Download [II] Molecular Techniques for Studying Control of Gene Expression (II).

[II] Molecular Techniques for
Studying Gene Expression
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Basics of recombinant DNA technology
Methods used to monitor the expression of genes
RT-PCR vs. Real-time RT-PCR
DNA microarray analysis
Transgenic analysis and gene inactivation
Chromatin immunoprecipiotation
Methods of analysis of proteins
Reading List II
 Cloning and Characterizing DNA
Molecule
**What is a recombinant DNA molecule?
**Cloning of a genomic gene
**Cloning of a complementary DNA (cDNA)
Why is it necessary to clone genes?
• Naturally occurring DNA molecules are very long and a single
molecule usually carrying many genes
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Genes may occupy only a small proportion of the
chromosomal DNA and the rest are noncoding sequences (a
human gene might constitute only 1/1,000,000 of a
chromosomal DNA)
Unlike enzymes, there is no convenient method to identify a
gene. Therefore, it is impossible to obtain a sizable amount of
DNA encoding a gene by the ordinary purification method
It is impossible to purify individual mRNA due to lacking
method to identify individual mRNA species. Clone individual
cDNA of the mRNA is the only solution
Technological Breakthrough in Molecular Biology
Leading to Cloning of DNA
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Discovery of restriction enzymes & modification enzymes --allowing cutting and manipulation of DNA molecules
Methods of isolating DNA molecules and agarose gel
electrophoresis for separating DNA molecules
Plasmids and bacteriophage as vectors for in vivo amplification of
DNA molecules
Methods of introducing DNA molecules into cells --“Transformation”
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Hybridization method for identifying specific DNA molecules
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Reading List:
 Restriction enzyme – a background paper
 Recombinant DNA and gene cloning
General Strategy of Recombinant DNA
Technology
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Recombinant DNA technology: cloning and manipulation of
DNA molecules (genomic DNA or cDNA)
Vector + DNA fragment
Recombinant DNA
Replication of recombinant DNA molecules in host cells
Isolation, sequencing, and manipulation of purified
DNA fragment
Several Enzymes that Digest DNA Molecule
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(a): Terminal phosphatase
(b): Nuclease: digest either
the first ester bond or
the second ester bond
(c): Endonuclease
(d): Exonuclease
Restriction Enzyme (I)
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Restriction enzymes were originally discovered in bacterial
cells serving to remove invading foreign DNA, but bacterial
DNA itself can be protected from restriction enzyme digestion
by adding methyl group (-CH3) to A or C on the DNA
Restriction enzymes were discovered by W. Arber, D. Nathans
and H. Smith in 1960’s. They were awarded with a Nobel Prize
in 1978
DNA fragments generated by digestion with some restriction
enzymes are with sticky ends
Restriction enzymes have been used to prepare DNA
molecules for cloning
Reading Assignment:
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Nobel lecture by H. Smith 1978
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Restriction Enzymes (II)
Restriction enzymes: Enzymes from bacterial cells that can cut
DNA molecules at specific nucleotide sequence
Restriction site
(palindromic sequence)
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Some enzymes digest DNA to produce sticky ends while others
produce blunt ends
Palindromic sequence; 4 cutters = 44 (256 bases); 6 cutters = 46
(4096 bases)
Restriction Enzymes (III)
(a). Some restriction
endonucleases that
cleave the restriction
sites to generate a
staggered cut
(b). Other restriction
endonucleases that
cleave the restriction
sites to generate blunt
ends
Restriction Map of a DNA Fragment
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A restriction map of a DNA fragment is a linear sequence of
sites separated by defined distances on DNA
The map shown above identifies three restriction sites cleaved
by restriction enzyme A and two sites by restriction enzyme B
Thus DNA fragment digested by restriction enzyme A alone
generates 4 fragments and digested by restriction enzyme B
alone generates 3 fragments
These DNA fragments can be resolved by electrophoresis on an
agarose gel and their determined
DNA Modification Enzymes
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DNA Modification Enzymes: Enzymes that can modify DNA
molecules
 DNA Ligase: An enzyme that can ligate two DNA molecules together
by making a phosphodiester bond
 DNA polymerase I: involves in synthesis of DNA molecules (identified
by Athur Kormberg)
 DNA phosphorylase: An enzyme that can remove phosphate group
from DNA molecules
 DNA kinase: An enzyme that can add a phosphate group onto the 5’end of a DNA molecule
 Terminal transferase: An enzyme that can add nucleotide on to 3’-end
of the DNA molecule
 Endo- and exo-nucleases: break a phosphodiester bond
 Reverse transcriptase: make a strand of DNA by copying RNA
template (identified by H. Temin and D. Baltimore independently)
 Tqa polymerase: synthesize DNA under high temperature. This
enzyme is isolated from Thermus aquatica (identified by T.D. Brock)
Cloning Vectors (I): Basic Components of a
Bacterial Plasmid Cloning Vector
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Plasmid cloning
vector in E. coli is a
circular DNA
molecule of 1.2 to 3
kb
Plasmid Vectors
contain:
 Selectable gene
such as ampr
 Replication origin
(ORI)
 A synthetic
polylinker with
unique restriction
enzyme
recognition site
DNA fragment from a few base pairs to about 10
kb can be inserted into the cloning vector and
manipulated
Construction of
a Recombinant
Plasmid
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Replication origin
Selection marker:
Ampicillin resistant
gene or others
MCS: mutiple
cloning site
DNA fragment of
interest can be
inserted at MCS for
amplification
DNA fragment
smaller than 10 Kb
can be amplified in
a bacterial plasmid
Cloning Vectors (II): Cloning Vectors for Cloning
Genomic DNA (the earliest version)
1. A Phage Cloning Vector: Lambda (l) phage DNA has been
developed into a cloning vector for cloning DNA fragment up to 20
kb. This is the earliest type of vector used to construct a genomic
library
Recombinant
l Phage
Cloning Vectors (III): Cosmid and Phagemid
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Cosmid is a type of hybrid plasmid (often used as a cloning
vector) that contains cos sequence. Cosmids (cos sites + plasmid
= cosmid) DNA sequences are originally from the Lambda phage.
