Download 2. recombinant gene

1.
Biotechnology
Gene technologybased approaches
Non-genetic
approaches
Knockout &
transgenic organisms
microchips
Recombinant
proteins
Live vaccines
New stem cell
techniques
Gene therapy,
immunotherapy
Industrial
fermentation
Monoclonal
antibodies
Cloned organisms
Traditional stem cell
technlogy
Live vaccines
etc.
2.
Intervention  Examination
Recombinant
gene
technology
Genomics
3.
Gene technology
Top danger
4.
Moratorium on genetic manipulations
- Asilomar: the conference of debate
Voluntary moratorium
The immediate reason: cloning of entire genome of SV40 in E. coli: contagious tumor?
Fears: human health and ecosystem of Earth
Asilomar Conference (1975): Something can be considered dangerous
if it is proved to be dangerous
Paul Berg
James Watson
Sidney Brenner
5.
Today’s fears
1. Human-made contagious diseases - AIDS from poliomyelitis vaccine?
2. We alter human evolution
3. We interfere to the Creator’s business
4. Genetically modified foods are unhealthy
5. We change the ecosystems
6.
Illegitimate recombination
- Random DNA integration
plasmid vector
transgene
chromosome
transgene
transgene
transgene
7.
Homologous recombination
- targeted DNA insertion
transfer vector
flanking seq.
foreign DNA
flanking seq.
chromosome
target sequence
chromosome
foreign DNA
Genetically modified sequence
Knock-in
Knock-out
8.
8.
Cultured cell types
1. Primary cell culture
2. Tumor cell culture
3. Immortalized cell culture
10.
Gene delivery techniques
– to cultured cells
Transfection
1. Liposome
2. Ca-phosphate
3. Electroporation
4. Gene gun
Virus vector
1. Retroviruses
2. Adenoviruses
3. Adeno-associated viruses
LIPOSOME
DNA
Protective
layer
crystallized
drog
Homing
peptides
Lipid Water-soluble
drog
bilayer
11.
Gene delivery methods
Liposome
electroporation
retrovirus
12.
Foreign gene (transgene)
promoter
transgene
T
transfection
13.
Foreign gene – transient gene expression
13.
Foreign gene – transient gene expression
14.
Foreign gene – stable gene expression
14.
Foreign gene – stable gene expression
15.
Genetically Modified
Organisms (GMOs)
Definition: addition of novel genetic material to the genome of an organism
1. Microorganisms
1. Basic research
2. Economic benefit
2. Plants
3. Animals
4. Human
GMO: Genetically Modified Organism
16.
Transgenic animals
What are they?
- Animals with one or more foreign genes in their genomes
What are they good for?
- (1) examination of gene function; (2) economically important GMOs, (3) gene therapy in future
17.
Gene delivery techniques – to living organisms
Germline gene delivery
 foreign gene is present in every cell
1. DNA microinjection zygote
2. Gene delivery to ES cells
3. Virus vectors (retroviruses)  zygote
Somatic gene delivery
1. Physico-chemical
2. Virus vectors
3. Stem cells
 foreign gene is NOT present in every cell
Direct: in vivo
Cell-mediated: ex vivo
Delivery of foreign genes
to zygote by pronuclear inoculation
promoter
foreign gene
Pronuclear microinjection
plasmid
foreign gene with promoter
+
pronuclear injection
reimplant injected embryos
to pseudopregnant females
transgenic founder
transgenic offspring
18a.
Foreign gene delivery
18b.
to the zygote by pronuclear microinjection
Pronuclei
Inject foreign DNA into the male pronucleus
+
Fertilized mouse egg prior to fusion of male and female pronuclei
Transfer injected
eggs into foster mother
About 10 to 30% of offspring contain injected foreign DNA. Foreign DNA is present in equal amounts
in all tissues
Mice expressing of foreign DNA are bred to continue DNA in germ line
Delivery of foreign genes
by retrovirus vector
19.
Delivery of foreign genes
to ES cells
Internal cell mass
Blastocyst
20.
21.
3.
1.
2.
Cloned gene
5.
4.
Gene delivery
to ES cells
6.
8.
9.
22.
Regulation of gene expression
A. Transgene expression (gain of function
1. Constitutive (continuous)
a. Ubiquitous expression
UP
transgene
b. “Semi-ubiquitous expression
PNP
transgene
c. Cell-specific expression
TSP
transgene
IP
transgene
2. Inducible
B. Gene inactivation (loss of function)
1. Knock-out technology
2. RNA interference
Cell-specific gene expression
BAC - technology
Regulatory elements
Genomial
DNA
BAC
100,000- 400,000 bp
RE digestion
Original gene
(exon + intron)
Ligation
Foreign gene
Recombinant
BAC
Transformation
Micro-inoculation
Transgenic mouse
23.
24.
Knock-out animals
Investigation paradigm: the function of the knocked out gene is the reverse as the obtained phenotype is
2007
M. Evans: development of ES cell technology (ES cells are used for knock-out technology)
M. Capecchi & O. Smithies: homologous recombination:
- knock-out technology
- replacement of defected genes with functional ones:
importance in medicine
25.
Generation of knock-out animals
Genomic DNA
Zygote
blastula
neo
flanking seq.
flanking seq.
„Targeting” plasmid construction
tk
Inner cell mass
Genomic DNA
ES cells
neo
Genomic DNA
Homologous recombination
Genomis DNA
neo
Genomic DNA
tk
Illegitimate recombination
Donor parents-1
Donor parents-2
x
1.

neo

x
tk
3.
2.
5.
Targeting sequence
4.
Microinjection
ICM
Blastocyst
ES cell culture
Mixing of ES cells with
the foreign DNA Selection of transgenecontaining cells
6.
Recipient parent
Transgenic ES cell
integration to the embryo
8.
Gene knock-out
in ES cells
Chimeric mice
9.
x
Chimera
Homozygote normal
Heterozygote
Normal
10.
x
Heterozygote mice
Homozygote knockout
26.
27.
In vivo RNA interference
3b.
lentivirus
adenovirus
What RNAi is good for?
(1) analysis of gene function
dsRNA
shRNA- expressing DNS
siRNA
3a.
1.
cell membrane
1.
2.
nuclear membrane
DICER
integration
shRNA
siRNA
(2)
shRNA
episome
DICER
therapy:
RISC
(a) blocking the expression of mutant gene
Stabile expression
RISC
Transient expression
siRNA
(b) blocking the overexpression of protooncogens in tumors
Activated RISC
mRNA
What do we deliver? Where to deliver?
LOCAL - SYSTEMIC
Intracerebral
Intravascular perfusion,
Hypodermal injection
(1) long dsRNA
(not applicable
in mammals, since it evokes apoptosis)
(2) siRNA
(systemic or local)
Intraperitoneal
(3) siRNA encoding DNA
(systemic or local)
(3a) Plasmid DNA
(3b) Virus vector
(lentivirus, adenovirus, adeno-associated virus)
Types of siRNAs
(2) In vitro
transcription
(1) Chemical synthesis
extracellular space
(3b) Virus vector
(3a) Plasmid vector
long dsRNA
Dicer
Dicer
cytoplasm
shRNA
Nucleus
shRNAcoding DNA
28.
Dicer
29.
RNA interference
nucleus
cytoplasm
shRNA
exportin
DICER
Degraded
mRNA
P body
target
mRNA
DICER
Acivated RISC
Single-stranded
siRNA
shRNA: small hairpin RNA
30.
The CRISPR system
CRISP: clustered regularly interspaced short palindromic repeats