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Chapter 17
Gene Technology
Central Dogma: DNA -> RNA -> Protein
DNA
CCTGAGCCAACTATTGATGAA
transcription
RNA
CCUGAGCCAACUAUUGAUGAA
translation
Protein
PEPTIDE
Genetic Recombination in Humans
There are three ways in which meiosis and fertilization
ensure that a child has a different combination of
genes from that of either parent:
1. Independent assortment of chromosomes during
metaphase I
2. Crossing-over during prophase I
3. Upon fertilization, recombination of chromosomes
from different individuals (via their gametes) occurs.
Recombinant DNA technology or genetic
engineering was developed in 1971-1973
Their core was gene cloning
 Lead to DNA sequencing techniques that
enabled the structures of individual genes to be
determined
 Lead to procedures for studying the regulation
of individual genes

Genetic recombination
-
transfer of DNA from one organism
(donor) to another recipient. The
transferred donor DNA may then be
integrated into the recipient's nucleoid
by various mechanisms (homologous,
non-homologous).
Types Of Recombination



Generalized or Homologous Recombination Occurs during prophase of meiosis I and involves
exchange between homologous strands of DNA
Site Specific Recombination - Short homologous
sections of bacterial and phage DNA serve as a site
for recombination and thus incorporation of phage
DNA into bacterial chromosomes
Transposition - Not truly recombination between
different genomes, but the movement of transposons
within a genome
Homologous recombination
homologous DNA sequences having nearly the same
nucleotide sequences are exchanged by means of Rec A
proteins. This involves breakage and reunion of paired
DNA segments as seen in Natural mechanisms of
genetic recombination in bacteria include:
a. transformation
b. transduction
c. conjungation
The Current Prokaryotic
Recombination Model
5´
3´
3´
5´
3´
5´
5´
5´
3´
3´
5´
3´
5´
5´
3´
5´
3´
3´
3´
3´
内切酶
(recBCD)
3´
5´
5´
3´
内切酶
(recBCD)
5´
3´
3´
5´
3´
5´
5´
3´ 5´
3´
DNA侵扰
(recA)
分支迁移
(recA)
DNA
连接酶
5´
3´
3´
5´
3´
5´
5´
3´
5´
3´
3´
5´
3´
5´
5´
3´
Holiday中间体
目录
5´
3´
3´
5´
3´
5´
5´
3´
Holiday中间体
3´ 5´
5´
3´
3´
5´
5´
3´
内切酶
(ruvC)
3´
3´
3´ 5´ 拼
5´
3´
5´
5´
3´
3´
5´
DNA
连接酶
5´3´
3´
5´
5´
3´
5´
5´
3´
5´
3´
内切酶
(ruvC)
接
重
组
体
体片
段
重
组
splice recombination
5´
3´
DNA
连接酶 3´
5´
3´
5´
3´
3´
5´
Patch recombination
5´
Genetic Transfer &
Recombination In Bacteria
Three kinds of genetic exchanges between
prokaryotes

Three kinds
Conjugation
Mediated by plasmids
Transformation
Mediated by free DNA
Transduction
Mediated by phages
Bacterial Conjugation
Bacterial Conjugation is genetic recombination in
which there is a transfer of DNA from a living
donor bacterium to a recipient bacterium. Often
involves a sex pilus.

The 3 conjugative processes
+
I. F conjugation
II. Hfr conjugation
III. Resistance plasmid conjugation
I. F+ Conjugation Process
F+ Conjugation- Genetic recombination in which there
is a transfer of an F+ plasmid (coding only for a sex
pilus) but not chromosomal DNA from a male donor
bacterium to a female recipient bacterium. Involves
a sex (conjugation) pilus. Other plasmids present in
the cytoplasm of the bacterium, such as those coding
for antibiotic resistance, may also be transferred
during this process.
Conjugal transfer of plasmid
The 4 stepped F+ Conjugation
1. The F+ male has an F+ plasmid
coding for a sex pilus and can serve
as a genetic donor
2. The sex pilus adheres to an Ffemale (recipient). One strand of the
F+ plasmid breaks
The 4 stepped F+ Conjugation (cont’d)
3. The sex pilus retracts and a
bridge is created between the two
bacteria. One strand of the F+
plasmid enters the recipient
bacterium
F
4. Both bacteria make a
complementary strand of the F+
plasmid and both are now F+ males
capable of producing a sex pilus.
There was no transfer of donor
chromosomal DNA although other
plasmids the donor bacterium
carries may also be transferred
during F+ conjugation.
http://www.cat.cc.md.us/courses/bio141/lecguide/unit4/genetics/recombination/conjugation/f.htm l
Transformation

Genetic recombination in which a DNA
fragment from a dead, degraded bacterium
enters a competent recipient bacterium and it
is exchanged for a piece of the recipient's
DNA.