Cosmids can be used to build genomic library.
A phagemid is developed as a hybrid of the filamentus phage M13
and plasmid to produce a vector that can grow as a plasmid, and
also be packaged as single stranded DNA in viral particles. It
contains an ORI for double stranded replication, as well as an f1
ori to enable single stranded replication and packaging into
phage particles.
Cloning Vectors (IV): Cloning vectors for
cloning a large piece of DNA
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Bacterial artificial
chromosome (BAC):
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A plasmid contains a
functional fertility
factor, replication
origin, partition factor,
selection factor and T7
and Sp6 promoters
This plasmid (Fplasmid) can
accommodate DNA
fragment of about 100300 Kb
The recombinant
plasmid can be
introduced into E. coli
cells by
electroporation
Cloning Vectors (IV): Cloning vectors for
cloning a large piece of DNA
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Yeast Artificial Chromosome:
 This cloning vector contains
Tel (telomere), CEN
(centromere) and ORI
(replication origin) of yeast
cells. It can accept a large
piece of DNA (100 kb to 3,000
kb)
 The recombinant cloning
vector is introduced into
yeast cells by electroporation
for amplification
Cloning Vectors for Different Purposes
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Detection of Nucleic Acids
 Agarose gel electroporation
 Hybridization, Southern blot and RNA
northern blot analyses
Agarose Gel Electrophoresis to
Separate DNA
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Agarose is an inert carbohydrate isolated from seaweeds. DNA
molecules of different sizes can be separated in an agarose gel by
electrophoresis and visualized by staining the gel with ethedium
bromide & observe under UV light. The DNA fragment can be
recovered from the gel by extraction with phenol and chloroform
DNA Fragments Visualized Under UV Light
DNA on agarose gel
is stained with
ethedium bromide
and observed under
UV light
Sizes of DNA Fragments Determined by
Agarose Eectroporation
Separation of Molecules by Gradient Centrifugation
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Macromolecules (DNA,
RNA and proteins) can be
separated by their sizes
or densities
Macromolecules of
different sizes can be
separated by
electrophoresis on
agarose or polyacrylamid
gels
Macromolecules of
different densities can be
separated by grant
centrifugation
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Sedementation
centrifugation
Equilibrium
centrifugation
Hybridization
• Hybridization: Two fragments of single-stranded
homologous DNA molecules can form hybrid through
hydrogen-bonding formation (base pairing)
5’-GTACTTAGGCAATTGGGCA-3’
3’-CATGAATCCGTTAACCCGT-5’
• If one of these two strands of DNA is labeled with
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radioactive isotopes, the hybrid will be easily visualized
by autoradiography
Hybridization can occur between two homologous DNA
molecules or a DNA molecule and a RNA molecule
“Hybridization” has been used in Southern blot and RNA
northern blot analyses
Southern Blot Hybridization
This method is developed by Edwin Southern
Results of Southern Blot Hybridization
Fluoerescence in situ Hybridization
This technique is used for cytological localization of molecules in the cell
Methods of Introducing DNA into Cells
• Recombinant DNA molecules
can be transferred into bacterial
cells, plant cells and animal cells
by transformation methods:
 Bacterial cells: regular
transformation method or
electroporation method
 Plant cells: Agrobacterium infection,
electroporation or particle gum
bombardment
 Animal cells: microinjection,
Ca3(PO4)2 precipitation method,
lipofection or electroporation
method
Electroporators
Electroporators can generate high frequency pulses to
create transient opening on cells by which the DNA
molecules enter into the cell
• Cloning of Genomic DNA and cDNA
Complementary (c) DNA Verses Genomic Gene
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Complementary DNA (cDNA): A reverse sequence of
an mRNA, synthesized by using mRNA as a template
and reverse transcriptase as the enzyme to catalyze
the reaction
Genomic Gene: A gene contains the coding region
(both intron and exon) and the control region
Why should one clone a cDNA or a genomic gene?
