza, Measles, Malaria,
TB and RSV
Tumor Immunology
Breast, Prostate, Melanoma, Colon, Lung, Cervical, Ovarian and
Leukemia
Autoimmune Disease
Diabetes, Lyme disease, Multiple sclerosis, Rheumatoid arthritis,
Autoimmune vitiligo
Transplantation
DTH detection: ‘OT’ test or PPD test
结核杆菌素、念珠菌素、麻风菌素、链激酶-链道酶、腮腺炎病毒
Immunization
Challenge (Ear/Foogpad)
Measurement (Calipers)
Apoptosis detection
Apoptosis detection
Flow cytometry of apoptotic cell death
Phospholipid redistribution
Apoptosis detection
In the TUNEL assay,
fragmented DNA is labeled by terminal
deoxynucleotidyl transferase (TdT)
to reveal apoptotic cells. When
Cytokines detection
1.Real-time PCR (mRNA Level)
2.ELISA/ELISPOT
3.Intracellular staining (FACS)
4. Biological Function
Intracellular Staining
Identification of different T
helper cells characterized by
different cytokine production
HLA detection
血清学方法：微量细胞毒试验或补体依赖
的细胞毒试验（CDC）
细胞学方法：混合淋巴细胞反应
分子生物学方法:PCR
HLA detection
HLA detection
Assessment of human immune function
Assessment of human immune function
Content
Laboratory methods using antibodies
Lymphocytes isolation
Methods for studying lymphocyte responses
Transgenic mice and gene targeting
Transgenic animals and knockout animals
Part 1: Transgenic animals:
Introduction to transgenic animals.
How to make transgenic animals?
Applications of transgenic animals.
Part 2: Knockout animals
Introduction to knockout animals.
How to make knockout animals?
How to make conditional knockout animals?
Applications of knockout animals.
Transgenic Animal
Animal has one or more foreign genes inserted into
chromosome DNA inside its cells artificially.
After injecting foreign gene into the pronucleus of a
fertilized egg or blastocyst, foreign gene is inserted in a
random fashion into chromosome DNA:
Randomly (Foreign gene may disrupt an endogenous
gene important for normal development, and the
chance is about 10%. )
multiple copies
Transgenic animals and knockout animals
Part 1: transgenic animals:
Introduction to transgenic animals.
How to make transgenic animals?
Applications of transgenic animals.
Part 2: Knockout animals
Introduction to knockout animals.
How to make knockout animals?
How to make conditional knockout animals?
Applications of knockout animal.
ES cell transformation
Injection of gene into fertilized egg
Method 1: ES cell transformation
vs. Method 2: Injection of gene into fertilized egg
1. ES cell transformation works well in mice only.
Other transgenic animals are produced by egg injection
2. ES cell transformation provides more control of the integration step
(selection of stably transfected ES cells)
3. Injection of gene into fertilized egg is less reliable
(viability of eggs, frequency of integration),
but it helps to avoids chimeric animals
Transgenic animals and knockout animals
Part 1: transgenic animals:
Introduction to transgenic animals.
How to make transgenic animals?
Applications of transgenic animals.
Part 2: Knockout animals
Introduction to knockout animals.
How to make knockout animals?
How to make conditional knockout animals?
Applications of knockout animal.
Applications of Transgenic Animals
Transgenic mice are often generated to
1. characterize the ability of a promoter to direct tissue-specific
gene expression
e.g. a promoter can be attached to a reporter gene such as
LacZ or GFP
2. examine the effects of overexpressing and misexpressing
endogenous or foreign genes at specific times and locations in
the animals
3 Study gene function
Many human diseases can be modeled by introducing the same
mutation into the mouse. Intact animal provides a more
complete and physiologically relevant picture of a transgene's
function than in vitro testing.
4. Drug testing
Example 1: Transgenic Mouse
The growth hormone gene has been engineered to be expressed
at high levels in animals.
The result: BIG ANIMALS
Mice fed with heavy metals are 2-3 times larger
Metallothionein
promoter
regulated by
heavy metals
Example 2: Transgenic Mouse
Trangenic mouse embryo in which the promoter for a gene expressed
in neuronal progenitors (neurogenin 1) drives expression of a betagalactosidase reporter gene. Neural structures expressing the reporter
transgene are dark blue-green.
Example 3: GFP transgenic mouse (Nagy)
9.5 day embryos GFP and wt
Tail tip
GFP transgenic mouse (Nagy)
Transgenic animals and knockout animals
Part 1: transgenic animals:
Introduction to transgenic animals.
How to make transgenic animals?
Applications of transgenic animals.