Involves 4 steps
http://www.cat.cc.md.us/courses/bio141/lecguide/unit4/genetics/recombination/transformation/transformation.html
The 4 steps in Transformation
1. A donor bacterium dies and is degraded
2. A fragment of DNA from the dead donor
bacterium binds to DNA binding proteins
on the cell wall of a competent, living
recipient bacterium
3. The Rec A protein promotes genetic exchange
between a fragment of the donor's DNA and the
recipient's DNA
4. Exchange is complete
Transduction

Genetic recombination in which a DNA
fragment is transferred from one bacterium to
another by a bacteriophage
Structure of T4 bacteriophage
Contraction of the tail sheath of T4
What are Bacteriophages?
Bacteriophage (phage) are obligate
intracellular parasites that multiply
inside bacteria by making use of some or
all of the host biosynthetic machinery
(i.e., viruses that infect bacteria
An
infection
cycle
Transduction
There are two types of transduction:
generalized transduction: A DNA fragment is
transferred from one bacterium to another by a
lytic bacteriophage that is now carrying donor
bacterial DNA due to an error in maturation
during the lytic life cycle.
specialized transduction: A DNA fragment is
transferred from one bacterium to another by a
temperate bacteriophage that is now carrying
donor bacterial DNA due to an error in
spontaneous induction during the lysogenic life
cycle
Site Specific Recombination

Short homologous sections of bacterial and
phage DNA serve as a site for recombination
and thus incorporation of phage DNA into
bacterial chromosomes
att = attachment site
O = center core of 15 bases
= the same in phage & bacterial
dsDNA
B,P = different in size and sequence in
bacterial & phage
XIS = Excisionase
INT = integrase
Integration of Lambda DNA-overview.
The control of INT & XIS activity
determines it latency or not.
Integration of Lambda DNA-Detail of crossover
例:细菌的特异位点重组
沙门氏菌H片段倒位决定鞭毛相转变
Transposition
-Movement of gene to a new site, on same
or a different chromosome
 Does not require extensive homology

Transposable elements
Insertion sequence
插
入
序
列
的
复
制
性
转
座
目录
Transposons: Mobile genetic elements that
enable genes to


move between non-homologous sites in DNA –
Transposable elements.
⋅Altered expression of genes in new environments
Gene technology

A set of methods and techniques used to study
biological processes on the molecular level

There have been considerable developments in
this field during the past two decades
Eg: new and powerful ways for the isolation,
analysis, and manipulation of nucleic acids
What is gene cloning
The basic steps in gene cloning experiment are as follows:
(1) A fragment of DNA is inserted into a circular DNA molecule
called a vector, to produce a chimera or recombinant DNA
molecule
(2) The vector acts as a vehicle that transports the gene into a
host cell
(3) Within the host cell the vector multiplies, producing
numerous identical copies
(4) When the host cell divides, copies of the
recombinant DNA molecule are passed to progeny
and further vector replication takes place
(5) After a large number of cell divisions, a colony, or
clone, of identical host cells is produced
Each cell in the clone contains one or more copies
of the recombinant DNA molecule
The gene carried by the recombinant molecule is
now said to be cloned
The basic steps
in gene cloning
Why gene cloning is so important

This technique can provide a pure sample of
individual gene, separated from all the other genes
in the cell