Purposes of Cloning Genomic DNA and cDNA
• Genomic DNA:
 Studying structures of genes, gene families
 Identifying promoters and other regulatory elements
 Studying evolution of genes
• cDNA:
 Studying structures of mRNA
 Measuring the levels of mRNA
 Studying developmental stage-specific and tissue-specific
expression of genes
 Studying processing and stability of mRNA
 Producing recombinant proteins
Construction of Genomic DNA Library
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Since the sizes of genomic genes vary from several
kbs to several hundred kbs, bacterial plasmid vector
is not very suitable for cloning a complete gene
Commonly used vectors for cloning complete genes
are:
 Lambda phage vector (up to 20-25 kb)
 Artificial bacterial chromosome (BAC) vector (up to 500 kb): it
contains a bacterial origin of DNA replication and genes
required to regulate their own replication
 Yeast artificial chromosome (YAC) vector (up to 1000 kb): it
contains all the elements of yeast chromosome i.e., elements
for replication of the chromosome during S phase,
segregation of chromosome, telomeres, genes for selection
and DNA fragment to be cloned
Genomic DNA library: A collection of DNA fragments
of a genome
Isolation of Genomic Genes from DNA Libraries
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Isolating gene clones from libraries by colony
hybridization or plaque hybridization
Characterizing the inserted DNA by:
 Restriction enzyme digestion (establishing restriction
maps)
 Determining the nucleotide sequence of the inserted DNA
 Expressing the gene product in E. coli or mammalian
cells
Colony
Hybridization
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This method is based
on the principle that
two homologous
strands of nucleic
acids can form hybrid
form
If one of the strands
of nucleic acid is
radio-labeled, the
hybrid can be
visualized by
autoradiography
Plaque
Hybridization
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This
technique is
suitable for
isolating
genes cloned
in a lambda
phage cloning
library
Restriction Enzyme Mapping
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Restriction enzyme recognition sites can be used to
make maps of DNA --- restriction mapping
Restriction map of a cloned DNA is made by
digesting the cloned DNA with a series of restriction
enzymes and determine their sites on the DNA
Restriction map of the catfish growth hormone gene
Structure of Dideoxynucleotide Triphosphate
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The nucleotide sequence
of a piece of DNA can be
determined by:
 Chemical sequencing
method developed by
Maxam Gilbert
 Dideoxy chaintermination method
developed by F. Sanger
Incorporation of
dideoxynucleotide
triphosphate into the
going chain of DNA will
stop the elongation of the
DNA chain
A Single Strand DNA to be Sequenced
Assigned Reading: Nobel lecture by F. Sanger on “DNA sequencing”, 1980
Separating the Products on a Denaturing
Polyacrylamid Gel
3’
Separating the Products on an Automated
Sequencing Machine
(1.8 x 106)
(1.2 x 107)
(9.7 x 107)
(1.0 x 108)
(1.8 x 108)
(3.2 x 109)
Strategies of Assembling Whole Genome
Sequence
Chromosome
Walking
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This technique
allows the
isolation of a long
eukaryotic gene
An alternative is to
construct a BAC
library that
contains long
piece DNA
molecules
Making cDNA
from an
Eukaryotic Gene
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To express an
eukaryotic gene into
its product in bacterial
cells, the mRNA is
converted into cDNA
The cloned cDNA can
be expressed into
protein product by
inserting it into a
plasmid containing a
functional promoter
(expression vector)
Construction of a
cDNA Library
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Synthesis of 1st strand
cDNA
Synthesis of double
strand cDNA
Ligate the double
stranded cDNA into a
plasmid vector or l
phage vector
Propagate the library
in E. coli cells
Lambda Phage Cloning Vector for Cloning cDNA
1. Phage Cloning Vector:
Lambda (l) phage DNA has also been developed for cloning
cDNA as well. This type of vector is suitable for cloning cDNA
banks. In this case, the non-essential region in the lambda
phage genome is not removed. If a functional promoter is
present in the mutiple cloning site, the cloned cDNA will also
express into it gene product
cDNA Library (Bank)
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Lambda gt10: for
cloning regular
cDNA that can
be identified by
hybridization
Lambda gt11: for
cloning cDNA
that can express
the protein
product, so the
cDNA clone can
be identified by
immunoblotting
Expression Vectors
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Expression vector: A cloning vector that contains a “functional
promoter”, if this vector also contains a gene that its expression
can be easily seen (detected), it is said that this vector contains a
“reporter gene”
Leuciferase gene derived from fireflies
is a popular reporter gene. An
expression vector containing
leuciferase gene can be used to define
the functional promoter and other
regulatory sequence
Expression of a lacZ gene
stained for b-galactosidase
in a mouse embryo
Visualization of Cancer Cell
Growth by GFP
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External and internal images of
bone metastasis of AC3488 GFP
(A) External images of tumors in
the skeletal system [including the
skull (arrow heads), scapula (thick
arrows), spine (fine arrows), and
liver metastasis (largest arrows) in
a dorsal view of live, intact nude
mouse
(B–I) Series of external
fluorescence images of metastatic
lesions in the skull, ribs, spine,
and tibia, (B, D, F, and H)
compared with corresponding
images of the exposed skeletal
metastases (C, E, G, and I) (Bars
=1280 mm).