Part 2: Knockout animals
Introduction to knockout animals.
How to make knockout animals?
How to make conditional knockout animals?
Applications of knockout animals.
knock-out Animal
One endogenous gene in an animal is
changed. The gene can not be expressed
and loses its functions.
DNA is introduced first into embryonic stem (ES) cells.
ES cells that have undergone homologous
recombination are identified.
ES cells are injected into a 4 day old mouse embryo: a
blastocyst.
Knockout animal is derived from the blastocyst.
Transgenic animals and knockout animals
Part 1: transgenic animals:
Introduction to transgenic animals.
How to make transgenic animals?
Applications of transgenic animals.
Part 2: Knockout animals
Introduction to knockout animals.
How to make knockout animals?
How to make conditional knockout animals?
Applications of knockout animals.
KNOCKOUT MICE
Normal (+) gene X
Isolate gene X
and insert it into vector.
Genome
Defective
(-)
Gene X
VECTOR
MARKER GENE e.g.(NeoR)
Inactivate the gene
by inserting a marker gene
that make cell resistant
to antibiotic (e.g. Neomycin)
Transfer vector
with (-) gene X
into ES cells
(embryonic stem cells)
Vector and
genome
will recombine
via homologous
sequences
Genomic gene
Homologous recombination
and gene disrution
Grow ES cells in
antibiotic containing media;
Only cell with marker gene
(without normal target gene)
will survive
Generation of
knockout mice
Transgenic animals and knockout animals
Part 1: transgenic animals:
Introduction to transgenic animals.
How to make transgenic animals?
Applications of transgenic animals.
Part 2: Knockout animals
Introduction to knockout animals.
How to make knockout animals?
How to make conditional knockout animals?
Applications of knockout animal.
Conditional knock-out animals
How to make FLOXed gene
Gene of interest
TK
NeoR
loxP
loxP
loxP
loxP
Gene flanked by loxP sites (floxed)
Electroporate targeting
vector into ES cells,
followed by +/selection
NeoR+/ HSVtkcells selected
Make mice and breed floxed allele to homozygousity.
Mate FLOXed mice with mice carrying a
Cre transgene
Marker gene
Promoter elements
Cre
IRES
GFP
intron
SV40 p(A)
Crucial element. Recombinase
would be expressed in accordance
with specificity of your promoter.
Promoter could be regulated !!!
artificailly or naturally
Conditional knock-out animals
inactivate a gene only in specific tissues
and at certain times during development and life.
Cre-lox technology
Your gene of interest is flanked by 34
bp loxP sites (floxed).
Cre – a site-specific recombinase enzyme from the P1 phage.
If CRE recombinase
expressed
Recognises a 34bp DNA sequence loxP =
Gene between loxP sites is
removed
Cre
Cre
Cre
Transgenic animals and knockout animals
Part 1: transgenic animals:
Introduction to transgenic animals.
How to make transgenic animals?
Applications of transgenic animals.
Part 2: Knockout animals
Introduction to knockout animals.
How to make knockout animals?
How to make conditional knockout animals?
Applications of knockout animal.
Applications of Knock-out animals
Find out if the gene is indispensable
(suprisingly many are not!)
Check the phenotypes of knockout animals
Determine the functions of knockout gene.