Gene isolation by cloning
Cloning allows individual fragments of DNA to be
purified
isolated long genes or those that have never been studied
before
Cloning
allows
individual
fragments of
DNA to be
purified
The basic concepts about gene cloning
Clone
A large population of identical molecules, or
cells that arise from a common ancestor.
Cloning
The process that produces a large number of
DNA or cell copies
vector
target DNA
Recombination DNA
Transformation
bacteria
Transfer the bacteria to
a solid culture plate
Screening the bacteria containing
the recombinant DNA
The process of gene cloning with the
plasmid as vector
DNA recombination
The process that two DNA molecules from different
source join together by covalent bond to form a new
DNA molecule is called DNA recombination.
Recombinant DNA
DNA recombination technique
By the application of some tool enzymes, the target
gene and vector are ligated together, then introduced
into the recipient cells which multiply and express the
protein products coded by the target gene, that is, DNA
recombination technique, DNA cloning or Gene
cloning, Molecular cloning.
Genetic engineering
All the work or methods used related to
the gene cloning and the target gene expressed
in host cells to produce the special protein or
polypeptide, or to change the character of an
organism, are called genetic engineering.
The requirements for the gene cloning
(1) The target genes
(2) The vectors
(3) The tool enzymes
(4) The host cells
Target DNA
• cDNA
The cDNA are synthesized by reverse
transcriptionase based on their mRNA templates of
a cell line or tissue.
• Genomic DNA
It represents whole DNA sequence of a genome
Purification of DNA from living cells

Preparation of total cell DNA (RNA)

Preparation of plasmid DNA

Preparation of bacteriophage DNA
Vectors
Cloning vector——Cloning vectors are DNAs
which can carry target genes, transfer them into
the recipient cells.
Cloning vector
classes
Plasmid DNA
Phage DNA
Virus DNA
As for the expression vectors, they can make the
proteins which are coded by the target gene expressed
in the host cell
Plasmids

Basic features of plasmids
Small (less than 10kb), Circular, duplex molecules of DNA
Exist at low or high copies within the bacteria, but useful
plasmid present in multiple copies
Replicate independently from the bacterial cell
Contain selectable markers, eg: the antibiotic resistance
capability conferred to bacterium
Possess at least one DNA sequence that act as an origin of
replication
Multiple RE sites ( multiple cloning sites, MCS)
plasmid (质粒 )
b
Go to pBR322
Fig
pBR322
Multiple
cloning site
(MCS)
Lac Z β-galactosidase gene
Ampicillin resistance gene
Origin of replication
Fig
pUC19
Go to pUC
Bacteriophages

Basic features of bacteriophages

Bacteriophages, or phages are viruses that
specifically infect bacteria

Simple in structure, merely of a DNA (or
occasionally RNA) carrying genes, including
several for replication of the phage, surrounded
by a protective coat or capsid made up of protein
The general pattern of infection

Attaches to the outside of the bacterium and injects its
DNA chromosome into the cell

The phage DNA is replicated, usually by specific phage
enzymes coded by genes on the phage chromosome

Other phage genes direct synthesis of the protein
components of the capsid, new phage particles are
assembled and released
The result of Infection
Lytic cycle:

With some phage types the entire infection cycle is
completed very quickly, possibly in less than 20
min. This type of rapid infection is called lytic
cycle.
Lysogenic infection:

Characterized by retention of the phage DNA
molecule in the host bacterium, possibly for
many thousands of cell divisions
Common used phages

Bacteriophage λ
A linear dsDNA approximately 49 Kb in length
After infection it forms circular structures
The phage DNA is inserted into the bacterial genome
The first two classes of vector to be produced were λ
insertion (λgt
phages)
phages) and λ replacement (EMBL
 Bacteriophage
M13
A circular ssDNA, and has been
used for sequencing of a cloned target
DNA fragment
Multiple
cloning site
(MCS)
Lac Z β-galactosidase gene
Ampicillin resistance gene
Origin of replication
Fig
pUC19
Go to pUC
Other vectors
Cosmid (粘性质粒)
Other vectors

Bacterial artificial chromosome (BAC) and
yeast chromosome

Viruse are used as vectors, eg: retro-virus,
adeno-virus, adenoassociated virus, etc
The tool enzymes





Nucleases—cut, shorten or degrade nucleic acid
molecules
Ligases—join nucleic acid molecules together
Polymerase—make copies of molecules
Modifying enzymes —remove or add chemical
groups
Topoisomerases —introduce or remove supercoils
from covalently closed-circular DNA
Nucleases

degrade DNA molecules by breaking the
phosphodiester bonds

There are two different kinds of nucleases
Exonucleases remove nucleotides one at a time
from the end of a DNA molecule
Endonucleases are able to break internal
phosphodiester bonds within a DNA molecule
ligases