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Methods used to monitor the
expression of genes
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Using Cloned DNA Fragments to Study Gene
Expression
 RNA northern blot analysis
 In situ hybridization
 RNase protection assay
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Measuring mRNA levels by RT-PCR or real-time
RT-PCR assay
Measuring the profiles of gene expression by
DNA microarray
Measuring the profiles of gene expression by
RNA sequencing analysis
Using Cloned DNA Fragments to Study
Gene Expression
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Hybridization techniques permit detection of specific
DNA fragments and mRNAs
 Southern blotting method: DNA-DNA hybridization
 RNA northern blotting method: RNA-DNA hybridization,
qualitative and semi-quantitative methods
 In situ hybridization
 RT-PCR
DNA microarrays can be used to evaluate the
expression of many genes at one time
E. coli expression systems can produce large
quantities of proteins from cloned genes
Cloned genes can also be introduced into yeast cells
and other animal cells for production of large
quantities of proteins for analysis
DNA Southern Blot & RNA Northern
Blot Techniques
RNA
RNA Northern Blot Analysis of b-Globin mRNA
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Total RNA was extracted
from erythrolukemia
cells grown but not
induced and in cells
induced to stop growing
and allowed to
differentiate for 48 h or
96 h
The RNA samples were
resolved in agarose gels
and transferred to a
nylon membrane
The membrane was
hybridized to radiolabeled b-globin cDNA
In Situ Hybridization to Detect of the mRNA
of Sonic Hedgehog in Mouse Embryos
Whole embryo
(10 days old)
A section of the
embryo
Drosophila
Embryo
In this experiment, the in situ hybridization technique is used to localize the
site of expression of sonic hedgehog gene in mouse embryos
Principle of RNase Protection Assay
AAAAAAA
mRNA
+
32P
DNA
Hybridization and digested
with RNase
The product is analyzed on a ureapolyaccrylamid gel
RNase Protection Assay to Measure IGF-I mRNA
Levels in the Liver of Rainbow Trout
CPM or OD600
If a standard curve is established using
known quantities of cRNA, the amount
of the unknown can be determined
mg cRNA
Nuclear Run-on
Transcription Assay
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Nuclear run-on assay can be
used to ascertain which gene is
active in a given cell allowing
transcription to continue in
isolated nuclei
Specific transcript can be
identified by their hybridization
to known DNAs on dot blot
It can also be used to determine
the effects of assay conditions
on nuclear transcription
Transcription activity of a
specific gene can be determined
It can also be used to measure
template activity
Nuclear Run-On Assay to Measure the
Transcription Rates of Genes in Various Tissues
Measuring levels of mRNA by RT-PCR or realtime RT-PCR assay
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Principle of Reverse
Transcriptioin (RT)PCR
End Point RT-PCR:
 Measuring the presence
of a particular mRNA
 Can be adapted to
perform semi-quantitative
determination of an
mRNA
Real-time RT-PCR: For
quantitative
measurement of the
level of an mRNA
 Absolute quantitative RTPCR
 Comparative quantitative
RT-PCR
PCR Amplification Program
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1 cycle at 95 oC for 3 min to denature all templates
40-50 cycles:
 95 oC for 15 sec for denaturation of PCR products
 At annealing temperature for 15 sec to anneal primers
to the templates
 72 oC for 30 sec for synthesis of DNA
Determine Levels of mRNA by Semiquantitative RT-PCR
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If levels of b-actin mRNA in
various tissues are
constant, then the levels of
mRNA of interest can be
expressed in terms of
relative to levels of b-actin
This assumption will only
work when the increase of
levels of mRNA is in the
linear range of the
detection
Example given in the left of
this slide is showing how
the levels of NPY mRNA is
determined by this method
Competitive Quantitative RT-PCR
AAAA
IGF-II mRNA to be determined
In vitro transcription
Competitor mRNA
Reverse
Transcription
Competitor cDNA
Reverse
Transcription
IGF-II cDNA
Various amounts of both templates and fixed amount of
amplification primers and proceed PCR amplification
Competitive Quantitative RT-PCR
When the ratio of
target/competitor
equals to 1, the levels
of competitor equals
the level of the target
Assigned Reading: Greene and Chen, 1999. Quantitation of IGF-I, IGF-II, and
multiple insulin receptor mRNAs during embryonic development in rainbow
trout. Molecular Reproductive Development 54: 348-361.
Real-time PCR Machine
Principle of Real-Time RT-PCR
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SYBR Green I (SG) is an asymmetrical cyanine dye used as a
nucleic acid stain in molecular biology. SYBR Green I binds to
DNA. The resulting DNA-dye-complex absorbs blue light (λmax =
497 nm) and emits green light (λmax = 520 nm). The stain
preferentially binds to double-stranded DNA, but will stain
single-stranded DNA with lower performance. SYBR green can
also stain RNA with a lower performance than DNA.
Since double stranded DNA can interact quantitatively with
SYBR Green 1 preferentially and thus can be used to monitor
the amount of DNA synthesized after each cycle of PCR
reaction.