Three genome editing technologies ：
1、Zinc-finger endonucleases (ZFNs)
2、Transcription activator–like effector
nucleases (TALENs)
3、RNA-guided CRISPR-Cas9 nuclease
system(CRISPR-Cas9)
癌症免疫疗法
大众基因显微手术
脑成像技术
人体胚胎克隆
迷你器官
2013年十大科技突破
宇宙射线的来源
太阳能新星
为什么睡觉
微生物与健康
疫苗设计
癌症免疫疗法
大众基因显微手术
脑成像技术
人体胚胎克隆
迷你器官
宇宙射线的来源
太阳能新星
为什么睡觉
微生物与健康
疫苗设计
CRISPR-Cas9 system introduction
无论是ZFN还是TALEN，这两种人工核酸酶的原理
是一样的，都是由DNA结合蛋白与核酸内切酶 Fok I
融合而成 。
CRISPR/Cas9
1987年，日本课题组在K12大肠杆菌的碱性磷酸酶基因附近发现
串联间隔重复序列，随后的研究发现这种间隔重复序列广泛存在于
细菌和古细菌的基因组中。
2002年，科学家将其正式命名为clustered regularly interspaced
short palindromic repeats(CRISPR)。
2005年，三个研究小组均发现CRISPR的间隔序列(spacer)与宿主
菌的染色体外的遗传物质高度同源，推测其功能可能与细菌抵抗外
源遗传物质入侵的免疫系统有关。
2007年，Barrangou等首次发现并证明细菌可能利用CRSPR系统
对抵抗噬菌体入侵。
2008 年 ，Marraffini等又发现细菌CRISPR系统能阻止外源质粒
的转移，首次利用实验验证了CRISPR系统的功能，由此科学家们
揭开了研究CRISPR系统作用机制的序幕。
CRISPR/Cas的基因座结构
基因座结构可分别三部分，5‘端为 tracrRNA基因，中
间为一系列Cas蛋白编码基因，包 括 Cas9、Cas1、Cas2
和Csn2，3'端为CRISPR基因座，由启动子区域和众多的
间隔序列(spacers)和重复序列(direct repeats)顺序排列组成。
Cas（CRISPR associated）：
存在于CRISPR位点附近，是一种双链DNA核酸酶，能在
guide RNA引导下对靶位点进行切割。它与folk酶功能类似，
但是它并不需要形成二聚体才能发挥作用。
Cas9蛋白包含两个功能结构域，
一个在N端，有类似于Ruc核酸
酶的活性，一个在中部有类似
HNH核酸酶的活性。
The crRNA and tracrRNA can be fused
together to create a chimeric, singleguide RNA (sgRNA)。
利用Cas9系统建立knock-out细胞系
1、 确定待敲除基因的靶位点
2 、设计识别靶位点的识别的一对DNA Oligo（引物）
3 、构建表达sgRNA的质粒
4 、sgRNA活性检测
5 、利用Cas9系统建立knock-out细胞系
1、 确定待敲除基因的靶位点
 根据基因ID在NCBI或ENSEMBLE中进行查找。
 找到该基因CDS区，分析相应的基因组结构，明
确CDS的外显子部分。
 对于蛋白编码基因，如果该蛋白具重要结构功能
域，可考虑将基因敲除位点设计在编码该结构域的
外显子；如果不能确定基因产物性质，可选择将待
敲除位点放在起始密码子ATG后的外显子上。
2 、设计识别靶位点的识别的一对DNA Oligo
（引物）
确定待敲除位点后，选择23-至250bp的外显子序
列输入到在线免费设计sgRNA的软件Input框中，然
后进行设计运算，软件会自动输出sgRNA序列。一
般为PAM前的20个bp。设计完成后要在NCBI上比对
一下，确定没有其他互补基因。
根据选择的sgRNA模板序列，公司合成一对序列互
补的DNA Oligos。
在线设计网址：http://crispr.mit.edu/
3 、构建表达sgRNA的质粒
将合成的Oligos以逐步降温的方法退火成双链，
然后与公司提供的质粒进行连接，连接后转化感
受态的大肠杆菌，再进行涂板。
挑单克隆，培养，提质粒，酶切鉴定并且测
序确认。
从公司买SgRNA载体，价格大概2250元/5T，
3380元/10T，Cas9质粒免费赠送。
4 、sgRNA活性检测
SSA sgRNA活性检测试剂盒1180元/10T,1980元/20T
5 、利用Cas9系统建立knock-out细胞系
将Cas9质粒和SgRNA转染细胞，通过荧光标记观
察转染效率，如果选择了带Neo抗性的质粒，则同
时用G418药物筛选。如果有FACS，可直接分选带荧
光的细胞。
分离阳性细胞，培养繁殖，然后提取部分细胞
的基因组DNA用作PCR模板。然后用对应的引物进
行PCR扩增。PCR产物先进行2%琼脂糖凝胶电泳，
如果出现条带，则将PCR产物直接送测序。
将携带有突变等位基因的阳性克隆扩增，保
存，稳转系建立成功。
PAM前20bp
Cas9
sg-RNA
帅选阳性克隆，构建成功
Cas9系统缺点：off target
在人类基因组中，平均每12.7bp就出现一个NGG PAM。
CRISPR-Cas系统前景分析
2013年4月12日在《cell stem cell》上发表的《Enhanced
Efficiency of Human Pluripotent Stem Cell Genome Editing
through Replacing TALENs with CRISPRs》一文中，作者
利用TALENs和CRISPRs对同一基因进行修饰，效率分别为
0%-34%和51%-79%。
而且从实际应用的角度来说，CRISPRs比TALENs更
容易操作，因为每一对TALENs都需要重新合成，而用于
CRISPR的gRNA只需要替换20个核苷酸就行。且Cas蛋白
不具特异性。
Cas9 movies:
http://v.youku.com/v_show/id_XODIzNj
AzNDI0.html