To repair single-stranded breaks(discontinuities)
that arise in double-stranded DNA molecules
during DNA replication

Join together two individual fragments of
double-stranded DNA
Polymerases

Synthesize a new strand of DNA complementary to
an existing DNA or RNA template

Four types of DNA polymerase are used routinely
in genetic engineering
 DNA polymerase I: from E.coli. Synthesizes dsDNA by formation of a
5’,3’-phosphodiester bond
 Klenow fragment : removes the first 323 amino acids from DNA
polymerase I , Synthesizes DNA by formation of a 5’,3’-phosphodiester
bond
 Reverse transcriptase: synthesizes DNA from RNA template
 Taq DNA polymerase: used in the PCR, it is the DNA polymerase I
from bacterium Thermus aquaticus
DNA modifying enzymes

Alkaline phosphatase
from E.coli, calf intestinal tissue or arctic shrimp
 removes the phosphate group present at the
5’terminus of a DNA molecule


Polynucleotide kinase
 from E.coli infected with T4 phage
 Has the reverse effect of alkaline phosphatase,
adding phosphate groups onto free 5’termini
DNA modifying enzymes

Terminal deoxynucleotidyl transferase
 from calf thymus tissue
 adds one or more deoxyribonucleotides onto the 3’
terminus of a DNA
Topoisomerases

Change the conformation of covalently closedcircular DNA by introducing or removing
supercoils.
Enzymes for cutting DNA-restriction
endonucleases

The initial observation that led to the
eventual
discovery
of
restriction
endonucleases (RE) was made in the early
1950s

Restriction occurs because the bacterium
produces an enzyme (called restriction
endonucleases) that degrades the phage
DNA

Three different classes of RE are
recognized, but the most important one is
RE II which is used in DNA manipulation

The discovery of these enzymes led to
Nobel prizes for W.Arber, H. Smith and D.
Nathans in 1978
Type II restriction endonucleases (RE) cut
DNA at specific nucleotide sequences

Generally, 4~8 bases be found, mostly 6 bases, a few of
8~10 bases

The sequences discriminated usually are palindrome
structure

To cut the double strands of DNA at special sites and to
yield two kinds of ends: blunt ends and sticky ends
Blunt ends and sticky ends:

Sticky or cohesive ends:
 the cleavage is staggered by two or four nucleotides
 the resulting DNA fragments have short single-
stranded overhangs at each end
 Base pairing between them can stick the DNA
molecule back together again
 Restriction endonucleases with different recognition
sequences may produce the same sticky ends eg:
BamH I (GGATCC) and Bgl II (AGATCT)
5’-sticky end (EcoR I )
5’-GGTGAATTCAGC…-3’
3’-CCACTTAAGTCG…5’
5’-GGTG
3’-CCACTTAA +
AATTCAGC…-3’
GTCG…5’
3’-sticky end ( Pst I )
5’-TTGCTGCAGAAG…-3’
3’-AACGACGTCTTC…5’
5’-TTGCTGCA
GAAG…-3’
+ ACGTCTTC…5’
3’-AACG
blunt end or flush end
Make a simple double-stranded cut in
the middle of the recognition sequence
5’-CCCGGG…-3’
3’-GGGCCC…5’
5’-CCC
GGG…-3’
+
3’-GGG
CCC…5’
Sma I
*The ligation efficiency between the blunt ends
is not as high as that of the stickly ends.
Naming of RE
REs are usually named after the bacterium
from which they are isolated.
Escherichia coli RY13 I
EcoR I
The genus name of bacteria
The order of the RE
found in bacteria
The strain name of bacteria
The species name of bacteria
The requirements for the gene cloning
(1) The target genes
(2) The vectors
(3) The tool enzymes
(4) The host cells
The basic process of recombination technique
* The preparation of target DNA
* The selection and preparation of vectors
* The ligation of DNA fragments in vitro
* Foreign DNA be transported into host cells
* The screening and identifying of target DNA
1. The preparation of target DNA
(1)To prepare from genomic library
genomic library contains a comprehensive DNA
fragments from genomic DNA cut by the
specific RE.
During the construction of the genomic library,
the DNA fragments and their vectors are ligated,
and then introduced into the recipient cells.
It represents whole DNA sequence of a genome
(2) To prepare from cDNA library or cDNA
Extracting total mRNA
Ligation
Reverse transcription
introduction
It represents the population of mRNAs coding
for gene and protein expression
(3) To prepare the gene fragment with
other methods
1) PCR amplification
2) To synthesize the DNA fragment by
chemical method
 it is typically used for those of
the small biologically active peptides
2. The selection and preparation of vectors
Plasmid
λ phage
< 10 kb
< 22 kb
Capacity
of cloning
cosmid
40~50 kb
M13 phage
< 1 kb
gDNA library
-
+
+
-
cDNA library
+
+
-
-
Subcloning
+
-
-
+
Sequencing
+
+
-
+
E coli expression +
+
-
-
3. Construction of Recombinant Molecules