Cycle threshold (CT): CT is defined as the number of PCR cycles
required for the fluoescent signal to cross the threshold (i.e.,
exceeds background level). This is usually determined
automatically by the real-time PCR machine set in the factory
Determination of Cycle Threshold (CT)
Sample Set Up in a 96-Well Plate
1
2
3
4
5
6
7
8
9
10
Unkn-1
Unkn-1
Unkn-1
Unkn-5
Unkn-5
Unkn-5
Unkn-9
Unkn-9
Unkn-9
82.20
82.00
82.00
82.20
82.00
82.00
82.00
82.00
82.00
Content
Sample
A
Peak 1
Peak 2
Content
Sample
B
Peak 1
Peak 2
None
None
None
None
None
None
None
None
None
Content
Unkn-2
Unkn-2
Unkn-2
Unkn-6
Unkn-6
Unkn-6
Unkn-10
Unkn-10
Unkn-10
Peak 1
82.20
82.00
82.00
82.00
82.00
82.00
82.00
82.00
82.00
Peak 2
None
None
None
None
None
None
None
None
None
Content
Unkn-3
Unkn-3
Unkn-3
Unkn-7
Unkn-7
Unkn-7
Unkn-11
Unkn-11
Unkn-11
Peak 1
82.20
82.00
82.00
82.00
82.00
82.00
82.00
82.00
82.00
Peak 2
None
None
None
None
None
None
None
None
None
Content
Unkn-4
Unkn-4
Unkn-4
Unkn-8
Unkn-8
Unkn-8
Unkn-12
Unkn-12
Unkn-12
Peak 1
82.20
82.00
82.00
82.00
82.00
82.00
82.00
82.00
82.00
Peak 2
None
None
None
None
None
None
None
None
None
Content
NTC-1
NTC-1
NTC-1
Peak 1
None
None
None
Peak 2
None
None
None
Sample
C
Sample
D
Sample
E
Sample
F
Content
Sample
G
Peak 1
Peak 2
Content
Sample
H
Peak 1
Peak 2
11
12
PCR Amplification Program
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1 cycle at 95 oC for 3 min to denature all templates
40-50 cycles:
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95 oC for 15 sec for denaturation of PCR products
At annealing temperature for 15 sec to anneal primers to
the templates
72 oC for 30 sec for synthesis of DNA
Melting Profile of RT-PCR Products
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From the melting profile, one can determine whether the PCR
product is amplified from a single target
Absolute Quantitative Real-time RT-PCR
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Construction of a standard curve:
 Construct a synthetic cRNA of your interest
 Reverse transcribe the cRNA into cDNA
 Set up Real-time PCR with known and increasing amount of
cDNA
 Plot Ct value vs. starting quantity of cDNA
Levels of Gene Expression Determined by
Absolute RT-PCR
NKX2.5
Norm.
GI,GII
Norm.
Norm.
GI,GII
β-actin
GATA 5
GI,GII
Norm.
Norm.
GI,GII
GI,GII
Norm.
GI,GII
Assigned Reading: Chun et al., 2006. Trout Ea4- or human Eb-peptide of proIGF-I disrupts heart, red blood cell, and vasculature development in zebrafish
embryos
Levels of Gene Expression Determined
by Absolute RT-PCR
GATA 2
GATA 1
Norm.
GI,GII
Norm.
GI,GII
Norm.
Norm.
Norm.
GI,GII
VEGF
GI,GII
GI,GII
Norm.
GI,GII
Reduction of mRNA Levels of Heart, Vasculature and
Blood Cell Development-Related Genes in rtEa4Peptide Injected Embryos
Molecules of mRNA/embryo at 36 hpf
Gene
Normal
Defective
embryos
Fold of
reduction
NKX2.5
2.77±2.04X106 8.02±5.96X103
242.1±1.0
GATA5
3.94±1.07X105 2.41±6.58X104
11.7±0.9
GATA1
1.63±0.46X106 2.27±0.12X105
5.5±1.2
GATA2
1.37±0.22X104 3.59±0.76X103
2.7±1.0
VEGF
2.87±0.96X105 3.09±0.41X104
6.4±1.1
β-actin
5.77±1.19X106 4.16±1.33X106
1.0±1 .0
Relative Quantitative RT-PCR
Calculation of Relative Expression Levels
∆CT= CT (target) – CT (normalizer)
∆∆CT = ∆CT (treated) - ∆CT (control)
Fold =1/[2-(∆ ∆ CT)]
GAPDH: glyceraldehyde-3-phosphate dehydrogenase or b-actin mRNA
is usually used as normalizer
Asigned Reading: Chen et al., 2007. Suppression of growth and cancerinduced angiogenesis of aggressive human breast cancer
cells (MDA-MB-231) on the chorioallantoic membrane
of developing chicken embryos by Epeptide of pro-IGF-I
Comparative Real-Time PCR Analysis of Genes
Up- and Down-Regulated by E-Peptide
Names of Genes
Relative Expression Levels
uPA
PAI 1
BCL 2
Cysteine-rich angiogenesis inducer
Tumor-associated Ca++ signal inducer
TYPO3 protein tyrosine kinase
Tumor rejection antigen (Gp96)
Heat shock protein 90 KDa protein 1a
Heat shock 70 KDa protein 10
Capase 3
Fibronectin 1
Laminin receptor 1
0.52 + 0.10
0.42 + 0.04
0.28 + 0.04
0.86 + 0.12
0.44 + 0.12
3.02 + 0.80
3.46 + 0.96
3.40 + 0.35
2.92 + 0.39
4.58 + 0.35
2.32 + 0.31
1.41 + 0.11
*Fold of reduction = 2-(∆ ∆ CT), where ∆ ∆ CT is ∆ CT sample - ∆CT control
where ∆ CT = CT (Target) – CT (normalizer)
• Microarray: a technique that allows
you to determine the expression of
many genes at one time
Why DNA Microarray Technology?
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Gene discovery: Examples
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Disease diagnosis:
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Drug discovery: Pharmacogenomics
 Profiling of cancer-specific expressing genes
 Tissue-specific expression of genes
 Developmental-specific expression of genes
 Collections of genes showing expression of genes specific
to certain types of diseases
 Examples: Specific cancer type, hematopoietic disease etc.
 To find correlations between therapeutic responses to drug
and gene profiles of patience
 Toxicological research: Toxicogenomics
•
To find correlations between toxic responses to toxicants
and changes in the gene profiles of the objects exposed to
such toxicants
Principle of DNA
Microarray
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•
•
•
Genes (cDNA or
oligonucleotides are
spotted on glass
plates
Messenger RNA is
reverse transcribed
into cDNA and labelled
with Cy3 (emission
570nm, green) or Cy5
(emission 670 nm, red)
Hybridization (mixing
Cy3-cDNA and Cy5
cDNA)
Scan the slide to
detect the
hybridization signals
Enlarged Photo of a Microarray Chip
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•
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This array has
2400 human
genes
Red indicates
increase of
expression;
yellow equal
expression and
green reduce of
expression
This technique
can help to
determine the
profiles of gene
expression
Interpreting the Scanned Image
•
•
•
•
The measured intensities from the two fluorescent
reporters have been false-colored red and green and
overlaid
Yellow spots have roughly equal amounts of bound
cDNA from each cell population and so have equal
intensity in the red and green channels (red + green
= yellow)
Spots whose mRNA’s are present at a higher level in
one or the other cell population show up as
predominantly red or green
The ratio of fluorescent intensities for a spot is
interpreted as the ratio of concentrations for its
corresponding mRNA in the two cell populations
Example
Novel Anti-cancer Activity of ProIGF-I E-Peptide
Assigned Reading:
Chen et al., 2007: Suppression of Growth and CancerInduced Angiogenesis of Aggressive Human Breast Cancer
Cells (MDA-MB-231) on the Chorioallantoic Membrane
of Developing Chicken Embryos by E-peptide of Pro-IGF-I
Insulin-Like Growth Factor (IGF) I
Primary translation product of IGF-I
B
S
C
A
D
E
Post-translational processing
Signal peptide
S
B
C
A
Mature IGF-I
D
E
E-peptide
E-Peptide of Pro-IGF-I
• The pro-hormone peptide of IGF-I
• It contains about 77 amino acid residues
• Presents in fish, human and other animals
• It is highly soluble in aquaous buffers
• Can be readily prepared by expressing the
gene in E. coli and other cell systems
Novel Anti-Cancer Activities of
the E-Peptide
•
•
•
•
•
Exerts dose-dependent mitogenic activities in
established non-transformed cell lines (Tian et al.,
1999)
Induces morphological differentiation and inhibits
anchorage-independent growth in oncogenic
transformed cell lines (Chen et al., 2002; Kuo and
Chen, 2002)
Inhibits tumor cell growth and invasion, and tumorinduced angiogenesis in developing chicken embryos
(Chen et al., 2007)
Induces programmed cell death in cancer cells (Chen
et al., 2011)
Up-regulate fironectin 1 and laminin receptor genes
and down-regulate uPA, tPA and TIMP1 (Siri and Chen
2006a, 2006b)
Is There Molecular Evidenced
•
•
If E-peptide of IGF-I can control cancer cell growth,
there must be genes that are up- or down-regulated
by E-peptide
Using microarray analysis technique, we determined
genes related to cancer cell activities that are up- or
down regulated by e-peptide in aggressive human
breast cancer cell (MBA-MD-231)
 Isolate RNA samples from MBA-MD-231 cells treated wit Epeptide and untreated cells
 Prepare cDNA from both RNA smples
 Labele E-peptide treated cDNA with Cy3 and the untreated
cDN with Cy5
 Combine both labeled cDNA and hybridized to a high
density human gene chip and scan the results
Results of the Microarray Assay
•
•
•
•
From repeat screening a gene chip contain 10,000
human genes, more than 1,000 genes were found to
be up- or down-regulated by E-peptide to a different
degree
Those genes with ratio of treated/untreated signals
over + or – 2 are considered real difference
Examples of up-regulated and down-regulated genes