Both purified DNA fragments and vectors
are digested with the same restriction
enzyme to give complementary cohesive
ends
•Analyzing the result of restriction endonuclease
cleavage



Separation of molecules by gel electrophoresis
Visualizing DNA molecules in a gel (EB staining)
Comparison with size markers
•Ligated by T4 ligase to recombinant molecules

joining together of the vector molecules and DNA
to be cloned
 The enzyme that catalyses the reaction is called
DNA ligase, which purified from E.coli bacteria
that have been infected with T4 phage
(1) Sticky-ended ligation
3’
GAATTC
CTTAAG
G
CTTAA
EcoR Ⅰ
5’
3’
GAATTC
CTTAAG
GAATTC
CTTAAG
5’
3’
5’
GAATTC
CTTAAG
G 3’
CTTAA 5’
ligation
GAATTC
CTTAAG
Bidirection insertions
AATTC
G
3’
5’ AATTC
3’ G
GAATTC
CTTAAG
5’
GAATTC
CTTAAG
(2) Blunt-ended ligation
Target gene
Vector
Restriction endonucleases
Restriction endonucleases
T4 DNAligase
15ºC
recombinate
Self-ligated
vector
Self-ligated
target gene
4. Introduction of DNA into living cells

Serves two main purposes:
 allows
a large number of recombinant DNA
molecules to be produced from a limited amount of
starting material
 Purification
-Methods
 Transformation
 Transfection
 Infection
Transformation ----The uptake of DNA
by bacterial cells

preparation of competent E.coli cells
50 mM CaCl2 is tranditionally used.
Another alternative is by electroporation
• In recent years, transformation has been extended to
include uptake of any DNA molecules by any type of cell
Whether the uptake results in a detectable change in the cell
Whether the Cells involved is bacterial, fungal, animal or plant
Introduction of phage DNA into bacterial cells

Two methods:
 Transfection
purified phage DNA, or recombinant
phage molecules, is mixed with competent
E.coli cells and DNA uptake induced by
heat shock
Transfection
Introduction of phage DNA into bacterial cells

Two methods:
 In vitro packaging
single strain system: the defective λphage
carries a mutation in the cos sites
 two strain system: two defective λphage
carries a mutation in a gene for one of the
components of the phage protein coat


Phage infection is visualized as plaques on the agar
medium
In vitro packaging
5. Screening and Identification of Recombinants

The problem of selection
 A restriction digest of total cell DNA produces not
only the fragment carrying the desired gene, but also
many other fragments carrying all the other genes
 Numerous different recombinant DNA molecules are
produced
 A variety of recombinant clones are obtained
Target Genes Carried by Plasmid
Target Genes
Restriction
Enzyme
DNA Recombination
Target Gene
Recombination
Chromosomal DNA
Restriction
Enzyme
Transformation
Host Cells
Recombinant
Plasmid
Transformation
1 plasmid
1 cell
Juang RH (2004) BCbasics
Amplification and Screening of Target Gene
1
Plating
1 cell line, 1 colony
Bacteria
Duplication
X100
Plasmid
Duplication
X1,000
Pick the colony
containing target gene
=100,000
Juang RH (2004) BCbasics
There are two basic strategies for obtaining
the clone you want