are summarized in the next two slides
To confirm the microarray results, the mRNA levels
of each gene in the treated and untreated RNA
samples should be determined by real-time
quantitative or comparative RT-PCR
Some Genes Down-Regulated by E-Peptide
•
•
•
•
•
•
Oncogenes: TC 21, P53, vav 1, v-myb, c-H-ras (p21),
sarc, v-erb 2 (high expression in tumors)
Angiogenesis: Cysteine-rich angiogenic inducer 61
IGF-II, IGF-IIR, IGFBP-3, -4 & -7
Cell adhesion: Catenin a-like-1, Integrin a-3, Integrin b3, Integrin b-4, Tight junction protein 1
Proteases: Cathepsin Z, Plasminogen activator
inhibitor type I, Urokinase plasminogen activator,
Tissue plasminogen activator
Cell cycle, growth & proliferation: Cyclin-independent
kinase inhibitor 1A, Latent transforming growth factor
b-binding protein 3, Serine inducible kinase, Colony
stimulating factor 1, Thymosin b-4
Cytoskeleton molecules: Keritin-7, -8, and -18
Some Genes Up-Regulated by E-Peptide
•
•
•
•
•
•
Cell adhesion: Fibronectin 1, Laminin receptor 1
Cytoskeleton molecules: Keratin-1, Restin
Proteases and inhibitors: Tissue inhibitor of
metalloprotease I, Cathepsin C, Zinc metalloprotease
Cell cycle, growth & proliferation: Cyclin A2, B1, B2,
Cyclin-dependent kinase 7, Topoisomerase II a
Cell signal transduction molecules: TyrO3 protein
kinase, Mitogen-activated protein kinase kinase 3, csar tyrosine kinase, Phosphotidylinositol-4-phosphate5 kinase type Ia
Tumor rejection antigen (gp96) 1, Cdc 14 phosphatase,
Protein tyrosine kinases, Heat shock proteins 70 and
90
Comparative Real-Time PCR Analysis of Genes
Up- and Down-Regulated by E-Peptide
Names of Genes
Relative Expression Levels
uPA
PAI 1
BCL 2
Cysteine-rich angiogenesis inducer
Tumor-associated Ca++ signal inducer
TYPO3 protein tyrosine kinase
Tumor rejection antigen (Gp96)
Heat shock protein 90 KDa protein 1a
Heat shock 70 KDa pr5otein 10
Capase 3
Fibronectin 1
Laminin receptor 1
0.52 + 0.10
0.42 + 0.04
0.28 + 0.04
0.86 + 0.12
0.44 + 0.12
3.02 + 0.80
3.46 + 0.96
3.40 + 0.35
2.92 + 0.39
4.58 + 0.35
2.32 + 0.31
1.41 + 0.11
RNA-Seq Technology
RNA-seq, also called "Whole Transcriptome Shotgun Sequencing"
("WTSS"), refers to the use of high-throughput technologies to
sequence cDNA or RNA in order to get information about a sample's
RNA content. The technique has been adopted in studies of diseases
like cancers. With deep coverage and base-level resolution, this
technology provides information on differential expression of genes,
including gene alleles and differently spliced transcripts; non-coding
RNAs; post-transcriptional mutations or editing; and gene fusions
Reading Assignment:
1. RNA-Seq
2. Wang et al., 2009. RNA-Seq: a revolutionary tool for
Transcriptomics. Nature Review: Genetics 10: 57-63.
•
Transgenic technology

Biotechnological application
Assigned Reading :
Application of recombinant DNA technology
Gene Transfer in Plants
Golden Rice
Transfer genes that can make vitamin A into rice
Transgenic Animal Technology
• Transgenic Animals: Animals with foreign gene(s)
integrated into their genomes
• The first transgenic animal was produced in mice by
Richard Palmiter and Ralf Brinster in early 1980s
Transgene: Human growth hormone gene driven by the
promoter of metallothionein gene
Procedure for Producing Transgenic Mice
Transgenic Aquatic Animals
•
•
•
We have been working on gene transfer
technology since mid 1980s
Over the past two decades, we have
produced several speccies of transgenic
aquatic animals by microinjection,
electroporation, infection with pantropic
retroviral vectors, and lipofection
References:
 Zhang et al., MRD 25: 3-13, 1990.
 Lu et al., Mol Marine Biol. Biotech 1:366375, 1991.
 Lu et al., PNAS 93: 3482-3486. 1996.
 Lu et al., Mol Marine Biol Biotecnnol 7: 289295, 1997.
 Sarmasik et al., Marine Biotechnol 3: 465473, 2001
 Sarmasik et al., Marine Biotechnol 3: S117S184, 2002
 Lu et al., Marine Biotechnol 4: 328-337,
2002.
Pore Formation on Bacterial Cell
Membrane by Cecropin
Cecropin
Pores Formation
Prototype of Transgene
CMV IgG-SP Cecropin P1
Promoter
Transfer the transgene construct into sperm of rainbow
trout by electroporation
Sperm-Mediated Gene Transfer
Transgene
Parameters: Dilution buffer, Dilution
factor, DNA/sperm, Voltage, Pulse #
Mortalities of F2 and F3 Cecropin P1 Transgenic
Fish Challenged with A. salmonicida
% Mortality (Mean ± S.D.)
Families
F2 Generation
S8#Y419
S7#375
S9#746
S7#342
S8#505
S9#638
S9#659
U6#768
Non-transgenic
12 ± 3
14 ± 5
20 ± 6
30 ± 3
20 ± 4
10 ± 2
25 ± 6
80 ± 3
80 ± 6
F3 Generation
15 ± 5
18 ± 8
30 ± 5
35 ± 4
26 ± 8
14 ± 8
40 ± 10
79 ± 12
85 ± 8
For each family, challenge was conducted with 50 fish/family (0.4 to
0.5 g body weight) in triplicates and the dose of A. salmonicida
used in each challenge study brings about 80% mortality in nontransgenic fish.
Some Key Terms
• Totipotent: Fertilized oocytes and blastomeres of 2, 4-, and
8-cell stage embryos. Could contribute to the development
of the embryo proper and the placental tissue.
• Pluripotent: Inner cell mass (ICM) of blastocysts, embryonic
ectoderm and primordial germ cells of fetal stages. Could
contribute to the development of embryo proper only.
• Embryonic germ cells:
Stem cells developed from
primordial germ cells. Could develop into all somatic cell
types of an embryo. These cells are also pluripotent.
• Somatic stem cell:
Stem cells could only develop into
restricted cell types. (e.g. Skin)
Stem cells: Stem cells are cells found in most, if not all, multicellular organisms. They are characterized by the ability to
renew themselves through mitotic cell division and
differentiation into a diverse range of specialized cell types.
Two types of mammalian stem cells are:
Embryonic stem cells: Isolated from the inner cell mass
of blastocyst
Adult stem cells: Found in adult tissues.
Assigned Reading:
 Embryonic stem cells.
Totipotent and Pluripotent Cells
Different Paths of Generating Human ES
Cells from In Vitro Fertilized Human Oocytes
Inactivation of Genes by Knock-Out Method
•
•
Gene knock-out: Interrupt a gene by inserting a
small piece DNA into it
To perform this technique, it requires:
 ES or EG cells
 Introduce the interrupting DNA into ES or EG cells and
isolate recombinants
 Injecting the recombinant cells into blastoceal cavity of
mouse embryos
 Transfer the embryos into pseudo-pregnant female
mice
Assigned Reading:
Elimination of a gene by the knock out technology
Construction of a Gene-Targeted Disruption
Construct
•
•
•
•
neor: a marker gene
that confers
resistant to G-418
tkHSV: a marker gene
confers sensitivity to
ganciclovir
When the above
construct is
introduced into
mouse stem cells,
stem cells with gene
been disrupted can
be isolated
The isolated ES cells
can be used to
generate gene knock
out animals
Isolation of Recombinant Stem Cells with a Gene
Been Interrupted
Production of Germ Line
Knock Out Animals
•
•
•
•
•
Introduction of knock out heterozygous
ES cells into the blastoceal cavity of
mouse embryos
Transfer embryos into pseudopregnant
female mice
Select chimeric mice
Mat chimeric mouse with homozygous
black mouse
Select brown mice for homozygous knock
out mice
Knock Out Genes in Somatic Cells by the loxP-Cre
Recombination System
•
•
loxP site:
site-specific
DNA
recombination site
Cre: enzyme
catalyzing
recombination
Inactivation of a Gene
by Using RNAi
•
•
In vitro or in vivo production of
siRNA
Inhibition of mex3 mRNA by
injecting siRNA into the cells
• Methods of analyzing proteins produced
in different tissues or composition of
proteins in different cells:




SDS-polyacrylamide gel electroporation
Immuno western blot analysis
Two dimensional electroporation analysis
Proteomic analysis
1
2 3 4
5 6 7 8 9
SDS Polyacrylamide gel stained
with coomassie brilliant blue dye
25.9kd
19.4kd
14.8kd
6.0kd
1
2 3 4 5 6 7
8 9
SDS polyacrylamide gel stained
with silver stain
25.9kd
19.4kd
14.8kd
6.0kd
Silver staining
1 2 3 4 5 6 7 8 9
Immuno western blot analysis
25.9kd
19.4kd
14.8kd
6.0kd
Anti Histag Antibody
Immuno Western Blot Analysis
Concept of Two Dimensional Gel Electrophoresis
Comparison of Two Protein Samples
Strategy of Sequencing a Protein by Mass-Spectrometry
Identification of Proteins in Organelles by Gradient
Centrifugation and LC-MS/MS Analysis
Chromatin
Immunoprecipitation
This technique can be
used to detect proteinDNA interactions in the
native chromatin context
in vivo. The associated
DNA is purified for
analysis by identifying its
specific sequence by PCR
or by labeling the DNA
and applying to a tiling
array to detect genome
wide interactions
Assigned Reading [II]
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
Restriction Enzymes: a background paper
Recombinant DNA and gene cloning
Nobel lecture by H. Smith, 1978
Nobel lecture by O. Shimomura on discovery of GFP
Nobel lecture by K. Mullis PCR, 1993
Nobel lecture by Sanger
Nobel lecture by Michael Smith
Elimination of a gene by the knock out technique
Greene and Chen, 1999. Quantitation of IGF-I, IGF-II……..
Chun et al., 2006: Trout Ea4 and human Eb-peptide ……..
Chen et al., 2007
Embryonic stem cells
RNA-Seq
RNA-Seq: a revolutionary tool for transcriptomics