Direct selection for the desired gene
the only clones that are obtained are clones of
the required gene
There are two basic strategies for obtaining the
clone you want
 Direct selection for the desired gene
the only clones that are obtained are clones of
the required gene

Identification of the clone from a gene library
entails an initial shotgun cloning experiment,
to produce a clone library representing all or
most of the genes present in the cell, followed
by analysis of the individual clones to identify
the correct one
Correct clone
A clone library
Direct selection

an antibiotic resistance gene
Direct selection

an antibiotic resistance gene

Marker rescue ---by αcomplementation
 plasmids contain sequence (lacZ) coding
for N-terminal amino acids (α fragment) of
β–galactosidase
 Mutant cells contain sequence (lacZ) coding
for C-terminal amino acids (ω fragment) of
β– galactosidase
By αcomplementation
 The
enzymatic activity is dependent on the
coexpression of the complete fragments, which can
hydrolyzes the specific substrate X-gal (5-bromo-4chloro-3-inolyl-β-D-galactoside) to turn to a blue
colored one under the induction of IPTG（isopropyl
thiogalactoside)
 The recombinant molecules have no this enzyme
activity because the insertion of target gene into the
lacZ region disturbs the expression of αfragment, and
therefor, the colour of the recombinant molecule
containing the colony is white.
 Based on this blue-white colony screening
α-mutual complement screening
Multiple cloning sites
Cleavage N end
Ampr
Ampr
The sequence coding
the N end fragment of
β-galactosidase
promoter
External DNA
Cleavage N end
transformation
transformation
Chromosome
The sequence coding
the C end fragment of
βgalactosidase
The growth of
bacteria on the
culture with X-gal
Recombinant pUC18
The growth of
bacteria on the
culture with X-gal
The white clone
containing the
recombinant
pUC18
The blue clone containing the pUC18
The blue clone
containing the
pUC18
White clone contains the
recombinant, but blue clone
not contain recombinant
Direct selection

an antibiotic resistance gene

Marker rescue ---by αcomplementation

Colony/plaque in situ hybridization is used for
positive colony screening
Colony Is Screened by Hybridization with Probe
Colony hybridization
Transferring …
Collect filter
paper
Dissolve cell
Autoradiography
DNA denatured
Add probe
Juang RH (2004) BCbasics
Cover with
filter paper
Direct selection

an antibiotic resistance gene

Marker rescue ---by αcomplementation

Colony/plaque in situ hybridization is used for
positive colony screening
Immunological Technology
Methods for clone identification
 Colony
PCR
 Enzyme digestion
 nucleic acid hybridization
 DNA sequencing
Using PCR to detect gene targeting events
Identification with restriction enzymes
M 1 2 3 4 5 6 7 8 9 10
M 1 2 3 4 5 6 7 8 9 10
1kb
Figure2a. The clones of positive plasmid
M----1Kb DNA ladder
1∽10, positive plasmid
A. before cut with RE
Figure2b.The positive plasmids after cutted
by EcoR I
M----1Kb DNA ladder
1∽10, positive plasmids cutted by EcoR
B. after cut with RE
To identify the target gene
band after cutted by RE
The basic process of recombination technique
* The preparation of target DNA
* The selection and preparation of vectors
* The ligation of DNA fragments in vitro
* Foreign DNA be transported into host cells
* The screening and identifying of target DNA
6. Expression of the cloned gene

Different vectors are selected for cloning
 cloning vectors are used for replicating
and amplifying genes
 Expression vector are applied to express
the gene product
Cloning vector
Antibiotics resistance, MCS and screening
Expression vector
Antibiotics resistance, MCS and screening
Contains regulatory sequences for
transcription
and
translation,
eg;
promoter, SD sequence for 16s rRNA
binding, and a terminator which is
ρfactor independent
Expression system
Prokaryote expression system
 E.coli (most popular)
 Its easy culture, fast proliferation, low
expense, large scale production
 Lack of the processing capability after
transcription and translation

Notes during expression
Infusion proteins maybe formed when other
sequences coding amino acids linked to a
target gene are co-expressed together with
target proteins
 Purified by affinity chromatography,
followed by the unnsecesary peptides cut
out

 eukaryote
expression system
 Mammalian cells are stable and
repeatable
 can process hnRNA to become mature
mRNA, as well as the post-translation
modifications
Application of Recombination DNA
Technology
DNA Recombination Medical Production
产
品
功
能
组织胞浆素原激活剂
抗凝
血液因子VIII
促进凝血
颗粒细胞-巨噬细胞集落剌激因子
剌激白细胞生成
促红细胞生成素
剌激白细胞生成
生长因子(bFGF, EGF)
刺激细胞生长与分化
生长素
治疗侏儒症
胰岛素
治疗糖尿病
干扰素( 1b, 2a,  2b, )
抗病毒感染及某些肿瘤
白细胞介素
激活、剌激各类白细胞
超氧化物歧化酶
抗组织损伤
单克隆抗体
利用其结合特异性进行诊断试验、肿
瘤导向治疗
乙肝疫苗(CHO, 酵母)
预防乙肝
口服重组B亚单位菌体霍乱菌苗
预防霍乱
目录
Gene diagnosis

It is recognized that the abnormal
structure and expression of the gene
are involved in the pathogenesis of
diseases

Gene diagnosis is the detection of
the abnormalities of the candidate
genes by ways of molecular biology
and molecular genetics
For examples：
Sickle cell anemia belongs to gene point
mutation
 The 6th codon GAG was changed to GTG
 Glu is changed to Val
 Abolish an MstII restriction site which
spans
codons 5-7
Sequencing HbS proteins revealed a single change:
Glu6Val in the β chain. Fiber formation (R) at low [O2]
causes sickling of RBCs (center).
Restriction mapping analysis of sickle cell anemia
MstⅡrestriction site (GCTNAGG)
5´
3´
1.15kb
Normal gene
×
5´
3´
1.35kb
Mutation gene
镰状红细胞贫血患者基因组的限制性酶切分析
﹣
1.35kb
1.15kb
0.2kb
+
Normal
Carrier
Sickle cell homozygote
Gene Therapy

Gene therapy is the way to transfer
genetic material which exerts the
biological function into the cells of
patients to treat the disease
Genetic material: normal gene, recombinant
DNA, RNA, synthetic oligonucletides
They may integrate into the chromosome or
express separately
The Strategies and technologies of gene therapy
 Gene
correction
The abnormal bases of a gene are
corrected
 Gene
replacement
The defected gene is replaced by the
normal one which can integrate into the
chromosomes by homologous
recombination or remain
extrachromosomal
The Strategies and technologies of gene therapy

Gene augmentation
The target gene is introduced to the
defected cells or other cells

Gene inactivation
the expression of the gene is intervened to
block or inhibit the inappropriate genes in
vivo on both transcriptional and
translational level
Applications of Gene Therapy

The first apparently successful
application was initiated on Sep. 14,
1990 for ADA deficiency

Results in a lymphopenic form of
SCID that is fatal in early childhood.
ADA SCID
(Severe Combined Immunodeficiency Diseases )
Autosomal recessive disorder
 ADA = adenosine deaminase (an enzyme
of purine metabolism)
 ADA is an important enzyme in the
purine catabolic pathway, catalyzing the
irreversible deamination of adenosine to
inosine.

SCID with ADA Deficiency
ADENOSINE
Purine Catabolism Pathway
Adenosine
Deaminase
INOSINE
GUANOSINE
Purine
Nucleoside
Phosphorylase
Purine
Nucleoside
Phosphorylase
GUANINE
HYPOXANTHINE
Xanthine
Oxidase
XANTHINE
Xanthine
Oxidase
URIC ACID
Guanase
SCID with ADA Deficiency

The enzyme deficiency inhibits the normal
catabolism of purines.

Results in the accumulation of metabolic
substrates that are toxic to lymphocytes,
particularly in the inhibition of lymphocyte
function.
GENE THERAPY IN ADA SCID
ADA deficiency was the first disorder to be
treated by gene therapy (Bordignon et al
1995)
 The initial targets for genetic manipulation
were bone marrow (BM) stem cells and
peripheral blood lymphocytes (PBLs)

GENE THERAPY IN ADA SCID
Vectors expressing human ADAcDNA (1.5
kbp) with their own promoters were
transfected into BM stem cells and PBLs in
vitro
 6 months after gene therapy ended, vectorderived DNA was found in the PBLs

The Cloning Procedure Used for Creating Dolly